Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms Pathiraja Mudiyanselage Gathanayana Pathiraja B.Sc. (Honours) in Microbiology M.Sc. in Environmental Science and Technology Submitted in Fulfilment of the Requirements for the Award of the Degree of Doctor of Philosophy Science and Engineering Faculty Queensland University of Technology 2018
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Bioremediation of Commercial
Polychlorinated Biphenyl Mixture
Aroclor 1260 by Naturally Occurring
Microorganisms
Pathiraja Mudiyanselage Gathanayana Pathiraja
B.Sc. (Honours) in Microbiology
M.Sc. in Environmental Science and Technology
Submitted in Fulfilment of the Requirements for the Award of the
Degree of Doctor of Philosophy
Science and Engineering Faculty
Queensland University of
Technology
2018
Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms i
1.2 Research Problem ........................................................................................ 2
1.3 Research Hypothesis ................................................................................... 3
1.4 Aims and Objectives .................................................................................... 4
1.5 Research Scope ............................................................................................ 4
1.6 Innovation and Contribution to Knowledge ................................................ 5
1.7 Research Design and Methodology ............................................................. 6
1.7.1 Critical review of research literature ...................................................... 9
1.7.2 Isolation, screening and identification of potential PCB degrading microorganisms .................................................................................... 9
1.7.3 Screening of bacterial isolates for their ability to produce biosurfactants to make hydrophobic PCBs soluble in aqueous media ...................... 10
1.7.4 Comparison of the individual facultative anaerobic bacterial isolates during PCB hydrolysis under aerobic, anaerobic and two stage anaerobic-aerobic conditions ............................................................. 11
1.7.5 Comparison of the bacterial consortium during PCB hydrolysis under two modes of combined anaerobic-aerobic treatments ................... 12
1.7.6 Analysis and characterization of proteins detected in the culture supernatants during PCB degradation. .............................................. 12
3.2.10 Screening tests for biosurfactant production ....................................... 73
3.2.11 Extracellular protein visualization, quantification, extraction and analysis ............................................................................................... 73
3.2.12 Storage of culture supernatant samples .............................................. 73
4.2.3.3 Identification of bacterial isolates using 16S rRNA full length gene sequencing ...................................................................... 81
4.2.3.4 Confirmation of the ability to grow on PCBs as sole carbon source ....................................................................................... 84
4.2.3.5 Growth profiles of bacteria ...................................................... 85
4.3 Results and Discussion ............................................................................... 86
4.3.1 Screening and Identification of PCB utilizing culture members ........... 86
4.3.1.1 16S rRNA gene based identification ........................................ 86
4.3.1.2 Morphological characteristics of identified bacteria ............... 93
4.3.1.3 Screening of microorganisms based on tolerance to atmospheric oxygen ................................................................. 96
4.3.2 Basic growth profiles using glucose as the carbon source ................... 98
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium ........................ 135
7.2.2 Frequency, collection and preservation of samples ........................... 137
7.2.3 Total PCB extraction and analysis ....................................................... 139
7.2.4 Carbon and nitrogen source utilization profiling of bacterial cultures 139
7.2.4.1 Procedure for Gram positive bacteria ................................... 140
7.2.4.2 Procedure for Gram negative bacteria .................................. 141
7.3 Results and Discussion ............................................................................. 142
7.3.1 Test for competition ........................................................................... 142
7.3.2 PCB degradation by the bacterial consortium under AN and TS treatments ........................................................................................ 143
7.3.2.1 Total PCB degradation ........................................................... 143
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 ................................................................ 163
9.1.1 Isolation, screening and identification of potential PCB degrading microorganisms ................................................................................ 201
9.1.2 Screening of bacterial isolates for their ability to produce biosurfactants to make hydrophobic PCBs soluble in aqueous media .................... 201
9.1.3 Comparison of the individual facultative anaerobic bacterial isolates during PCB hydrolysis under aerobic, anaerobic and two stage anaerobic-aerobic conditions ........................................................... 202
9.1.4 Comparison of the bacterial consortium during PCB hydrolysis under two modes of combined anaerobic-aerobic treatments ................. 203
9.1.5 Analysing and characterization of proteins detected in the culture supernatants during PCB degradation ............................................. 204
9.2 Practical applications of research outcomes ........................................... 206
9.3 Recommendations for future research ................................................... 207
Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xi
Appendix B: PCB data related to bacterial consortium study ....................... 251
Appendix C: Extracellular protein analysis .................................................. 255
Supplimentary Material .................................................................................... 291
Supplementary Material 1: Abstracts of conference papers relevant to the thesis .......................................................................................... 293
Supplementary Material 2: Publications relevant to the thesis ................... 295
xii Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms
List of Figures
Figure 1.1 Schematic representation of the research methodology ........................... 8
Figure 2.1 Structural form of PCB. Clx and Cly are number of chlorines attached to each benzene ring and x + y = 1 to 10. Adapted from Wiegel and Wu (2000). .......................................................................................................... 17
Figure 2.2 Concentrations of total PCBs in surface soils at global background sites (Li et al., 2010). .................................................................................... 22
Figure 2.3 Potential pathway for anaerobic dechlorination of highly chlorinated congeners. Adapted from Borja et al. (2005). ............................................. 31
Figure 2.4 The upper biphenyl degradation pathway. Modified from Field and Sierra-Alvarez (2008). ................................................................................... 35
Figure 2.5 The lower biphenyl degradation pathways (a) Mineralization of 2-hydroxypenta -2,4-dienoate. Modified from Field and Sierra-Alvarez (2008). (b) Mineralization of chlorobenzoic acid. Modified from ATSDR (2000). .......................................................................................................... 38
Figure 2.6 Overview of factors affecting microbial remediation ............................... 40
Figure 2.7 Variation of the bacterial community at genus level in the bulk, top and rhizosphere soils. The phylum of each genus is reported in brackets (Acid-Acidobacteria; Act-Actinobacteria; Alph-Alphaproteobacteria; Beta-Betaproteobacteria; Chlo-Chloroflexi; Firm-Firmicutes; Gamm-Gammaproteobacteria; Gemm-Gemmatimonatedes). Adapted from Stella et al. (2015). ........................... 45
Figure 2.8 The omics pyramid. Modified from NASEM (2016). ................................. 52
Figure 2.9 Organism based and community based “omic” approaches for assessing bioremediation approaches. Modified from Chovanec et al. (2011). .......................................................................................................... 54
Figure 3.1 Standard plate count technique ............................................................... 67
Figure 3.2 Recovery of Aroclor 1260 soluble in the aqueous minimal salt medium as a percentage of total PCBs added to the mixture, using diethyl ether (DEE) and hexane as the extraction solvents. Error bars represent the standard deviation of mean values (n = 3). .......................... 70
Figure 3.3 The Coy anaerobic chamber with the connecting airlock to the right, used in this research. ................................................................................... 74
Figure 4.1 Selective enrichment of potential PCB degrading bacteria, under aerobic and anaerobic conditions at 28 °C. ................................................. 80
Figure 4.2 Agarose gel electrophoresis of genomic DNA isolated from bacterial isolates using Isolate II genomic DNA kit (Bioline). A1 to A5 were from
Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xiii
aerobic selective enrichments and AN1 to AN6 were from anaerobic selective enrichments. ................................................................................. 87
Figure 4.3 Blue-white screening on LB/ampicillin/IPTG/X-Gal plate after the transformation into high efficiency E. coli JM109 competent cells. White colour colonies representing the positive transformations. ............ 88
Figure 4.4 Agarose gel electrophoresis of purified recombinant plasmids. 5 µL from each sample was loaded into the corresponding well. A1 to A5 were from aerobic selective enrichments and AN1 to AN6 were from anaerobic selective enrichments. Instead of plasmids, sterile MilliQ water was used in the negative control. ..................................................... 88
Figure 4.5 Pairwise alignment of forward and reverse DNA sequences of bacterial isolate AN2 using Emboss Needle software. Total length after the alignment was 1459 and number of similarities between two sequences were 738/1459 (50.6%). ............................................................ 89
Figure 4.6 Colony morphology of the bacterial cultures on nutrient agar after 48 h incubation at 28 °C. Culture A to F were under aerobic conditions and culture G was under anaerobic conditions. .......................................... 94
Figure 4.7 Gram stained bacterial cultures under the light microscope (100x magnification). ............................................................................................. 95
Figure 4.8 Bacterial colonies on minimal salt agar with 50 mg/L Aroclor 1260 as sole source of carbon after 48 hrs at 28 °C (A) under aerobic conditions, (B) facultative anaerobic cultures under anaerobic conditions, (C) negative controls under aerobic conditions (D) negative controls under anaerobic conditions. ........................................................................ 97
Figure 4.9 Growth of obligate anaerobic Novosphingobium sp. NP07 on 25 mg/L and 50 mg/L Aroclor 1260 containing minimal salt agar after 48 h incubation at 28 °C under anaerobic conditions. ........................................ 98
Figure 4.10 Basic bacterial growth profiles of the six bacterial isolates grown in minimal salt medium over a nine hour period at 28 °C and 150 rpm using glucose as the carbon source. Error bars represent the standard deviation of mean values (n = 3). ................................................................ 99
Figure 4.11 Variation of pH in the culture medium during the bacterial growth profile studies. Error bars represent the standard deviation of mean values (n = 3). ............................................................................................. 101
Figure 5.1 Variation of the total solubility of Aroclor 1260 in the aqueous minimal salt media inoculated with bacterial cultures. Error bars represent the standard deviation of mean values (n=3 for bacterial cultures and n=2 for abiotic controls). ...................................................... 107
Figure 5.2 Bacterial growth as optical density (OD600) in the batch mesocosms at 28 °C and 150 rpm. Error bars represent the standard deviation of mean values (n=3). ..................................................................................... 109
xiv Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms
Figure 5.3 Chloride ion accumulation in the batch mesocosms after six weeks of incubation at 28 °C and 150 rpm. The background values from the controls of (1) minimal salt medium only and (2) seed cultures only were subtracted first. Error bars represent the standard deviation of mean values (n=3). ..................................................................................... 110
Figure 5.4 pH variation in the batch mesocosms at 28 °C and 150 rpm. Error bars represent the standard deviation of mean values (n=3). .................. 112
Figure 5.5 Drop collapse test. 1% (w/v) sodium dodecyl sulphate (SDS) solution was used as the positive control. The phosphate buffered saline (PBS) solution and abiotic control (minimal salt medium only) were used as negative controls. ....................................................................................... 114
Figure 5.6 Haemolysis of sheep blood in Tryptone soya agar after incubation at 28 °C for 48 hours (A) Chryseobacterium sp. NP01, (B) Delftia sp. NP02, (C) Achromobacter sp. NP03, (D) Ochrobactrum sp. NP04, (E) Lysinibacillus sp. NP05, (F) Pseudomonas sp. NP06, (G) 1% SDS as positive control, and (H) abiotic control. ................................................... 115
Figure 6.1 Growth of the four facultative anaerobic bacterial strains under (a) aerobic, (b) anaerobic, and (c) two stage anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values (n = 3). ....... 125
Figure 6.2 PCB solubility under (a) aerobic, (b) anaerobic, and (c) two stage anaerobic-aerobic conditions. Prior to the addition of microbes, samples were removed and analysed for Initial soluble PCBs measurement and then after adding microbes, samples were removed immediately and represent week 0. Error bars represent the standard deviation of mean values from triplicates. ................................................ 128
Figure 6.3 Chloride ion accumulation in the culture media after six weeks. The background values from the controls of (1) minimal salt medium only and (2) seed cultures only were subtracted first. Error bars represent the standard deviation of mean values from triplicates. .......................... 130
Figure 6.4 Variation of pH and chloride ion concentrations after six weeks under aerobic, anaerobic and two stage anaerobic-aerobic conditions (Initial pH was adjusted to 7.0). Error bars represent the standard deviation of mean values from triplicates. ................................................................ 132
Figure 6.5 Growth profile, PCB hydrolysis and pH variation of Lysinibacillus sp. NP05 under two stage anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values from triplicates. .......... 133
Figure 7.1 Inoculation of batch mesocosms with bacterial seed cultures inside the anaerobic chamber. ............................................................................. 137
Figure 7.2 Growth of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 on minimal salt-Aroclor 1260 agar at 28 °C after 72 h (a) under aerobic conditions, (b) under anaerobic conditions. ......... 143
Figure 7.3 Total PCB degradation as a percentage and bacterial growth as OD600 under (a) alternating and (b) two stage anaerobic-aerobic treatments
Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xv
by the bacterial consortium. Error bars represent the standard deviation of mean values (n = 3). .............................................................. 145
Figure 7.4 Variation of PCB homolog groups following AN treatment; (a) lower chlorinated congener groups (mono to tetra), and medium to highly chlorinated congener groups, (b) penta to hepta, (c) octa and nona. Error bars represent the standard deviation of mean values (n = 3). ....... 148
Figure 7.5 Variation of PCB homolog groups following TS conditions; (a) lower chlorinated congener groups (mono to tetra), and highly chlorinated congener groups, (b) penta to hepta, (c) octa and nona. Error bars represent the standard deviation of mean values (n = 3). ........................ 150
Figure 7.6 Chloride ion accumulation under alternating (AN) and two stage (TS) anaerobic-aerobic conditions. The background chloride values from the minimal salt medium were first subtracted from experimental values and media controls. Error bars represent the standard deviation of mean values (n=3 for experimental values and n=2 for media controls). .................................................................................................... 151
Figure 7.7 Measured chloride ion buildup in the culture medium and calculated chloride ion removal from the PCB mixture based on homolog group reductions under; (a) alternating (AN), and (b) two stage (TS) anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values (n = 3). .............................................................. 153
Figure 7.8 pH trends relative to chloride ion concentration. (a) AN and (b) TS anaerobic-aerobic treatments. Error bars represent the standard deviation of mean values (n = 3). .............................................................. 155
Figure 8.1 The secretome and exoproteome of a Gram negative bacterial cell. (Armengaud et al., 2012) ........................................................................... 164
Figure 8.2 Role of extracellular enzymes in insoluble compound metabolism. ...... 165
Figure 8.3 SDS-PAGE analysis of Lysinibacillus sp. NP05 containing controls (minimal salt medium with no added PCBs) under anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6....................................................... 173
Figure 8.4 SDS-PAGE analysis of extracellular proteins of Achromobacter sp. NP03 under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6. ...................................................................................... 174
Figure 8.5 SDS-PAGE analysis of extracellular proteins of Ochrobactrum sp. NP04 under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6. ...................................................................................... 175
xvi Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms
Figure 8.6 SDS-PAGE analysis of extracellular proteins of Lysinibacillus sp. NP05 under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6. ...................................................................................... 176
Figure 8.7 SDS-PAGE analysis of the extracellular proteins of the bacterial consortium consisting of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 under (A) AN, and (B) TS anaerobic-aerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 2 to 5, at fortnightly intervals up to week 6. ................................................................................................................. 177
Figure 8.8 Proportions of classically and non-classically secreted proteins in the culture supernatant of the bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05. ..................... 183
Figure 8.9 Functional groupings of proteins identified as classically secreted proteins. ..................................................................................................... 184
Figure 8.10 The concentration of the sulfate transporter protein detected in the culture supernatant over time, under the alternating anaerobic-aerobic (AN) conditions. ............................................................................ 188
Figure 8.11 Venn diagram of the distribution of proteins identified as classically secreted proteins among (A) alternating (AN) anaerobic-aerobic, and (B) two stage (TS) anaerobic-aerobic conditions. ...................................... 190
Figure 8.12 Functional groupings of proteins identified as non-classically secreted proteins. ...................................................................................... 191
Figure 8.13 Variation of glutamine synthetase concentration and pH level in the culture supernatant under alternating anaerobic-aerobic conditions. ..... 192
Figure 8.14 Distribution of non-classically secreted proteins under (A) AN anaerobic-aerobic treatment, and (B) TS anaerobic-aerobic treatment. .................................................................................................................... 193
Figure 9.1 A schematic representation of the summary of major findings of the research study. ........................................................................................... 200
Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xvii
List of Tables
Table 2.1 PCB homologs and their chlorine substitutions ......................................... 18
Table 2.2 Comparison of PCB levels (µg/kg dry weight) in soils based on selected international studies. ................................................................................... 23
Table 2.3 Plant–microbial relationships associated with PCB degradation .............. 27
Table 2.5 Types and microbial origin of biosurfactants. ............................................ 49
Table 3.1 Summary of physicochemical properties of Aroclor 1260 ........................ 57
Table 3.2 Average weight percent of PCB homolog groups and chlorines in Aroclor 1260. ................................................................................................ 72
Table 4.1 Comparison of closest relatives of isolated bacteria based on NCBI and RDP databases ...................................................................................... 91
Table 4.2 Final nomenclature and identification of the pure bacterial isolates ....... 92
Table 4.3 Specific growth rate of bacterial cultures during the growth profile studies using 2 g/L glucose as the carbon source. ..................................... 100
Table 5.1 Summary of biosurfactant screening tests .............................................. 113
Table 6.1 Bacteria cell count in overnight Luria–Bertani liquid medium at 28 °C..................................................................................................................... 123
Table 7.1 Ingredients for the nutrient additive solution, 12x (PM additive) ........... 140
Table 7.2 Preparation of final inoculation fluid to inoculate the Biolog plates....... 141
Table 7.3 Carbon source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05. .................... 158
Table 7.4 Nitrogen source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05. ............... 159
Table 8.1 Standard dilutions preparation for BCA assay ......................................... 167
Table 8.2 Analysis of extracellular proteins identified in culture supernatants of consortium under AN and TS conditions. .................................................. 178
Table 8.3 Functional classification of 319 identified proteins that were predicted to be non-secretory proteins by the SignalP 3.0 and SecretomeP 2.0 servers. ............................................................................ 179
Table 8.4 Heat map showing the relative abundance of ten highly secreted extracellular proteins detected in the culture supernatant of the bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05. .............................................................. 186
xviiiBioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms
List of Abbreviations
Abbreviations
AN Alternating anaerobic-aerobic treatment
CFU Colony forming units
DEE Di ethyl ether
DNS Dinitrosalicylic acid
GCMS Gas chromatography-mas spectroscopy
gDNA Genomic DNA
IPTG Isopropyl β-D-1-thiogalactopyranoside
LB Luria–Bertani medium
LCMS Liquid chromatography–mass spectrometry
NA Nutrient Agar
NCBI National Centre for Biotechnology Information
MSM Minimal salt medium
OD600 Optical density at 600 nm
PBS phosphate buffer saline
PCBs Poly chlorinated biphenyls
PCR Polymerase chain reaction
RDP Ribosomal database project
RSDV Relative standard deviation
SDS Sodium dodecyl sulphate
SOC Super optimal broth with catabolite repression
SRM Selective reaction monitoring
SWATH Sequential window acquisition of all theoretical mass spectra
TSA Tryptone soya agar
TS Two stage anaerobic-aerobic treatment
Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xix
Units
bp Base pair(s)
g Gram(s)
g Relative centrifugal force in units of gravity
h Hour(s)
kb Kilobase
kDa Kilodalton
min Minute(s)
mol Mole(s)
rpm Revolutions per minute
s Second(s)
v/v Volume per volume
w/v Weight per volume
xx Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms
Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the
best of my knowledge and belief, the thesis contains no material previously published
or written by another person except where due reference is made.
Signature:
Date: November 2018
QUT Verified Signature
Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xxi
Acknowledgements
The successful completion of my thesis would not have been possible without the
guidance, support, encouragement and help from my supervisory committee,
technical staff, scholarship from the Australian Government, my family and friends.
I am extremely thankful to my principal supervisor, Associate Professor V. S. Junior
Te'o, for his invaluable guidance and support throughout my PhD research. I would
also like to express my sincere thanks to my associate supervisor, Professor Ashantha
Goonetilleke for his guidance, support and encouragement to complete my research
study successfully. My sincere thanks is extended to my associate supervisor, Dr.
Prasanna Egodawatta for giving me the opportunity to undertake my PhD study at
QUT and for his continuous support and guidance.
I sincerely acknowledge Queensland University of Technology (QUT) for providing me
financial support through a RTPSD scholarship to conduct this doctoral research
study. I would also wish to acknowledge the staff of Central Analytical Research
Facility (CARF) of QUT, especially Mr. Vincent Chand and Mr. Shane Russell for their
technical support, Ms. Silvia Gemme for supporting me with the GCMS analysis, Dr.
Pawel Sadowski for helping me with proteomics. I am very thankful to Mr. Tony Tuong
Ngo, Powerlink Queensland Oil testing services, for supplying me transformer oil
samples without any hesitation to initiate my experiments.
I would like to extend my thanks to my fellow HDR colleagues for their support,
friendship, and encouragement. I am very grateful to my husband Samanola and kids
Vinuri, Mindulie and Biman for their love, support, patience, and encouragement to
complete my PhD study. Finally, my parents are remembered with love and gratitude
for their guidance and blessings throughout my life.
xxii Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms
List of thesis associated publications
Conference Papers
• Pathiraja P.M.G., Egodawatta P., Goonetilleke A., Te'o V.S. J. Degradation of
commercial polychlorinated biphenyl mixture by naturally occurring facultative
microorganisms.
Nineteenth International Conference on Environmental Biodegradation
Rates, Sydney, Australia, 2017.
Journal Papers (published)
• Pathiraja, G., Egodawatta, P., Goonetilleke, A., & Te'o, V. S. J. (2019). Solubilization
and degradation of polychlorinated biphenyls (PCBs) by naturally occurring
facultative anaerobic bacteria. Science of The Total Environment, 651, 2197-2207.
Journal Impact Factor: 4.9, SJR Rank Q1.
Journal papers (Under review)
• Effective degradation of polychlorinated biphenyls by three facultative anaerobic
bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium.
Submitted to Science of the Total Environment, Journal Impact Factor: 4.9, SJR Rank
Q1.
Chapter 1: Introduction 1
Chapter 1: Introduction
1.1 Background
The diversity and magnitude of man-made toxic chemicals released into the
environment are creating long-term ecological impacts. Polychlorinated biphenyls
(PCBs) are one such toxic chemical group, consisting of 209 different chlorinated
organic compounds. Due to their unique chemical and physical properties, PCBs had
been used widely in many industrial applications, especially as insulating fluids in
transformers, capacitors, hydraulic systems, and as flame retardants. As a result,
since 1930, PCBs have been manufactured commercially in large scale as complex
mixtures.
The low reactivity, high chemical stability and complex nature of commercial PCB
mixtures have made them highly persistent and less environmentally desirable than
many other organic chemicals (Beyer & Biziuk, 2009). Their affinity to build up in living
organisms though bioaccumulation over time, biomagnification along the food chains
and resistance to biotransformation have led to numerous health implications in
humans and animals. As a result, PCBs were categorized as one of the original twelve
worldwide priority persistent organic pollutants (POPs) covered by the Stockholm
Convention (Bedard et al., 2007). Even though commercial production and use of
PCBs were banned or restricted decades ago, there is still a substantial amount of
PCBs present in the environment due to the continuing use and disposal of
equipment containing PCBs, recycling of PCB-contaminated products, emissions from
combustion of PCB contaminated waste, contaminated sites and disposal areas
(Breivik et al., 2007).
Treating environmental media such as soil contaminated with PCBs is of vital
importance to safeguard human and ecosystem health. In this regard, a range of
physical, chemical and biological treatment technologies have been investigated to
identify new remediation possibilities (Gomes et al., 2013). Bioremediation, the use
of microorganisms or microbial processes to degrade environmental contaminants,
2 Chapter 1: Introduction
is one of the methods intensively investigated for treating PCB-contaminated soils
(Boopathy, 2000).
1.2 Research Problem
Developing an environmentally sustainable and economically viable biological
treatment method as an alternative to existing physical and chemical treatment
methods for treating soil contaminated with PCBs is of critical importance. Over the
last decades, some aerobic and anaerobic microorganisms capable of degrading a
broad range of PCBs have been identified (Dercova et al., 2008; Sowers & May, 2013).
However, the application of microbial based bioremediation to treat PCB
contaminated soil is not yet widely practiced. The effectiveness of bioremediation is
determined by a number of factors, such as the lack of suitable microorganisms in
the contaminated environment, difficulties in maintaining the PCB degrading
bacterial communities in real-world environmental conditions, the nature of the PCB
mixture and the severity of contamination, low bioavailability of PCBs and the
characteristics of the contaminated environment.
As noted by Passatore et al. (2014), complete degradation of commercial PCB
mixtures that consist of compounds with varying degree of chlorination by
microorganisms can be achieved through a combination of anaerobic and aerobic
processes. It was recognized that the anaerobic dechlorination of more highly
chlorinated congeners followed by the aerobic degradation of those dechlorinated
products are the most likely degradation pathways occurring in the environment
(Payne et al., 2013). However, due to the complexity of commercial PCB mixtures
with the varying degree of chlorination, a single bacterium is not capable of degrading
all or even most of the PCB congeners present in contaminated environments (Pieper,
2005). Therefore, the search for appropriate microorganisms with the ability to
survive and degrade complex PCB mixtures under both anaerobic and aerobic
conditions would be a potential solution to achieve an efficient and effective process
for the biodegradation of PCBs.
Chapter 1: Introduction 3
Stella et al. (2015) noted that the extremely hydrophobic nature of PCBs makes them
poorly soluble in aqueous media, and this attribute can lead to PCBs being less
bioavailable for microbial degradation. Therefore, an increase in solubilisation would
enhance the bioavailability and subsequent biodegradation of PCBs (Ohtsubo et al.,
2004). However, the application of chemical and biological surfactants to increase
PCB solubility is limited due to various factors, such as chemical toxicity and high cost.
Therefore, the identification of suitable microorganisms that are capable of surviving
under a PCB contaminated environment, while producing biosurfactants would
accelerate PCB solubility and subsequent degradation.
Moreover, both anaerobic and aerobic PCB degradation pathways so far identified in
microorganisms were found to have occurred intracellularly (Wiegel & Wu, 2000;
Pieper, 2005; Agullo et al., 2017). However, the mechanism of transportation of PCB
molecules across the cytoplasmic membrane of microorganisms is not clear (Parales
& Ditty, 2017). The proteins or enzymes released and/or secreted by the
microorganisms to the extracellular environment may provide novel insights into the
mechanisms involved in modifying and transporting hydrophobic PCB molecules into
the cell (Basak & Dey, 2015). Therefore, identification of proteins released by
microorganisms into their surrounding extracellular environment and the functional
relationship with the PCB uptake into the bacterial cells would be important in order
to accelerate the degradation process.
Accordingly, this study aimed to discover microorganisms that can survive and
effectively degrade PCBs under both anaerobic and aerobic conditions, while
facilitating aqueous solubility and cellular uptake.
1.3 Research Hypothesis
The research study was based on the following hypotheses:
• Complete degradation of complex PCB mixtures can be achieved through a
combination of anaerobic-aerobic treatments.
4 Chapter 1: Introduction
• Microbial mixtures with varied capabilities for increasing the aqueous
solubility of PCBs, dechlorinating highly chlorinated congeners and complete
degradation of lower chlorinated congeners are required for comprehensive
degradation of PCBs.
• Extracellular proteins or enzymes released and/or secreted by the
microorganisms cultivated in minimal salts based medium with PCBs as a
carbon source, facilitate the uptake of PCB molecules into the bacterial cells
for degradation.
1.4 Aims and Objectives
The primary objective of this research was to identify suitable microorganisms from
the natural environment, which are capable of solubilizing and degrading complex
PCB mixtures under varying anaerobic and aerobic conditions in order to effectively
treat soil contaminated with PCBs.
To achieve this objective, the study aimed to:
• Isolate, screen, identify and characterize naturally occurring microorganisms
and select the best ones for further work, based on their ability to effectively
degrade PCBs under aerobic and anaerobic conditions.
• Screen the identified microorganisms for biosurfactant production in order to
increase the water solubility of PCBs.
• Determine the PCB degradation potential of facultative bacterial cultures
under anaerobic and aerobic conditions both individually and as a consortium.
• Analyse the extracellular proteins released by microorganisms into the
external environment.
1.5 Research Scope
The scope of this research study was as follows:
Chapter 1: Introduction 5
• The PCB source used in the present study was limited to Aroclor 1260, one of
the most commonly used and highly chlorinated commercial PCB mixtures
(USEPA, 2013). Its abundant usage has resulted in contamination of many
sites worldwide and has proven to be difficult to biodegrade due to its high
levels of chlorination (Bedard et al., 2007). Aroclor 1260 contains 60 to 90
different PCB congeners out of 209 possibilities (ATSDR, 2000; Breivik et al.,
2007). The outcomes of the study may not be applicable to PCB congeners
outside of the congeners available in Aroclor 1260.
• The research study was confined to treating soil contaminated with Aroclor
1260. However, the knowledge created in relation to the PCB solubility,
uptake and degradation by microorganisms is applicable to other
contaminated media such as sediments.
1.6 Innovation and Contribution to Knowledge
Investigating the performance of facultative anaerobic bacteria on PCB degradation
is expected to provide new insights to the knowledge base and research field. The
use of facultative anaerobic bacteria under anaerobic and aerobic conditions has led
to opportunities for further research based on their ability to degrade PCBs under
both aerobic and anaerobic conditions. Though a range of studies have so far
discussed PCB degradation potential of aerobic and anaerobic bacteria, their practical
use in the real-world environment is limited. Therefore, the innovative outcomes
from this study can be effectively utilized to treat contaminated soil under varying
anaerobic aerobic conditions in the field without losing the degradability and viability
potential.
The study also created new knowledge on the ability of PCB degrading bacteria to
produce biosurfactants. This finding is essential for designing an effective process to
enhance the bioavailability of PCBs in remediation applications, without addition of
toxic chemical surfactants or costly biological surfactants. Furthermore, the new
knowledge created relating to the identification of the alternating anaerobic-aerobic
treatment method as a rapid and efficient treatment option over the conventional
6 Chapter 1: Introduction
long-term two stage anaerobic-aerobic treatment can be successfully applied in
order to increase the effectiveness and reduce the cost of lengthy treatments. These
discoveries will contribute towards enhancing current microorganism based
bioremediation approaches to treat soils contaminated with PCBs and other similar
toxic chemical contaminants, in order to reduce and eventually eliminate their
harmful impacts on ecosystems and human health.
1.7 Research Design and Methodology
Effective biodegradation of PCBs is a complex process that involves a combination of
factors such as having suitable microbial cultures, complexity, concentration and
bioavailability of PCB congener mixtures, and environmental conditions. Therefore, the
research methodology was formulated in order to first, isolate, screen and identify
appropriate microorganisms with PCB degradation potential, second, react selected
microorganisms with PCBs, and third, analyse the results and characterize the key
contributing factors influencing the extent of PCB degradation by the microorganisms.
This section outlines the research methodology adopted.
The research methodology consisted of the following phases:
• Critical review of research literature.
• Isolation, screening and identification of potential PCB degrading
microorganisms.
• Screening of bacterial isolates for their ability to produce biosurfactants to make
hydrophobic PCBs soluble in aqueous media.
• Comparison of PCB degradation rates of facultative anaerobic bacterial isolates
individually under aerobic, anaerobic and two stage anaerobic-aerobic
conditions.
• Comparison of PCB degradation efficiency of selected bacterial isolates as a
consortium under two modes of combined anaerobic-aerobic treatments.
• Detection, identification and bioinformatics analysis of peptides produced from
proteins found in the culture supernatants during PCB hydrolysis indicative of
protein or enzyme secretion.
Chapter 1: Introduction 7
• Assessment of the types of proteins and a discussion of their possible roles in PCB
hydrolysis, and potential mechanisms of protein externalization by the
microorganisms.
The critical review of the research literature was first undertaken to identify the
knowledge gaps and research questions in PCB bioremediation, and thereby to
formulate the approaches to investigate the research problem. Figure 1.1 shows the
schematic of the research methodology describing the development of key study steps,
which are discussed in Chapters 4 to 8.
8 Chapter 1: Introduction
Figure 1.1 Schematic representation of the research methodology
Switzerland (0.86 – 12)f n=23, (10cm) (Schmid et al., 2005)
South Sweden (2.3 - 986) n=66, (5 cm) (Backe et al., 2004)
Dalian, Liaoning Province, China
2.8 (1.3 - 4.8)g n=14, (5 cm) (Wang et al., 2008)
KwaZulu-Natal, South Africa
109.64±116.07h
19.22±33.23i
n=15, (0.5 cm)
n=15, (1–2 cm)
(Batterman et al., 2009)
Notes:
n – number of samples, NM – Not mentioned a 31.89% hexa, 23.98% penta, 18.47% tri, 13.67% tetra & 11.99% hepta PCBs b All PCB congeners were present except PCB 18 c Sum of 33 PCB congeners with 9% PCB 153, 8.6% PCB18, 8.8% PCB28 & 7% PCB138. d 16.6% PCB 56, 9.8% PCB 52, 9.6% PCB 44 e 20.9% PCB 56 f Sum of 7 PCBs g Sum of 57 PCBs h, i As wet weight of 38 congeners, most prevalent PCBs - 41+ 71, 153+132, 138 +163
24 Chapter 2: Bioremediation of Polychlorinated biphenyls
2.3.2 Remediation of PCB contaminated soil
Bioremediation is defined as a process that uses microorganisms, green plants or
their enzymes to treat the polluted sites for regaining their original condition (Glazer
and Nikaido, 1995). It is considered very promising as natural biological activities are
utilized either to partially degrade the contaminants to less harmful products or to
completely mineralize them (Tomei & Daugulis, 2013; Sharma et al., 2014). Generally,
biological treatments are long-term processes as they take a long time to show
satisfactory performance. However, in comparison to the other physical and chemical
treatment methods, it is less expensive and environmentally friendly while having
higher public acceptance (Busset et al., 2012; Tomei & Daugulis, 2013; Sharma et al.,
2014). In bioremediation approaches, in-situ (on site) bioremediation is preferred
over ex-situ (off site) as it involves the treatment of polluted material at the site
without removing the contaminated soil for ex-situ treatment.
2.3.3 In-situ bioremediation
In-situ bioremediation consists of the use of green plants (phytoremediation),
microorganisms (fungi and bacteria), or their enzymes to treat the polluted materials
at the site and is discussed in section 2.3.3.1 to 2.3.3.3.
2.3.3.1 Phytoremediation
In phytoremediation, plants and their associated microorganisms are used for the
treatment of contaminated soil (Aken et al., 2009). Extensive laboratory and
greenhouse studies have been undertaken to remediate PCB contaminated soils
through different phytoremediation approaches that are summarized below.
Phytoextraction is to grow suitable plant varieties capable of accumulating significant
amounts of the contaminant from the soil and storing it in the plant shoots. These
plants with high PCB levels can then be harvested and treated using suitable method
such as high temperature incineration (Aslund et al., 2007; Anyasi & Atagana, 2014;
Luo et al., 2015). This method cannot be regarded as a direct treatment option as it
use plants as a vector to physically transfer the contaminants from one place to other.
Chapter 2: Bioremediation of Polychlorinated biphenyls 25
In phytotransformation, once the pollutants are absorbed by the plant through the
root system, enzymatic breakdown of pollutants takes place inside the plant tissues
(Aken et al., 2009). However, plants generally lack the enzymes that are necessary to
achieve complete degradation of recalcitrant organic compounds and therefore,
results in slow and incomplete degradation (Eapen et al., 2007).
In contrast, rhizoremediation is more effective as some secondary plant metabolites,
which are structurally similar to contaminants are released by plant roots. They
stimulate the growth of PCB degrading microbial community associated with the root
zone (Meggo et al., 2013; Liang et al., 2014; Jha et al., 2015; Liang et al., 2015).
Microorganisms utilize these secondary plant metabolites such as naringin, myricetin
and flavonone as their growth substrate while inducing PCB cometabolism (Leigh et
al., 2006; Musilova et al., 2016). In cometabolism, contaminants which cannot serve
as the primary substrate to provide energy for the microorganisms are degraded
cometabolically while utilizing some other substrate as their primary substrate
(Musilova et al., 2016). Fully developed roots and rhizospheres in switchgrass
(Panicum virgatum) and poplar (Populus deltoids x nigra) planted microcosms have
shown better biotransformation potential than the unplanted reactors (Meggo &
Schnoor, 2013). Table 2.3 summarises recent studies based on plant-microbe
combinations in PCB degradation. Plant growth-promoting rhizobacteria can survive
and multiply under extreme weather and climatic conditions while improving the
plant biota against stress imposed by contaminants and increase their
biomass/efficiency to take up contaminants (Asad, 2017).
Higher degradation rates in the mesocosms of switchgrass rhizospheres
bioaugmented with PCB degrading bacteria suggest that the use of phytoremediation
and bioaugmentation in combination could be an efficient and sustainable strategy
for the treatment of PCB contaminated soil (Liang et al., 2015). However, success in
remediation relies on several factors such as selection of suitable plant - microbial
combinations based on soil conditions, level of contamination and bioavailability of
PCBs as well as maintenance of appropriate conditions such as pH, moisture, and
other growth requirements. Most of the available information are based on small-
scale research studies conducted in confined environments such as hydroponics and
26 Chapter 2: Bioremediation of Polychlorinated biphenyls
greenhouses. Performance under full scale is not yet known as full scale studies are
limited to a few trials and are not yet completed due to the long time span required
for plant based remediation approaches (Passatore et al., 2014). Furthermore, as a
contaminant, PCBs are frequently associated with other environmental contaminants
like dioxins and heavy metals. Therefore, an in-depth knowledge is needed to
understand the effects exerted by these co-contaminants on microorganisms and
plants associated with PCB remediation (Wang & He, 2013a).
Chapter 2: Bioremediation of Polychlorinated biphenyls 27
Table 2.3 Plant–microbial relationships associated with PCB degradation
Microorganism/s Plant PCB reduction % Type of treatment Reference
Pseudomonas putida
Stenotrophomonas maltophilia
Mustard (B. juncea) 7.2–30.3% after 2
months
Greenhouse pots with
biochar as bio-carrier
(Pino et al., 2016)
Actinobacteria sp.
Chloroflexi sp.
Alfalfa 31.4% in 1 yr.
78.4% in 2 yrs.
Contaminated site (Tu et al., 2011)
Rhodococcus sp.
Burkholderia sp.
Austrian pine (P. nigra)
Goat Willow (S. caprea)
- Contaminated site (Leigh et al., 2006)
Geobacter sp. Switchgrass (Panicum
virgatum)
30–40 % after 6 months Soil microcosms (Liang et al., 2015)
Bioaugmentation with Burkholderia
xenovorans LB400
Switchgrass (P. virgatum) 47.3 ± 1.22% after 6
months
Soil microcosms (Liang et al., 2014)
Pseudomonas sp.
Agrobacterium sp.
Ochrobactrum sp.
Horseradish (A. rusticana)
Goat willow (S. caprea)
28.0 %
31.8 % after 6 months
Open air pots
(Ionescu et al., 2009)
Mixed microbial consortium Poplar > 90% after 14 months Contaminated site (Ancona et al., 2016)
28 Chapter 2: Bioremediation of Polychlorinated biphenyls
2.3.3.2 Fungal bioremediation
Knowledge on the potential of applying fungi for PCB breakdown and their
occurrence in contaminated soils is limited. Penicillium chrysogenum, Penicillium
digitatum, Scedosporium apiospermum, and Fusarium solani have been found to
display rapid growth in mineral media containing PCB and glucose while degrading
PCBs (Tigini et al., 2009). However, none of the taxa was able to grow on PCBs as their
sole source of carbon. In microbial community analysis of soil historically
contaminated with high concentrations of PCBs, the microbial community was found
to consist of both bacteria and fungi although their individual contribution towards
PCB degradation was not known (Stella et al., 2015). Fate of some selected mono (2-
chlorobiphenyl and 4-chlorobiphenyl), tri (3,4,5-trichlorobiphenyl) and penta
(3,3`,4,4`,5-pentachlorobiphenyl) chloro biphenyls in a pure culture of Aspergillus
niger were determined. Only 2-chlorobiphenyl and 4-chlorobiphenyl were
transformed to hydrophilic metabolites with 38 and 52 % reductions, respectively,
while there was very low reduction of trichlorobiphenyl (2%) and no observable
reduction of pentachlorobiphenyl concentration (Kim et al., 2016). This result
suggests that the degradation ability of A. niger is limited to simple monochlorinated
PCB congeners.
Federici el al (2012) reported 33.8% overall PCB removal in soils contaminated with
Aroclor 1260 after bioaugmentation with maize stalk-immobilized Lentinus tigrinus.
However, the incubation control which only consisted of residential microflora also
demonstrated 28% overall PCB degradation (Federici et al., 2012). It is not clear that
the 5.8% difference between the L. tigrinus bioaugmented and control microcosms
was due to the degradation ability of L. tigrinus itself, its indirect contribution to
proliferate the residential soil microflora or direct absorption of PCBs into the fungal
mycelium. Other than the degradation, translocation of PCBs into fruiting bodies
were observed in white rot fungus Pleurotus ostreatus (oyster mushroom) growing
on straw spiked with a Delor 103 commercial PCB mixture (Moeder et al., 2005).
Pandey et al (2016) suggest that the metabolism of PCB compounds and their
metabolites by the ligninolytic enzymes secreted by white rot fungi is due to the
Chapter 2: Bioremediation of Polychlorinated biphenyls 29
structural similarities between PCB molecules and lignin molecules (Pandey et al.,
2016).
It can be concluded that the fungal action takes place primarily in the extracellular
environment due to the secretion of enzymes to the external environment (Passatore
et al., 2014). Moreover, fungal based degradation seems to be most effective against
the lower chlorinated PCBs. Assessing the applicability of some fungal strains, which
have the ability to live in symbiosis with plant roots, for remediating PCB
contaminated soils will be beneficial, as they can survive under extreme
environmental conditions which would be an added advantage in remediation
applications.
2.3.3.3 Bacterial bioremediation
When compared to plants (Section 2.3.3.1) and fungi (Section 2.3.3.2), bacteria are
the most widely studied microorganisms in PCB bioremediation due to their
ubiquitous distribution in the environment and the ability to degrade lower and
highly chlorinated congeners by numerous bacterial strains through different
anaerobic metabolism, aerobic co-metabolism and aerobic metabolism pathways
(Passatore et al., 2014). Though the findings of many research studies are available,
bacteria based bioremediation of PCBs is not yet widely practiced as a field scale
treatment technique. Therefore, bacterial bioremediation of PCBs was chosen in the
current study as a way forward to minimize the gaps in available knowledge.
In the following sections, different types of biodegradation pathways, known types
of bacteria with PCB degradation potential, and their applications are discussed.
2.4 Biodegradation pathways
In PCB degradation, two distinctive microbial processes have been identified:
anaerobic reductive dechlorination; and aerobic oxidative degradation. The higher
chlorinated biphenyls are often subjected to anaerobic dechlorination while the
lower chlorinated biphenyls are often subjected to aerobic oxidation (Demirtepe et
al., 2015).
30 Chapter 2: Bioremediation of Polychlorinated biphenyls
2.4.1 Anaerobic reductive dechlorination
The conversion of highly chlorinated congeners (congeners with five or more chlorine
atoms) to less chlorinated congeners (congeners with four or less chlorine atoms) by
replacing a single chlorine atom by a hydrogen atom is termed as dechlorination
(Hughes et al., 2009). This process reduces the potential toxicity and bioaccumulation
of highly chlorinated congeners. As illustrated in Equation 2.1, in the reductive
dechlorination process, PCBs are used as electron acceptors and the chlorine is
replaced with hydrogen (Borja et al., 2005).
R⎼Cl + 2e¯+ H+→ R⎼H + Cl¯ Equation 2.1
Anaerobic dechlorination of PCBs was detected in the sediments of the Hudson River,
Massachusetts (Furukawa & Fujihara, 2008). An escalation in the lower chlorinated
congeners and a reduction in the highly chlorinated congeners were observed when
analyzing the PCBs inanaerobic sediments (Borja et al., 2005). a similar trend was
exhibited during a laboratory study based on electronic waste contaminated soil
using dissimilatory Fe(III) reducing and arylhalorespiring bacteria (Song et al., 2015).
Specialized populations of microorganisms with distinct dehalogenating enzymes
have the potential to carry out reductive dechlorination. So far, several anaerobic
dechlorinating bacteria have been isolated. The microorganisms frequently belong to
the Dehalorespiring Chloroflexi group, such as Dehalococcoides spp. (Adrian et al.,
2009; Praveckova et al., 2015) and Dehalobium spp. (Payne et al., 2011). In addition,
Bacteroides and Betaproteobacterium, Pseudomonas (Bedard et al., 2006),
All four strains performed better under anaerobic conditions compared to aerobic
conditions. However, the two stage anaerobic-aerobic conditions produced the best
overall results when assessed on cell growth, PCB solubility and chloride build up.
Importantly, it was found that these microorganisms can carry out these reactions
relatively rapidly within the first one to two weeks. This is despite the fact that a
number of research studies carried out so far have shown comparatively long
durations ranging from three to six months for the reactions to be completed (Master
et al., 2002; Chen et al., 2014).
The presence of chloride ions under all the three conditions tested found that the
highest chloride ion concentrations was achieved under two stage anaerobic-aerobic
conditions which suggest that the combined anaerobic-aerobic conditions could
further enhance the chlorine removal from the biphenyl structure. Therefore, in field
scale soil remediation applications, facultative microorganisms have the potential to
be better candidates as they can survive and degrade PCBs under both anaerobic and
aerobic conditions, while achieving relatively higher degradation rates.
The limitations of biological breakdown due to the characteristic hydrophobic
properties of PCBs can be overcome by the use of suitable bacterial strains, which
can simultaneously solubilize and breakdown PCBs. During this study, Lysinibacillus
sp. NP05 was found to have high potential as a candidate to effectively
decontaminate environmental pollutants such as highly chlorinated complex PCB
mixture, Aroclor 1260. Therefore, based on these results there is an opportunity to
produce and apply tailored-made consortia for future process designs and
applications resulting in shorter time frames, while effectively hydrolyzing PCBs.
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 135
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
7.1 Background
Reductive dechlorination and oxidative degradation are the two main processes
involved in the biodegradation of PCBs. Therefore, a combination of anaerobic
dechlorination and aerobic oxidation is essential to effectively degrade these
complex polychlorinated biphenyl mixtures to less toxic products. Due to the complex
metabolic network responsible for PCB degradation, a single bacterium does not
possess the enzymatic capability to degrade all or even most of the PCB congeners
present in polluted environments (Fritsche & Hofrichter, 2005; Pieper, 2005). Based
on the availability of genes encoding enzymes that degrade PCBs (Seeger & Pieper,
2010; Hassan, 2014; Wang et al., 2014), types of degradation products (Petric et al.,
2007; PetricBru et al., 2011) and information on how well individual microorganisms
degrade PCBs, a consortium of carefully selected microbial species is expected to
perform better than the application of individual microbes (Liz et al., 2009; Chen et
al., 2015a).
Currently, two modes of combined anaerobic dechlorination and aerobic oxidation
applications have been reported in PCB bioremediation studies, and have been
labelled as: (1) two stage (or sequential) anaerobic-aerobic degradation; and (2)
concurrent (or alternating) anaerobic-aerobic degradation (Master et al., 2002;
Payne et al., 2013; Chen et al., 2014; Long et al., 2015). Based on the results discussed
in Chapter 6, three individual strains Achromobacter sp. NP03, Ochrobactrum sp.
NP04 and Lysinibacillus sp. NP05 provided positive indicators as potential candidates
to undergo further tests as a consortium.
136 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
Accordingly, Chapter 7 primarily focuses on testing a consortium made up of
facultative anaerobic Achromobacter sp. NP03, Ochrobactrum sp. NP04 and
Lysinibacillus sp. NP05 based on their ability as individuals to degrade the PCB mixture
Aroclor 1260 under both, anaerobic and aerobic conditions. The rates and efficiencies
of PCB biodegradation by the selected three strains as a consortium were compared
under; (1) alternating (AN), and (2) two stage (TS) anaerobic-aerobic modes.
7.2 Materials and Methods
Three facultative anaerobic bacterial strains Achromobacter sp. NP03, Ochrobactrum
sp. NP04 and Lysinibacillus sp. NP05 capable of degrading PCBs under both, anaerobic
and aerobic conditions were used as a consortium in the present study. Initially they
were streaked on solid minimal salt medium containing 50 mg/L Aroclor 1260 and
duplicate plates were incubated anaerobically and aerobically at 28 °C to see whether
there was any visible competition among them for the growth substrate.
The two modes of treatments were tested at 28 °C for six weeks in order to compare
PCB degradation rates under; (1) two stage anaerobic-aerobic (TS), and (2)
alternating anaerobic-aerobic (AN) conditions. The TS experiment continued for four
weeks under anaerobic static conditions (as described in Chapter 6.2.1) and then
switched to aerobic mixing conditions at 150 rpm for the last two weeks. Under AN
conditions, the experiment was kept under anaerobic static conditions in the first
week and switched to aerobic mixing conditions in the second week. Switching from
one week of anaerobic to one week of aerobic incubations was continued throughout
the six-week study. In anaerobic stages, flasks were kept at 28 °C in an incubator kept
inside the anaerobic chamber with occasional gentle shaking by hand. Hydrogen gas
(H2) at 4.92% (BOC Australia) was provided as the electron donor during the
anaerobic phases.
Additionally, three culture members used in the consortium were checked for their
carbon and nitrogen source preferences. Understanding their preferences is
important so that cost, availability and applicability of carbon and nitrogen sources
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 137
during inoculation preparation, scale up and on-site remediation applications can be
determined.
7.2.1 Laboratory microcosm batch experiments
The liquid microcosms were prepared using 250 mL Erlenmeyer flasks containing 20
mL of sterile minimal salt medium (see Section 3.1.2.1A). 50 mg/mL Aroclor 1260
stock solution in GCMS grade acetone was added to each flask as sole source of
carbon to give 50 mg/L Aroclor 1260 final concentration.
For seed culture preparations, single bacterial colonies were picked and inoculated
into 250 mL of sterile Luria-Bertani (LB) broth and grown overnight at 28 °C, 150 rpm.
The seed cultures were collected by centrifuging at 5000 x g for 10 min, washed twice
in minimal salt medium, and the resulting cell pellets were resuspended in 1 mL of
sterile minimal salt medium. Previously prepared batch mesocosms were inoculated
with seed cultures under anaerobic conditions (Figure 7.1).
Figure 7.1 Inoculation of batch mesocosms with bacterial seed cultures inside the
anaerobic chamber.
7.2.2 Frequency, collection and preservation of samples
In order to obtain the total PCBs concentrations, the entire content in each flask was
extracted and tested. This was done as PCBs are poorly soluble in water and taking
138 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
aliquots may result in unrepresentative samples. Due to this, the experiment was set
up to sacrifice six whole flasks at each sampling from each TS and AN experiment.
These six flasks included one set of triplicate flasks for total PCB extraction and
another set of triplicate flasks for the analyses of bacterial growth, pH, chloride ion
concentration and extracellular proteins. Sampling was done initially (at time zero)
and at fortnightly intervals (at week 2, 4 and 6) for both, AN and TS treatments.
Two controls were also prepared to test parallel to the samples. First control was an
abiotic control without bacterial seed culture additions (i.e. Minimal salt medium
spiked with 50 mg/L Aroclor 1260) and the second control was a growth medium
control (i.e. minimal salt medium with bacterial seed culture addition, but no Aroclor
1260 was added). These controls were conducted in parallel to the experiments
under each TS and AN treatments, and duplicate flasks from each abiotic and growth
medium controls were sacrificed at each sampling time for analyses.
The following is the summary of the test set up of the mesocosms at each sampling
under AN and TS conditions:
• Three flasks for extractions for total PCBs and PCB homolog groups;
• Three flasks for bacterial growth, pH, chlorides and proteomic analyses;
• Two flasks as abiotic controls (minimal salt medium spiked with 50 mg/L
Aroclor 1260, but no seed culture added) ; and
• Two flasks as media controls (minimal salt medium spiked with an equal
volume of acetone used to dissolve Aroclor 1260 and inoculated with seed
culture, but no 50 mg/L Aroclor 1260 added).
Additionally, to see the difference when switching from one week of anaerobic to
one week of aerobic under AN treatment conditions, additional three flasks from AN
treatment and two flasks from each control were used for each consecutive week (at
week 1, 3 and 5) to take samples for cell growth, chloride, pH and extracellular
proteins.
Bacterial growth, pH and chloride ion concentrations were measured as per Section
3.2.4A, Section 3.2.5 and Section 3.2.7, respectively. Final chloride ion concentrations
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 139
were determined by subtracting the chloride concentrations of the controls. To see
whether there was any contribution of chloride from the cell lysis was also tested by
comparing the chloride levels of samples before and after being subjected to 10 min
sonication for cell disruption. Samples collected for protein analysis were stored at -
80 °C freezer until subsequent analysis. Analytical processes and outcomes of
extracellular protein analysis are discussed in Chapter 8.
7.2.3 Total PCB extraction and analysis
PCB extraction was performed according to the method described by Murinova et al.
(2014) using GC grade n-hexane as the extraction solvent. Further details of the
extraction method is as described in Section 3.2.8B. Recovery of the surrogate
standard was obtained at 77.0% ± 4.3% (n = 61) and found to be within the USEPA
recommended recovery limits of 70-130% (Eichelberger et al., 1995). A method blank
was also carried out in parallel to each batch of PCB extractions through the entire
procedure using sterile distilled water to evaluate any presence of contamination
from the complete preparation and analytical procedure.
PCB extracts were diluted four fold using n-hexane and analyzed using gas
chromatography (GC) as per the method described in Section 3.2.9 to determine the
total and homolog group specific PCB levels. PCB degradation rates were calculated
as a percentage using Equation 7.1.
PCB degradation (%)
=(Initial PCB concentration - PCB concentration in culture suspension)
Initial PCB concentrationx 100
Equation 7.1
7.2.4 Carbon and nitrogen source utilization profiling of bacterial cultures
Phenotype microarray plates (PM1 and PM3B, Biolog) containing 95 separate sole
carbon and nitrogen sources were used to identify carbon and nitrogen utilization
preferences of the three facultative bacterial consortium members. Depending on
the ability of bacteria to oxidize carbon and nitrogen sources in the wells, initially
140 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
colourless tetrazolium dye was reduced to form a purple colour (Miller & Rhoden,
1991). The colour intensity ranged from dark purple to colourless depending on the
intensity of bacterial growth. Biolog phenotype microarray (PM) procedures for Gram
negative and positive bacteria were followed during the cell suspension preparation
and inoculation.
7.2.4.1 Procedure for Gram positive bacteria
(A) Bacterial cell suspension preparation
Gram positive Lysinibacillus sp. NP05 was grown overnight at 30 °C on nutrient agar
to produce pure and isolated colonies. A cell suspension was prepared by transferring
bacterial colonies from the nutrient agar plate into 5 mL 1x inoculating fluid (Biolog
IF-0a GN/GP base) using a sterile cotton swab until the transmittance of 81% (0.0915
absorbance at 600 nm) was reached.
(B) Nutrient additive solution preparation
Nutrient solutions as indicated in Table 7.1 were prepared to use in the final
inoculation fluid, filter sterilized through 0.2 µm filters and stored at 4 °C until use.
Table 7.1 Ingredients for the nutrient additive solution, 12x (PM additive)
Ingredient Gm/50 mL PM1 (mL) PM3 (mL)
Sodium succinate 4.0515 - 3
MgCl2.6H2O
CaCl2.2H2O
2.44
0.88 1 1
L-arginine, HCl
L-glutamate, Na
0.0315
0.0505 1 -
L-cystine, pH 8.5 0.006 3 -
Yeast extract 0.3 1 1
Tween 80 0.3 1 1
Sterile water 3 4
Total 10 10
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 141
(C) Final inoculation fluid preparation
The recipe as indicated in Table 7.2 was used to prepare the final inoculating fluids.
Duplicate PM1 and PM3 Biolog plates were inoculated with respective final
inoculation fluid (100 µL/well). Plates were incubated at 30 °C and colour
development was monitored for 24 h in 30 min intervals and measured at 595 nm
wavelength using a plate reader (Synergy HTX). Equivalent volumes of bacterial cell
suspensions with no added carbon and nitrogen sources were used as negative
controls.
Table 7.2 Preparation of final inoculation fluid to inoculate the Biolog plates
Description mL/PM1 Biolog plate
(For carbon sources)
mL/PM3 Biolog plate
(For nitrogen sources)
1.2X stock inoculation fluid
(Biolog IF-0a GN/GP base)
10.0 10.0
Redox dye mix D, 100x (Biolog,
for Gram positive bacteria)
0.12 0.12
Nutrient additive solution, 12x
(PM additive)
1.0* 1.0**
Cell suspension (with 81% T) 0.88 0.88
Total 12.0 12.0
* From PM1 nutrient solution from Table 7.1.
**From PM3 nutrient solution from Table 7.1.
7.2.4.2 Procedure for Gram negative bacteria
(A) Bacterial cell suspension preparation
Gram negative bacteria, Achromobacter sp. NP03 and Ochrobactrum sp. NP04 were
separately grown overnight at 30 °C on nutrient agar to obtain pure colonies. Cell
suspensions were prepared by transferring the bacterial colonies from the nutrient
agar plates into 5 mL 1x inoculating fluid (Biolog IF-0a GN/GP base) using sterile
cotton swabs until the transmittance of 42% (0.3768 absorbance at 600 nm) was
reached.
142 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
(B) Final inoculation fluid preparation and inoculation of PM1 and PM3 plates
0.6 mL redox dye H (Biolog, for Gram negative bacteria) and 7.734 mL sterile water
were added to a sterile 50 mL falcon tube containing 41.666 mL of 1.2X stock
inoculation fluid (Biolog IF-0a GN/GP base) and thoroughly mixed to form a uniform
solution. To prepare the final inoculation fluid, 40 mL of the mixture was transferred
into a sterile 50 mL falcon tube and mixed gently with 8 mL of previously prepared
cell suspension having 42% Transmittance. 24 mL from the final inoculation fluid was
transferred to a sterile reservoir and PM1 plates were inoculated in duplicate for each
bacterial culture (100 µL/well). Then, 240 µL of the previously prepared 2M sodium
succinate solution was added to the remaining 24 mL of the final inoculation fluid.
The mixture was gently mixed to make a uniform suspension and used to inoculate
in duplicate PM3 Biolog plates (100 µL/well). Similar to the plates for the Gram
positive bacterium, plates for the two Gram negative bacteria were incubated at 30
°C and the colour development was monitored for 24 h in 30 min intervals and
measured as absorbance at 595 nm wavelength using a plate reader (Synergy HTX).
Bacterial cell suspensions with no added carbon or nitrogen sources were used as
negative controls.
7.3 Results and Discussion
7.3.1 Test for competition
At the start of the experiment, the three chosen bacterial cultures Achromobacter sp.
NP03, Ochrobactrum sp. NP 04 and Lysinibacillus sp. NP05 were streaked on to minimal
salt agar plates spiked with 50 mg/L of Aroclor 1260 as the sole source of carbon and
grown under both, aerobic and anaerobic conditions to test for growth inhibition. All
three strains grew well without any visible growth inhibition from the neighbouring
organism after 72 hours, and in particular, no obvious zones of inhibition were observed
at the areas where each strain merged with each other (Figure 7.2).
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 143
Figure 7.2 Growth of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and
Lysinibacillus sp. NP05 on minimal salt-Aroclor 1260 agar at 28 °C after 72 h (a) under
aerobic conditions, (b) under anaerobic conditions.
7.3.2 PCB degradation by the bacterial consortium under AN and TS treatments
7.3.2.1 Total PCB degradation
Typically, anaerobic processes have been shown to be slow and require long
durations in order to achieve effective and higher degradation of PCBs. Adrian et al.
(2009) reported that 64% total PCB reduction was observed after four months of
incubation under anaerobic conditions using anaerobic Dehalococcoides sp. CBDB1,
whereas 36% reduction of total PCBs by a consortium of anaerobic microorganisms
after 10 months of anaerobic incubation was reported by Praveckova et al. (2015) .
Differences in rate of PCB degradation could be due to the differences in types of
microorganisms used and the application or treatment conditions. However, as
discussed in Chapter 6, when facultative anaerobic bacterial strains were tested
during this work as individuals for their PCB degradation potential under aerobic,
anaerobic and combined anaerobic-aerobic conditions, the highest results were
obtained under the combined anaerobic-aerobic conditions.
There are only limited research studies on conventional combined anaerobic-aerobic
treatments (Master et al., 2002; Chen et al., 2014; Long et al., 2015). Most of these
144 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
studies consist of lengthy (70 to 180 days) anaerobic phase followed by short aerobic
phase (28 to 60 days). Based on these earlier studies, a two stage (TS) treatment
process was designed for this study, which included a four week anaerobic stage
followed by a two week aerobic stage. In the alternating (AN) treatment mode,
cultures were placed in anaerobic conditions for one week and then switched to one
week long aerobic conditions. Alternation of anaerobic and aerobic conditions were
repeated throughout the six week testing period.
The results of PCB degradation in relation to cell growth under the AN vs TS
conditions are shown in Figure 7.3. According to Figure 7.3, the rate of PCB
degradation occurred faster under AN compared to TS conditions, with 49.2±2.5%
total PCB reduction reached during the first two weeks under AN (Figure 7.3a). In
comparison, only about 25% of PCB degradation was obtained in the first two weeks
of just anaerobic conditions under TS (Figure 7.3b). The results from the AN
conditions showed better degradation efficiency within a short period than that of
previous combined anaerobic-aerobic studies. In the study conducted by Master et
al. (2002), it took four months of anaerobic treatment followed by 28 days of aerobic
treatment to achieve 66% PCB reduction. Total PCB reduction reported by Long et al.
(2015) after 70 days of anaerobic treatment and 28 days of aerobic treatment was
only 25%. Therefore, the results of this study are superior in terms of treatment
efficiency compared to past studies.
According to Figure 7.3, though 49.2±2.5% total PCB reduction occurred after the first
two weeks under AN, only a 4.9% further reduction occurred during the last four
weeks reaching up to 54.1±0.49% degradation by week 6. However, despite the fact
that there was no considerable increase in PCB reduction after the second week,
bacterial growth continued to rise from week 2, demonstrated by the optical density
increase from 0.59±0.01 to 2.49±0.16 (Figure 7.3a). This suggests the possibility of a
consortium utilizing the intermediate products produced during the degradation of
Aroclor 1260 as their growth substrates. This hypothesis was confirmed by the fact
that there was no other carbon source added to the medium other than Aroclor 1260
and the increase in growth cannot be expected while there is no substantial PCB
reduction. However, the types of intermediate products produced and their fate
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 145
during the degradation process needed to be confirmed through further
investigation.
Figure 7.3 Total PCB degradation as a percentage and bacterial growth as OD600 under
(a) alternating and (b) two stage anaerobic-aerobic treatments by the bacterial
consortium. Error bars represent the standard deviation of mean values (n = 3).
The results from the two stage (TS) anaerobic-aerobic approach are shown in Figure
7.3b. Under TS conditions, the total PCB degradation rate after two weeks of
anaerobic treatment was 24.44±2.46% compared to 49.2±2.5% achieved during the
AN conditions (Figure 7.3b vs Figure 7.3a). From week 2 to 4 under continuing
anaerobic conditions, PCBs were further degraded up to 34.89±2.26%. Anaerobic PCB
dechlorination is a reductive process that uses PCBs as electron acceptors, but the
rings are not usually cleaved as reported by Borja et al. (2005). Therefore, PCB
dechlorinating only microorganisms require additional carbon and electrons sources
for their growth. During the first four weeks of anaerobic growth, the gradual
increase in the growth based on values of OD600 0.18±0.01 to 0.66±0.14,
corresponded well with total PCB concentration reduction (Figure 7.3b). These
results indicate that the consortium members were able to utilize PCBs as their
carbon source under anaerobic conditions. Furthermore, these results were
comparable to the findings from the comparative growth studies including the same
146 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
three microorganisms when tested individually under similar conditions as described
in Chapter 6.
Under TS conditions, when the cultures were switched from anaerobic to aerobic
conditions during the last two weeks, the PCB degradation rate increased from
34.89±2.26% (in week 4) to 47.99±1.55% (in week 6, Figure 7.3b). Meanwhile the
optical density value increased nearly three times from 0.66±0.14 in week 4 to
1.56±0.03 in week 6. Microbial growth thrived once it changed from anaerobic to
aerobic conditions. The increased PCB degradation and growth rates were highly
likely due to ring cleavage and hydrolysis of lower chlorinated PCB congeners
generated during the initial anaerobic phase.
7.3.2.2 PCB Homolog analysis
PCB congeners are categorized into ten homolog groups and labelled from
monochlorobiphenyls to decachlorobiphenyls based on the degree of chlorination in
the biphenyl molecule (ATSDR, 2000). However, decachlorobiphenyls were not
included in the analysis as they were present in trace quantities (Less than 0.05% of
the total PCB mixture). According to the existing literature, highly chlorinated
congeners (with 5 or more chlorines) are converted into lower chlorinated (four or
less) congeners under anaerobic conditions. Under aerobic oxidations, lower
chlorinated congeners breakdown into intermediate products such as
chlorobenzoates and benzoates based on upper biphenyl pathway (Borja et al.,
2005). Benzoate is a growth substrate for a broad range of bacteria and it can be
further mineralized to CO2 and H2O via lower biphenyl pathways such as the
chlorocatechol or 3-oxoadipate pathways (Pieper, 2005).
To determine the capability of the consortium to degrade the different PCB homolog
groups under alternating (AN) and two stage (TS) anaerobic-aerobic conditions,
samples were removed, extracted and analysed as described in Sections 7.2.2, 7.2.3
and 7.2.4, respectively. The results are shown in Figure 7.4 for the AN treatment and
in Figure 7.5 for the TS treatment. Since the range of homolog concentrations were
too broad to illustrate in a single graph, three graphs are plotted for each of the AN
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 147
and TS treatments in three different scales as: (a) homologs containing lower
chlorinated congeners (from mono to tetra); and (b) penta, hexa, hepta groups; and
(c) octa, nona groups of highly chlorinated congeners.
As shown in Figure 7.4, the concentrations of all the PCB homolog groups decrease
significantly during the first two weeks under AN conditions. Similar to total PCBs,
there was no considerable reduction in the concentrations of homolog groups after
the second week indicating the ability of the consortium to reach their optimum
degradation within the first two weeks under AN conditions. However, when
individual homolog groups were considered, the rate of degradation was negatively
correlated with the increasing number of chlorine atoms in the PCB molecules.
Congeners with single chlorine atoms (monochlorobiphenyl) displayed the highest
reduction rate reaching a 98.69±0.05% overall reduction at the end of week 6 (Figure
7.4a), whereas the nonachlorobiphenyls showed the lowest reduction rate of
47.12±1.64% at the end of week 6 (Figure 7.4c). Based on these results, it can be
concluded that the higher the number of chlorines attached to the biphenyl rings, the
more resistant the PCBs are to microbial biodegradation. A similar observation was
noted by Furukuwa et al (2006). When compared to other homolog groups,
hexachloribiphenyls represent 50.69±0.47% (by weight) of Aroclor 1260 mixture.
Therefore, reduction of initial 19.73±1.12 mg/L hexachlorobiphenyl level to
10.46±0.05 mg/L by week 2 is nearly 47% reduction of the original
hexachlorobiphenyl concentration and this is the main contribution to 49.2±2.5%
overall total PCB reduction (Figure 7.4b).
148 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
Figure 7.4 Variation of PCB homolog groups following AN treatment; (a) lower
chlorinated congener groups (mono to tetra), and medium to highly chlorinated
congener groups, (b) penta to hepta, (c) octa and nona. Error bars represent the
standard deviation of mean values (n = 3).
In the TS treatment, the first four weeks were kept under anaerobic conditions and
switched to aerobic conditions in the last two weeks. When lower chlorinated
congener groups were analyzed, the concentration of each group slightly increased
during the anaerobic phase with the highest increase observed in dichlorobiphenyl
from 0.21±0.02 mg/L in week 0 to 1.55±0.11 mg/L by the end of week 4 (Figure 7.5a).
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 149
However, once conditions changed from anaerobic to aerobic after week 4,
concentrations of all the lower chlorinated congener groups reduced far below the
initial concentrations (Figure 7.5a). These results indicate that the microorganisms
utilized the lower chlorinated PCBs as an energy source under the aerobic conditions.
The subsequent increase in cell density during the last two weeks under aerobic
conditions as demonstrated in Figure 7.3b would support this conclusion.
Furthermore, these findings are in agreement with the study outcomes by Borja et
al. (2005).
In contrast, the concentrations of highly chlorinated congeners gradually decreased
during the four week anaerobic phase as shown in Figure 7.5. The highest reductions
were observed for penta, hexa and octa homolog groups at the end of anaerobic
phase with 48.2±3.85%, 42.09±1.8% and 39.28±1.62% reductions, respectively (see
Figure 7.5b and Figure 7.5c). These reductions in the highly chlorinated congener
groups suggested the possibility of dechlorination during the anaerobic conditions. It
can also be pointed out that if these highly chlorinated congener groups were
dechlorinated into lower chlorinated congeners, increase in the lower chlorinated
groups need to be higher than what is shown in Figure 7.5a. As there is no significant
increase in lower chlorinated congeners during the anaerobic phase as expected, this
was due to the ability of consortium members to utilize the lower chlorinated
congeners as their carbon and energy source under anaerobic conditions. This can be
further confirmed by the increase in cell density during the anaerobic phase as shown
in Figure7.3b. After the conditions changed from anaerobic to aerobic, there was no
substantial reduction of any highly chlorinated congener groups. However, the
overall reduction in homolog groups was higher in the AN treatment with nearly 90%
of the total reductions achieved within the first two weeks, when compared to the
TS treatment.
150 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
Figure 7.5 Variation of PCB homolog groups following TS conditions; (a) lower
chlorinated congener groups (mono to tetra), and highly chlorinated congener
groups, (b) penta to hepta, (c) octa and nona. Error bars represent the standard
deviation of mean values (n = 3).
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 151
7.3.3 Chloride ion accumulation and pH variation
7.3.3.1 Chloride ion analysis
Monitoring of chloride ion accumulation in the culture medium was performed to
further confirm the removal of chlorines from the PCB mixture. It was expected that
the chloride ion build up in the culture medium would increase proportionately to
the decrease in PCB homolog groups over time. Accordingly, the presence and
buildup of chloride ion concentrations in both AN and TS treatments were measured
as described in Section 7.2.2, and the results are shown in Figure 7.6.
Figure 7.6 Chloride ion accumulation under alternating (AN) and two stage (TS)
anaerobic-aerobic conditions. The background chloride values from the minimal salt
medium were first subtracted from experimental values and media controls. Error
bars represent the standard deviation of mean values (n=3 for experimental values
and n=2 for media controls).
152 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
Primary contributors of chloride levels in the experimental setup are minimal salt
medium used for the experiments and dechlorination of PCBs. In order to eliminate
the contributions of chlorides from minimal salt medium, chloride concentrations of
controls were first examined. The chloride ions in the abiotic controls were expected
to come from chloride containing compounds in the basal minimal salt medium and
the average chloride level in the abiotic control was 37.36±0.47 mg/L. To remove the
contribution from the minimal salt medium, chloride values of the abiotic controls
were first subtracted from the experimental values and media controls before final
values were plotted as shown in Figure 7.6. There was no significant difference of the
chloride levels before and after sonication of the samples and the difference is less
than 1 mg/L. This is indicative of no significant contribution of chlorides to the final
chloride concentration from cell lysis.
As shown in Figure 7.6, increasing levels of chloride concentrations occurred over the
six weeks under both AN and TS treatments. However, higher yields of chlorides were
detected in AN compared to TS at the end of week 4. By the end of week 6, a yield of
17.63±0.91 mg/L chlorides were measured under AN conditions, compared to 11.79±
1.28 mg/L measured under TS conditions.
In addition to the measurement of chloride ion build up in the culture medium, the
theoretical chloride ion removal from Aroclor 1260 present in the medium was also
calculated based on the PCB homolog group reduction rates as discussed in Section
7.3.2.2. Calculated and measured chloride ion levels under each treatment condition
are shown in Figure 7.7.
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 153
Figure 7.7 Measured chloride ion buildup in the culture medium and calculated
chloride ion removal from the PCB mixture based on homolog group reductions
under; (a) alternating (AN), and (b) two stage (TS) anaerobic-aerobic conditions. Error
bars represent the standard deviation of mean values (n = 3).
It can be postulated that the release of chlorines is proportional to the homolog
group reduction rates and released chlorides are accumulating in the culture
medium. This leads to an ideal scenario where calculated chloride ions based on
homolog group reductions is equal to the measured chloride levels in the culture
medium. As shown in Figure 7.3a, Figure 7.4b and Figure 7.4c, nearly 50% reduction
in total PCBs and highly chlorinated penta to nona homolog groups were recorded in
week 2 under AN conditions and this would result in high chloride ion concentration
in the medium. Based on the PCB homolog group reduction rates, the calculated
chloride ions released at week 2 was 14.09±0.41 mg/L. However, measured chloride
ion concentration in the culture medium was only 3.9±0.8 mg/L in week 2 (Figure
7.7a) and it was significantly lower than that of week 4 and the calculated chloride
level. This suggests the possibility of transformation of PCBs into chlorinated
intermediate products such as chlorobenzoates and chlorocatechols rather than
releasing chlorine from biphenyl molecules as suggested by Petric et al. (2007).
However, by week 4, measured chloride level increased significantly up to 16.03±0.7
mg/L, a similar value in comparison to calculated chloride level of 14.19±0.65 mg/L.
This indicates the breakdown of intermediate products and release of bound
chlorides. This was further confirmed by the continued growth demonstrated by the
154 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
bacterial consortium from week 2 to week 4 with increased optical density from
0.59±0.01 at week 2 to 1.11±0.21 at week 4 as demonstrated in Figure 7.3a, even
when there was no significant increase in the PCB reduction after the second week.
When compared to AN treatment, there was no significant difference between the
calculated and measured chloride concentrations in TS treatment. As both values
were lower than that of AN treatment, it indicates that AN treatment is more
effective than TS treatment.
7.3.3.2 pH analysis
The pH of the culture medium was also measured as described in Section 7.2.2 and
the results are shown in Figure 7.8. As expected, the controls remained almost
constant throughout the study period (7.09±0.1) and were not included in Figure 7.8.
Overall, the pH in both AN and TS treatments under anaerobic conditions
demonstrated negative trends, while the pH trend was positive under aerobic
condition (Figure 7.8). In the AN treatment, the pH dropped from 7.0±0.04 to
6.67±0.09 at the end of each anaerobic phase. However, when switching from
anaerobic to aerobic, the pH appeared to have self-recovered and adjusted back to
its original neutral condition irrespective of the increasing chloride ion concentration
(Figure 7.8a). According to White (1986), some bacteria can undergo metabolic
processes that can lead to the secretion of compounds with the ability to alter the pH
of the medium . Under the aerobic conditions, it is possible that the consortium
members have metabolized the salts in the minimal salt medium such as ammonium
sulfate as their nitrogen source that resulted in the generation of ammonia (Park &
Lee, 1998). Moreover, under aerobic conditions, the flasks are shaken at 150 rpm, in
the presence of oxygen. It is common that microbes under respiration produce CO2,
which can be converted to HCO3 that will also contribute to the alkalinity of the
medium (Hem & Survey, 1989).
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 155
Figure 7.8 pH trends relative to chloride ion concentration. (a) AN and (b) TS
anaerobic-aerobic treatments. Error bars represent the standard deviation of mean
values (n = 3).
In the TS treatment, the initial pH dropped from 7.0±0.04 to about 5.51±0.27 by the
end of week 4 (Figure 7.8b). In contrast, the chloride ions from PCB degradation
increased linearly reaching 20.76±0.46 mg/L by the end of week 4. When compared
between the two conditions, it was of interest to see that during the first four weeks
under AN conditions, the buildup in chloride concentration was more gradual
compared to the linear and exponential increase under TS conditions (see Figure 7.8a
vs Figure 7.8b). However, once the conditions switched from anaerobic to aerobic
after week 4, pH level gradually recovered back to its original neutral value of 7.05.
Although, under TS conditions three consortium members individually were not able
to change the pH back to its original neutral position as shown in Figure 6.4 in Chapter
6. When used as a consortium, the ability to self-recover pH from acidic to neutral
condition is an added advantage in soil remediation applications as low pH severely
inhibit PCB biodegradation while.prolonged acidic pH can lead to leaching of acid
soluble toxic compounds present in the soil (Chen et al., 2015a).
156 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
7.3.4 Carbon and nitrogen wide substrate utilization tests
Availability of different carbon and nitrogen sources can be a major growth
requirement for microorganisms and play a significant role in the metabolism and
hydrolysis of environmental pollutants such as PCBs. Anaerobic dechlorination is a
reductive process and biphenyl rings are not cleaved unless the microorganisms are
capable of utilizing PCBs as a carbon source as reported by Wiegel and Wu (2000).
Therefore, supplementing a suitable additional carbon source would be essential for
the microorganisms for their survival while dechlorinating PCBs, under anaerobic
conditions. It was apparent that during the first four weeks, the growth rate under TS
conditions was much slower than the AN treatment (see Figure 7.3). Therefore,
providing an additional carbon source, which can be utilized by all the consortium
members, could enhance the PCB dechlorination rate. Addition of a desirable carbon
source was also shown to enhance the mineralization of PCBs under aerobic
conditions through co-metabolism of biphenyl as reported by Beyer and Biziuk
(2009). In the present study, (NH4)2SO4 in the minimal salt medium was provided as
the inorganic nitrogen source for the microorganisms. However, addition of an
economical organic nitrogen source which can be utilized by all the consortium
members would be important to maintain bacterial growth, either under laboratory
conditions, or in on-site applications.
Therefore, as part of further strain characterization and potential future applications,
the three strains Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus
sp. NP05 were subjected to a carbon and nitrogen-wide substrate utilization screen.
The Biolog system PM1 for carbon and PM3B for nitrogen substrate were used as
described in Section 7.2.4.
The results for different carbon substrate tests are shown in Table 7.3. Likewise,
results for the different nitrogen substrate tests are summarized in Table 7.4. If
bacteria were able to utilize particular carbon or nitrogen source, the initially
colourless tetrazolium dye is reduced to purple colour compound. The intensity of
colour change is proportional to the the degree of substrate utilization. Based on the
colour intensities, L-proline was identified as the best substrate that all three
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 157
consortium members, Achromobacter sp. NP03, Ochrobactrum sp. NP04, and
Lysinibacillus sp. NP05, could utilize as their sole source of carbon and nitrogen. L-
Proline is one of the twenty amino acids used by living organisms as the building
blocks of proteins (Al-Mailem et al., 2018). L-proline is produced at industrial scale
and some of the raw materials rich with L-proline are algae Chlorella (Leavitt, 1983)
and keratin separated from waste biomass (Sharma & Gupta, 2016). In addition to L-
proline, all three consortium culture members were able to utilize L-lactic acid and
methyl pyruvate as carbon sources (Table 7.3) and L-Glutamic acid, Ala-His, Ala-Leu
and Gly-Gln as nitrogen sources (Table 7.4) at high rates.
However, it should be further confirmed by additional research as the presence of
other carbon sources could either stimulate or inhibit PCB dechlorination by
facilitating out-competing the non-PCB dechlorinators or providing more preferred
electron acceptors other than PCBs to the dechlorinators in the natural environment
(Wiegel & Wu, 2000; Yang et al., 2008; Parnell et al., 2010). Additionally, potential for
the use of these chemical compounds as nutritional supplements to the soils in on-
site applications need to be carefully decided based on the associated cost and their
impact on the soil ecosystem.
158 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
Table 7.3 Carbon source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05.
A - Achromobacter sp. NP03, O – Ochrobactrum sp. NP04, L – Lysinibacillus sp. NP05
High Moderate Low Negative
Carbon Source A O L Carbon Source A O L Carbon Source A O L Carbon Source A O L
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 159
Table 7.4 Nitrogen source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05.
Nitrogen Source A O L Nitrogen Source A O L Nitrogen Source A O L Nitrogen Source A O L
A - Achromobacter sp. NP03, O – Ochrobactrum sp. NP04, L – Lysinibacillus sp. NP05
High Moderate Low Negative
160 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
7.4 Conclusions
Based on an extensive search of published literature, this appears to be the first
comparative study assessing PCB degradation efficiency of three facultative
anaerobic bacteria as a consortium under two different combined anaerobic-aerobic
modes; alternating (AN) and two stage (TS). Based on the literature, microbial
degradation of PCBs under anaerobic conditions has been shown to be a long-term
process ranging from 70 to 180 days. Therefore, the two stage (TS) process was set
up to have an extended anaerobic phase of four weeks followed by a short aerobic
phase of two weeks. In contrast, weekly intervals of anaerobic and aerobic
conditions, and vice versa was applied in the alternating (AN) study, over a six week
period. The study found that the alternating approach was more efficient compared
to the two stage treatment conditions with nearly 50% reduction in total PCBs
achieved within the first two weeks compared to only 24% from the two stage
treatment. This is significant because one of the limiting factors in bioremediation
applications is the long time span required to achieve a satisfactory degradation rate.
During both treatments, the consortium exhibited a greater ability to degrade the
lower chlorinated PCB homolog groups under aerobic conditions compared to highly
chlorinated homolog groups. However, the total PCB reductions were always higher
under alternating conditions, suggesting the weekly switching between anaerobic
and aerobic conditions favored the consortium to change between dechlorination
and oxidation activities. Even after the PCB degradation appeared to have reached a
plateau at week two, the bacterial cell density and chloride ion concentration in the
culture medium under alternating conditions kept increasing, suggesting the ability
of the consortium to further break down the intermediate products as their sole
carbon source and release the bound chlorides.
L-proline was found to be the best substrate to be utilized by all three consortium
members as their carbon and nitrogen source. Additionally, all three bacterial strains
were able to utilize L-lactic acid and methyl pyruvate as their carbon source and L-
Glutamic acid, Ala-His, Ala-Leu and Gly-Gln as their nitrogen source. However,
applicability of the addition of such chemical compounds as nutritional supplements
Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 161
to soil in bioremediation applications needs to be considered only after additional
research on their suitability for the soil ecosystem.
Finally, it can be concluded that the alternating (AN) anaerobic-aerobic treatment
would be a preferred approach compared to the two stage (TS) anaerobic-aerobic
process in PCB remediation applications when compared to degradation efficiencies
and time. Importantly, the use of facultative anaerobic bacteria such as
Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05 as a
consortium would be advantageous as they have the ability to survive and degrade
PCBs under both, aerobic and anaerobic conditions more efficiently than the
individual organisms.
162 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 163
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
8.1 Background
Recent research literature provide confirming evidences to suggest that the
intracellular degradation of PCBs occurs via a series of enzymes through anaerobic
dechlorination and aerobic oxidation (Wiegel & Wu, 2000; Pieper, 2005; Agullo et al.,
2017). However, there is no real knowledgeabout specific proteins involved in active
transport of hydrocarbons across the biological membranes (Parales & Ditty, 2017).
As PCBs are insoluble in aqueous media, it is a prerequisite to transform PCBs into
soluble and / or smaller molecules to diffuse more easily into the cells across the cell
membranes (Hearn et al., 2008). Extracellular enzymes released by microorganisms
into their surrounding environment may play a major role in transformation of
hydrophobic pollutants (Basak & Dey, 2015).
According to Desvaux et al. (2009), actively secreted and non-secreted proteins by
an organism are collectively called as exoproteome (see illustration in Figure 8.1).
However, the total proteins or enzymes exported from inside the cells to the external
environment by an organism is defined as the secretome. The secretome consists of
proteins that are actively transported to outside the cell through the cytoplasmic
membrane via classical or non-classical secretion mechanisms, proteins shedding
from the cell membrane, proteins released from the membrane vesicles, as well as
from the secretion machinery itself (Caccia et al., 2013).
164 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Figure 8.1 The secretome and exoproteome of a Gram negative bacterial cell.
(Armengaud et al., 2012)
As illustrated in Figure 8.2, oxidoreductases and hydrolases are some of the secreted
proteins from microorganisms with the ability to convert polymeric chemical
substances such as poly aromatic hydrocarbons to partially degraded or oxidized
products that can be easily absorbed by microorganisms (Gianfreda & Rao, 2004;
Basak & Dey, 2015). Hydrolases or hydrolytic enzymes facilitate the cleavage of major
chemical bonds such as C–C, C–O, C–N in toxic molecules and help to reduce their
toxicity (Karigar & Rao, 2011). However, there is limited knowledge about the role of
extracellular proteins or enzymes secreted by that can attacking and hydrolysing PCB
molecules to produce more easily diffusible intermediates. Proteins that facilitate
uptake of those intermediates into cells for further degradation, and subsequent
conversion to energy are also poorly known.
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 165
Figure 8.2 Role of extracellular enzymes in insoluble compound metabolism.
Chapter 8 focuses on extracellular proteins detected in the culture supernatants of
Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 both
individually as described in Chapter 6 and as a consortium reacting with PCBs under
AN and TS conditions, as described in Chapter 7. This chapter also discusses; (1) mass
spectrometry based proteomics approaches for the identification of proteins present
in bacterial culture meda (2) bioinformatics analyses of these protein sequences, and
(3) their possible roles during the degradation of PCBs by the three facultative
anaerobic bacteria acting as a consortium.
8.2 Materials and Methods
Supernatant samples (1 mL) were collected from individual culture experiments of
facultative anaerobic members Achromobacter sp. NP03, Ochrobactrum sp. NP04
and Lysinibacillus sp. NP05 (as described in Chapter 6), and from the bacterial
consortium based experiments (as described in Chapter 7). These were used for the
166 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
analysis of extracellular proteins found in the culture supernatants. The samples were
centrifuged at 10,000 × g for 15 min at 4 °C to remove the bacterial cells, and the
resulting cell free culture supernatants containing proteins were used for protein
visualization, quantification and extraction prior to mass spectrometry analyses.
8.2.1 Protein visualization
8.2.1.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was used to visualise the proteins in the culture supernatants. Protein
sample (32.5 µL) was mixed with 12.5 µL of 4x Laemmli loading buffer (Thermo Fisher
Scientific) and 5 µL of 10x Bolt reducing agent (Thermo Fisher Scientific). The mixture
was then denatured by boiling at 95 ̊ C for 5 min. Samples were resolved by SDS-PAGE
using Bolt 4-12% Bis-Tris Plus SDS-PAGE gels (Invitrogen, Thermo Fisher scientific).
SeeBlue Protein standard (Thermo Fisher scientific) was used as the protein
molecular weight (MW) marker. A 40 µL aliquot was loaded to each well and
electrophoresis was performed under constant voltage of 200 V for 30 min in mini
gel tanks (Life Technologies) using 1x Bolt MES SDS running buffer (Novex).
8.2.1.2 Coomassie blue staining
Following electrophoresis, gels were removed from tank and fixed by adding 50 mL
fixing solution (10% (v/v) acetic acid and 40% (v/v) ethanol) and agitating gently for
five minutes in a shallow tray. Gels were then rinsed with deionised water and stained
in QC Coomassie stain (Biorad) overnight at room temperature with gentle agitation.
The next day, gels were destained in deionized water for 1 to 3 hr with gentle
agitation at room temperature until the background cleared, and the protein bands
became clearly visible.
8.2.2 Protein quantification using Bicinchoninic acid assay (BCA)
The total protein concentrations in the culture supernatants were quantified using a
microplate bicinchoninic acid assay as per the Pierce BCA Protein Assay Kit protocol
(Catalog number 23227, Thermo Scientific). The protein concentrations were
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 167
determined relative to the triplicates of standard dilutions (0-2000 ng/μL) of bovine
serum albumin (BSA, stock solution of 2 mg/mL, Sigma-Aldrich (Table 8.1). 25 μL of
each sample and 200 μL working reagent (50 parts of BCA reagent A with 1 part of
BCA reagent B) was mixed to give 1:8 ratio in a 96-well microtiter plate and incubated
at 37 ˚C for 30 min. The plate was cooled to room temperature and the absorbance
was measured at 562 nm using a microplate reader (FLUOStar Optima, BMG Labtech).
Table 8.1 Standard dilutions preparation for BCA assay
Vial Volume of
Diluent* (μL)
Volume and Source of BSA
(μL)
Final BSA concentration
(μg/mL)
A 0 300 of Stock 2000
B 125 375 of Stock 1500
C 325 325 of Stock 1000
D 175 175 of vial B 750
E 325 325 of vial C 500
F 325 325 of vial E 250
G 325 325 of vial F 125
H 400 0 0 = Blank
*Sterile MilliQ water was used as the diluent.
8.2.3 Trypsin digestion of extracellular proteins
A modified in-filter digestion (FASP) protocol based on the work of Wisniewski et al.
(2009) was used to digest the proteins in the culture supernatant into peptides prior
to mass spectroscopy. Based on the protein concentration measurements in Section
8.2.2, supernatants containing 20 µg of proteins were used for the trypsin digestion.
Parallel to each batch of samples, 10 µg of BSA was also analyzed as a quality control
measure. Buffers and chemicals needed for the protein extractions were prepared as
per Section 3.1.2.6 of Chapter 3.
During sample preparation, protein samples were combined with 200 μL of
168 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
tubes (catalogue number MRCF0R030, Millipore) and incubated at room
temperature for 60 min in an agitator. Filters were centrifuged at 14,000 x g, 21 °C
for 15 min and the filtrate was discarded. Filters were washed with 200 μL Urea-Tris
buffer and centrifuged at 14,000 x g, 21 °C for 15 min, and the filtrate was discarded.
100 μL Iodoacetamide-Urea buffer was then added to each of the filters and
incubated at room temperature for 20 min in an agitator. Filters were centrifuged at
14,000 x g, 21 °C for 10 min and the filtrate was discarded. The filters were washed
twice with 100 μL of Urea-Tris buffer and centrifuged at 14,000 x g, 21 °C for 15 min
each, with the filtrate discarded after each time. The filters were then equilibrated
by washing twice in 100 μL of 100 mM ammonium bicarbonate buffer and
centrifuged at 14,000 x g, 21 °C for 10 min. The filtrate was removed after both
centrifugation steps.
Trypsin stock (10 µL of 1 µg/µL in 100 mM ammonium bicarbonate buffer) was
defrosted on ice and the working trypsin solution was prepared by adding 90 μL of
100 mM ammonium bicarbonate buffer to the trypsin stock. The working trypsin
solution was added to the tubes containing filters to achieve an enzyme : protein
ratio of 1:50 and the tubes were placed in a humidified chamber and incubated
overnight at 37 °C with gentle agitation.
Following protein tryptic digestions, the filters were transferred to clean 1.5 mL
Eppendorf tubes and the resultant peptides were eluted by centrifugation at 14,000
x g, 21 °C for 15 min. An additional elution step was performed with one wash of the
filters with 20 μL of 100 mM ammonium bicarbonate buffer and collected at 14,000
x g, 21 °C for 15 min. Samples containing tryptic peptides were vacuum dried using a
rotational vacuum concentrator (RVC 2-33IR, John Morris Scientific) for 30 min at 40
°C and reconstituted in 10 μL of 2% (v/v) Acetonitrile (ACN) / 0.1% (v/v) trifluoroacetic
acid (TFA) solution, before proceeding to desalting.
8.2.4 Desalting of samples prior to mass spectroscopy analysis
Following elution, the peptide samples were further purified using solid phase
extraction as described by Rappsilber et al (2003). In particular, 200 µL pipette tips
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 169
were filled with single SCX membrane disc (Empore 3M) as described in Section
3.1.2.6 J in Chapter 3 and activated by passing 30 μL of 100% ACN through using a
centrifugal force (2 min spin at 2,000 rpm). Next, 30 μL of 5% (v/v) ammonium
hydroxide/80% (v/v) ACN was added to the tips just before the last remainder of ACN
left the tip and collected for 2 min at 2,000 rpm. Finally, 30 μL of 0.2% TFA (v/v) was
added and collected for 2 min at 2,000 rpm. Peptide samples were then loaded onto
the tips and centrifuged for 2 min at 2,000 rpm. A 30 μL 0.2% TFA solution was added
and the tips were centrifuged for 2 min at 2,000 rpm and this step was repeated.
Peptide samples were eluted to clean 1.5 mL Eppendorf tubes by adding 30 μL of 5%
ammonium hydroxide/80% ACN to the tips followed by 2 min spin at 2,000 rpm. The
eluted peptide samples were vacuum dried as before using a rotational vacuum
concentrator and the pellets were reconstituted in 10 μL iRT buffer (see Section
3.1.2.6 G for preparation) ready for liquid chromatography - mass spectroscopy (LC-
MS) analyses.
8.2.5 Secretome analysis
8.2.5.1 Liquid chromatography – mass spectroscopy
(A) Chromatography
The peptide samples were analysed using the TripleTOF® 5600+ mass spectrometer
(SCIEX) in either DDA or DIA mode as described below. Approximately 400 ng – 1 µg
of peptide material was injected to the instrument. Peptides were separated by
performing reversed-phase chromatography using an Eksigent ekspert™ nanoLC 400
System directly interfaced to the MS/MS instrument. The LC platform was set up in a
trap and elute configuration with a 10 mm × 0.3 mm trap cartridge packed with
ChromXP C18CL 5 µm 120 Å material and a 150 mm × 75 µm analytical column packed
with ChromXP C18 3 µm 120 Å (Eksigent Technologies, Dublin, CA). The mobile phase
solvents were composed of mobile phase A: water/0.1% formic acid (FA); mobile
phase B: ACN/0.1% FA; and mobile phase C: water/2% ACN/0.1% FA. Trapping was
performed in mobile phase C for 5 min at 5 µL/min followed by an elution
configuration across either 9.5 min or 40 min gradient (depending whether 25 min or
170 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
65 min method was used) using mobile phases A and B at a conserved flowrate of
300 nL/min. The proportions of both solvents (A and B) were adjusted at specified
time-points of:
a) 0, 30, 35, 40, 49, 50 and 60 min corresponding to 98, 60, 35, 10, 10, 98 and 98 % of
solvent A in the case of the 65 min method.
b) 0, 5, 7, 9.5, 10.2 and 20 min corresponding to 95, 60, 10, 10, 95 and 95 % of solvent
A in the case of the 25 min method.
To minimise retention time drift, the analytical column was maintained at 40 °C.
(B) Data-dependent acquisition (DDA)
For spectral library generation, peptides were analysed by data-dependent
acquisition (DDA) using 25 min (for BSA QC samples) and 65 min method (for culture
supernatant samples). The DDA mode of the instrument was set to obtain high
resolution (resolving power: 30,000) TOF-MS scans over a range of 350-1350 m/z,
followed by up to 15 (in the case of 25 min method) or 40 (in the case of 65 min
method) high sensitivity MS/MS scans of the most abundant peptide ions per cycle
over the range of 100-2000 m/z. The selection criteria for the peptide ions included
intensity greater than 150 cps and charge state of 2-5. The dynamic exclusion
duration was set at either 3 s or 9 s to account for the difference in chromatographic
peak width between the two methods used. Each survey (TOF-MS) scan lasted 250
ms and the product ion (MS/MS) scan was acquired for 50 ms, resulting in a total
cycle time of either 1 s or 2.3 s depending on the method. The ions were fragmented
in the collision cell using rolling collision energy, and collision energy spread (CES) was
set to 5. The collected peptide ion fragmentation spectra were stored in two files per
sample with extensions format of .wiff and .wiff.scan (SCIEX).
(C) Data-independent acquisition (DIA)
For quantitation, eluted peptides were subjected to a cyclic data-independent
acquisition (DIA) using even isolation windows SWATH-MS™ acquisition 65 min
method (SCIEX) based on its earlier description (Gillet et al., 2012). In particular, a
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 171
survey scan data (MS) was acquired for 80 ms, followed by MS/MS on all precursors
within a particular isolation window in a cyclic manner using an accumulation time of
80 ms per individual SWATH-MS window. 36 overlapping windows, each 26 m/z units
wide, were used to cover the peptide ions in a range of 350 – 1250 m/z which resulted
in the cycle time of 3 s. Fragment ions were recorded in a high sensitivity mode and
in a range of 100 – 1800 m/z. Given the peptide chromatographic peaks were
approximately 18 s wide, the above parameters allowed the collection of at least 6
data points for each chromatographic peak to ensure accurate quantitation. The
collected data were stored in two files per sample with extensions format of .wiff and
.wiff.scan (SCIEX).
8.2.5.2 Protein identification using ProteinPilot v5
All DDA data files of individual culture samples of Achromobacter sp. NP03,
Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 (as discussed in Chapter 6) were
submitted together for peak picking and database search through ProteinPilot (V5.0,
ABSciex) software using Paragon algorithm (Shilov et al, 2007). The database was
created using the existing protein sequences of the three microorganisms used in the
consortium study at genus level and protein sequences of the microorganisms
identified with PCB degradation potential in the present and previously published
studies downloaded (on 09.08.2017) from the NCBI protein database. The resulting
protein list was stored as a single .group file.
8.2.5.3 Protein quantitation using PeakView
The resulting .group file generated from ProteinPilot software via SWATH microapp
to create a spectral library with false discovery rate set to 1% for protein identification
and requirement of only unmodified peptides to be used. Next, SWATH™-MS data
files were imported into PeakView and SWATH microapp was used for targeted
extraction of fragment ion signals specific to peptides represented in the library from
each individual SWATH™-MS run. This peak detection step was conducted under
were performed in triplicate for each sample analyzed. At least four different
172 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
peptides were required per protein and their signals were averaged at a protein level
inside SWATH microapp. A protein was considered valid when it was detected in all
three replicates, and absent in the media controls. Protein level data were normalized
using the Most Likely Ratio (MLR) method using MarkerView software (V1.3.1, SCIEX)
which was also used to statistically identify the significantly changed proteins through
principal component analysis (PCA) before any further analysis using bioinformatics.
Finally, using Excel, relative abundance of significantly changed proteins were
determined as heat maps.
8.2.5.4 Bioinformatics analysis of peptide sequences
Once the identity of significantly changed proteins was established, the
corresponding peptide sequences were analysed whether that protein would fall
under a ‘secreted’ or ‘non-secreted’ protein using bioinformatics tools. The proteins
that would fall under classically secreted extracellular proteins were identified using
SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP) (Armengaud et al., 2012).
Default cut-off values were used during the search for both Gram positive and Gram
negative bacteria. In contrast, the proteins classified as non-classical secreted
proteins were predicted with SecretomeP 2.0
(http://www.cbs.dtu.dk/services/SecretomeP) (Vazquez-Gutierrez et al., 2017). A
protein without a signal peptide was considered positive as a non-classically secreted
protein when the SecP score was above 0.5. Analysis using SignalP first, followed by
SecretomeP, was considered as the preferential order when the same protein was
found positive under both prediction tools. All protein sequences were further
scrutinized through the redundancy check using Geneious (R10.2.3, Biomatters Ltd.).
Proteins that were grouped under secreted and non-secreted proteins were also
analysed according to their potential cellular and biological functions, following NCBI,
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 173
8.3 Results and Discussion
8.3.1 SDS-PAGE analysis: Extracellular protein detection and visualization
8.3.1.1 Individual cultures
As an initial screening step, 1 mL samples were collected at weekly intervals from the
supernatants of individual batch culture experiments of the three facultative
anaerobic culture members Achromobacter sp. NP03, Ochrobactrum sp. NP04 and
Lysinibacillus sp. NP05 as described in Chapter 6. Following separation of supernatant
from cells by centrifugation at 10,000 × g for 15 min at 4 °C, a total of 32.5 µL of clear
culture supernatants was checked from each sample, in order to visualize the
presence or absence of proteins in the supernatant over time (see SDS-PAGE protein
analysis in Section 8.2.1). Parallel to each set of samples, separate SDS-PAGE gels
were run for control samples collected at weekly intervals from flasks containing
minimal salt medium inoculated with bacterial seed cultures, containing no PCBs and
there were no visible proteins detected in any of the control SDS-PAGE gels (see
Figure 8.3 for Lysinibacillus sp. NP05 controls under anaerobic conditions).
salt medium with no added PCBs) under anaerobic conditions at 28 °C. Lane 1,
SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0
immediately after addition of seed culture; lane 3 to 8, week 1 to week 6.
174 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
The SDS-PAGE results are shown in Figure 8.4 for Achromobacter sp. NP03, Figure 8.5
for Ochrobactrum sp. NP04 and Figure 8.6 for Lysinibacillus sp. NP05, respectively.
Notably, none of the three strains had any visible protein bands present in week 0,
directly after the addition of the seed cultures.
According to Figure 8.4, the main proteins detected in the supernatants of
Achromobacter sp. NP03 cultures are shown running at ~ 40 kDa that first appeared
in week 1 and were present up to week 6, during anaerobic conditions (Figure 8.4 B).
In contrast, lower yield proteins running at about 50 kDa appeared at weeks 1-6
under aerobic conditions. However, more proteins around the 90 and 40 kDa were
present during week 5 (Figure 8.4 A).
Figure 8.4 SDS-PAGE analysis of extracellular proteins of Achromobacter sp. NP03
under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein
standard as the protein molecular weight markers; lanes 2, time 0 immediately after
addition of seed culture; lane 3 to 8, week 1 to week 6.
Similar to Achromobacter sp. NP03, low amounts of proteins were detected for
Ochrobactrum sp. NP04 under aerobic conditions during weeks 1 to 4 and 6, with
even low to no proteins appearing in week 5 (Figure 8.5 A). More proteins were
present under anaerobic conditions in weeks 2 to 6, with no obvious proteins
detected in week 1 (Figure 8.5 B).
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 175
Figure 8.5 SDS-PAGE analysis of extracellular proteins of Ochrobactrum sp. NP04
under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein
standard as the protein molecular weight markers; lanes 2, time 0 immediately after
addition of seed culture; lane 3 to 8, week 1 to week 6.
The proteins detected in the Lysinibacillus sp. NP05 culture supernatants are shown
in Figure 8.6. Under aerobic conditions, a dominant high molecular weight proteins
running at about 100 kDa appeared during week 1 and was show continuing presence
from weeks 2 to 6 (Figure 8.6 A). In contrast, more proteins were detected under
anaerobic conditions, with the yield and pattern of proteins appearing similar in
weeks 1 and 3, and, weeks 4 and 6 (Figure 8.6 B). It was of interest that high amounts
of proteins running approximately at 100 kDa appeared in weeks 1 and 3, while lesser
amounts in weeks 2, 4 and 6. When compared to other weeks, relatively low amounts
of proteins were present in the supernatant in week 5.
176 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Figure 8.6 SDS-PAGE analysis of extracellular proteins of Lysinibacillus sp. NP05 under
(A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard
as the protein molecular weight markers; lanes 2, time 0 immediately after addition
of seed culture; lane 3 to 8, week 1 to week 6.
Despite the fact that the samples were normalized to the volume and not protein
amount, and therefore, change in the protein signal likely correlated with bacterial
density/growth as well as increased/decreased secretion. In summary, Lysinibacillus
sp. NP05 under anaerobic conditions showed the highest number of protein bands
present in the culture supernatants among all the samples (compare Figures 8.4, 8.5
and 8.6). In addition, protein levels in the extracellular environment under aerobic
conditions seemed lower than under anaerobic conditions. This may be due to the
low concentration of lower chlorinated PCB congeners present in the PCB mixture
(see Table 3.2 in Chapter 3) as the only available carbon source for the
microorganisms to utilize under aerobic conditions.
8.3.1.2 Consortium study under AN and TS treatment conditions
In the consortium experiments as described in Chapter 7, supernatant samples
collected at fortnightly intervals from the alternating (AN) and the two stage (TS)
anaerobic-aerobic experiments were also analysed using SDS-PAGE protein analysis.
The results are shown in Figure 8.7. Similar to the individual cultures (see Figures 8.4,
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 177
8.5 and 8.6), no obvious proteins can be seen in week 0, straight after the addition of
the seed comprising of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and
Lysinibacillus sp. NP05, as a consortium. However, obvious proteins at around 38-39
kDa are present in week 2, and lesser amounts of the similar sized proteins in weeks
4 and 6 during the AN conditions (Figure 8.7 A). Another protein at about 29 kDa is
present in higher amounts in week 2, under the AN conditions compared to the week
2 results under the TS conditions (Figure 8.7 B). Incidentally, the high levels of
extracellular proteins in week 2 under the AN conditions coincides with the high PCB
degradation rate of 49.2±2.5% observed in week 2 under AN treatment conditions as
discussed in Section 7.3.2.1 in Chapter 7.
Figure 8.7 SDS-PAGE analysis of the extracellular proteins of the bacterial consortium
consisting of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp.
NP05 under (A) AN, and (B) TS anaerobic-aerobic conditions at 28 °C. Lane 1, SeeBlue
Protein standard as the protein molecular weight markers; lanes 2, time 0
immediately after addition of seed culture; lane 2 to 5, at fortnightly intervals up to
week 6.
In comparison, similar sized proteins (~38-39 and 29 kDa) are also present in weeks
2 and 4 under anaerobic conditions in TS treatment (Fig. 8.7 B). However, it appeared
that the same proteins are not present in the culture medium, during the last 2 weeks
of cultivation when the condition was changed from anaerobic to aerobic in TS
conditions (Figure 8.7 B).
178 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
8.3.2 Proteomics analysis
The proteins in the culture supernatants from the consortium study were further
analysed using mass spectrometry in order to enable their identification. Proteins
isolated from the culture supernatants were digested into peptides, purified using
solid phase extraction and resultant peptides were submitted for mass spectrometric
analysis. After quantification using SWATH, a total of 618 proteins representing the
extracellular protein fractions in both AN and TS conditions were identified for
further analyses, following quality control measures as described in 8.2.3.4.
All the 618 total proteins were then subjected to bioinformatics analyses in order to
determine: (1) the presence of signal peptides characteristic of classically secreted
proteins using the SignalP 3.0 server (Petersen et al., 2011); and (2) proteins
characterized as non-classically secreted proteins using the SecretomeP 2.0 server, a
prediction method for identifying the proteins using the signal peptide independent
secretion pathways (Bendtsen et al., 2005). The remaining proteins were grouped as
non-secretory proteins. Analysis of 618 total proteins from AN and TS conditions over
the six weeks period is shown in Table 8.2. The highest number of total proteins (n-
542) detected in week 6 under AN conditions coincides with the maximum PCB
degradation rate (54.1±0.49%), optical density (2.49±0.16 (see Figure 7.3a in Chapter
7) and chloride ion accumulation (17.63±0.91 mg/L) (see Figure 7.6 in Chapter 7).
Table 8.2 Analysis of extracellular proteins identified in culture supernatants of
consortium under AN and TS conditions.
Category
Number of proteins
AN Treatment (weeks) TS Treatment (weeks)
2 4 6 2 4 6
Non secretory 265 220 273 261 249 263
Secretory 260 254 269 266 278 254
Total 525 474 542 527 527 517
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 179
8.3.2.1 Non-secretory proteins
Out of the 618 total proteins, 319 (52%) of the proteins found in the culture
supernatant were predicted to be non-secretory by the SignalP 3.0 and SecretomeP
2.0 servers. Both the highest (n=273) and lowest (n=220) number of non-secretory
proteins were detected in week 6 and week 4 under AN conditions as summarized in
Table 8.2. The predicted 319 non-secretory proteins were grouped according to their
potential functional activities (Table 8.3) and the detailed functional classification of
predicted non-secretory proteins in the culture supernatant is given in Table C.3 in
Annex C.
Table 8.3 Functional classification of 319 identified proteins that were predicted to
be non-secretory proteins by the SignalP 3.0 and SecretomeP 2.0 servers.
Function Number of proteins
Amino acid metabolism 43
Aromatics & Xenobiotic degradation 16
Carbohydrate metabolism 32
Carboxylic acid metabolism 15
Elongation factors and chaperones 11
Genetic Information Processing 41
Hypothetical 20
Lipid metabolism 22
Membrane related 6
Metabolism of cofactors and vitamins 6
Nucleotide metabolism 21
Signal transduction and chemotaxis 11
Stress and redox signalling 26
Transport and secretion 8
Other (uncategorised) 41
Total 319
The functional classification of these predicted non-secretory proteins revealed that
the large proportion matched to proteins involved in major metabolic processes
essential for the survival of cells such as amino acid metabolism (n=43), genetic
information processing (n=41) and carbohydrate metabolism (n=32) as shown in
180 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Table 8.3. There were also 16 proteins related to aromatics and xenobiotic
degradation including 4-oxalocrotonate tautomerase, phenyl phosphate carboxylase,
3-oxoadipate CoA transferase, carboxymuconolactone decarboxylase, 2-
aminobenzoate-CoA ligase, and glutathione S-transferase.
4-Oxalocrotonate tautomerase enzyme is part of the bacterial plasmid-encoded
pathway, which allows the microorganisms harbouring the plasmid to use various
aromatic hydrocarbons as their sole sources of carbon and energy (Whitman, 2002).
Phenylphosphate carboxylase has been found to be responsible for catalysing the
para carboxylation of phenylphosphate to 4-hydroxybenzoate in anaerobic
metabolism of phenol (Schuhle & Fuchs, 2004). During aerobic degradation, aromatic
compounds are transformed to intermediates like catechol and protocatechuate
(Altenschmidt & Fuchs, 1992). However, there is a limited number of bacterial species
which are able to both, convert the chloroaromatics into catechol and
protocatechuate intermediates and further mineralize them, due to the lack of
chlorocatechol pathway enzymes (Pieper & Reineke, 2004). Both 3-oxoadipate CoA-
transferase and carboxymuconolactone decarboxylase detected here, are two such
enzymes involved in the catechol and protocatechuate branches of the 3-oxoadipate
pathway, which are important for the degradation of aromatic compounds (Eulberg
et al., 1998). 2-aminobenzoate-CoA ligase was found to be able to catalyse the first
step of aerobic metabolism of 2-aminobenzoate, an intermediate of PCB degradation
into the coenzyme A thioester of 2-aminobenzoate (Altenschmidt & Fuchs, 1992).
Bacterial glutathione transferases (GSTs) are one of the superfamily of enzymes that
play an important role in cellular detoxification against toxic xenobiotics via
conjugation of reduced glutathione and protection against chemical and oxidative
stresses (Nebert & Vasiliou, 2004; Allocati et al., 2009). Glutathione S-transferase is
also known as BphK and catalyzes the dechlorination of 3-chloro-2-hydroxy-6-oxo-6-
phenyl-2,4-dienoates (HOPDAs) compounds that are produced during the
degradation of PCBs in some bacterial biphenyl catabolic (bph) pathways. The
enzyme also catalyzed the dechlorination of 5 chloro HOPDA and 3,9,11-trichloro
HOPDA (Fortin et al., 2006). Additionally, GSTs are also involved in the reductive
dechlorination of pentachlorophenol (Van Agteren et al., 2013).
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 181
Oxidoreductases were present in culture supernatant throughout the study period
under both AN and TS conditions with the maximum concentration observed at week
2 under AN conditions. These enzymes are responsible for bacterial mediation of
detoxification of toxic organic compounds through oxidative coupling.
Microorganisms extract energy via energy-yielding biochemical reactions mediated
by oxidoreductases to cleave chemical bonds and to assist in the transfer of electrons
from a reduced organic substrate (donor) to another chemical compound (acceptor)
(Karigar & Rao, 2011). As a result of such oxidation-reduction reactions, the toxic
contaminants are oxidized to harmless compounds.
In addition to these results, the release of certain proteins without known peptide
secretion signals have also been reported in some comparative proteomics studies of
extracellular proteins of Escherichia coli. (Li et al., 2004; Xia et al., 2008). Li et al.
(2004) reported the presence of some enzymes that are involved in metabolic
processes such as nucleotide metabolism, glycolytic pathway, amino acid metabolism
as well as some outer membrane, periplasmic, and cytosolic proteins in the
extracellular proteome of some virulent Escherichia coli strains. Enzymes related to
metabolism and cellular processes such as isocitrate lyase, malate synthase without
signal peptide sequences were also detected in the extracellular proteome of some
other Escherichia coli (9BL21 and W3110) strains (Xia et al., 2008).
Importantly, the potential for cell lysis could not be excluded as a cause for the
release of these proteins, since increased concentrations of some major cytoplasmic
proteins such as chaperonin GroEL, DnaK suppressor protein and thioredoxin TrxA
were detected in the culture medium over time under both AN and TS treatments.
However, occurrence of cell lysis leading to cell death is less likely since there is a
strong trend in increasing cell density and subsequent PCB degradation rates in the
culture medium over time (see Figure 7.3 in Chapter 7).
Selective leakage of some cytoplasmic proteins by unidentified mechanisms was
previously reported (Xia et al., 2008). Therefore, alterations to the cell membrane
and cell wall structures is likely to occur rather than complete cell lysis due to various
stresses such as osmotic shock-like response (Kaakoush et al., 2010). Vazquez-Laslop
et al. (2001) reported that the increased permeability of the outer membrane due to
182 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
sudden drop in osmolarity in the medium leads to selective release of some small
cytoplasmic proteins. Thioredoxin and DnaK, which were present in high
concentrations in the exoproteome of the present study, were also among the highly
released proteins due to the osmolarity shock (Vazquez-Laslop et al., 2001).
Furthermore, Cl- ions build up during PCB dechlorination (see Figures 7.6 in Chapter
7) may have contributed to changing the osmolality of the culture medium and may
have some impact on the release of non-secretory proteins. However, this needs to
be further investigated.
Overall, the presence of enzymes that are responsible for the intermediate steps of
PCB degradation pathways and detoxification of toxic chemicals in the extracellular
environment by cell lysis or by selective straining provides strong support for the
occurrence of PCB degradation by the bacterial consortium.
8.3.2.2 Secretory proteins: Secretome
Out of 618 total proteins found in the exoproteome, 299 (48%) were
bioinformatically predicted to be secreted. Of the 299 total proteins identified to be
secreted in the culture supernatant, 71% (212 proteins) were predicted to have signal
peptides and grouped to be classically secreted proteins, while only 29% (87) proteins
were predicted to be secreted by non-classical secretion pathways (Figure 8.8).
Detailed information related to secretory proteins under AN and TS conditions are
given in Table C.1 in Appendix C.
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 183
Figure 8.8 Proportions of classically and non-classically secreted proteins in the
culture supernatant of the bacterial consortium Achromobacter sp. NP03,
Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05.
4.3.2.2.1 Classically secreted proteins
Classically secreted proteins were further categorized based on their functional
groups in Figure 8.9. Some proteins such as superoxide dismutase that were found to
have a specific function in the cytoplasm were also recognized to be actively involved
in some biological processes in the extracellular environment (Bendtsen et al., 2005;
Henderson & Martin, 2011). The functions they perform in the cytoplasmic and
extracellular environments are not always identical. Such proteins involved in
multiple functions are known as moonlighting proteins (Jeffery, 2003).
184 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Figure 8.9 Functional groupings of proteins identified as classically secreted proteins.
As shown in Figure 8.9, most abundantly detected classically secreted proteins were
transporters and they represented 58% (122 proteins) of the total classically secreted
proteins. Transporter proteins are one of organism’s primary interfaces with the
environment. Bacterial transport proteins comprise a heterogeneous group
representative of their diverse biological functions (Giuliani et al., 2011). The ability
of bacteria to occupy specific environments mainly depends on their capacity to
obtain sufficient supplies of nutrients that are essential for their growth (Vazquez-
Gutierrez et al., 2017). Bacterial transport proteins play an important role in
facilitating the uptake of essential nutrients, regulate the cytoplasmic concentrations
of metabolites, export large molecules to the outer surface of the cell and prevent
toxic effects of toxins by catalyzing their active efflux (Yen et al., 2009). These proteins
comprise a heterogeneous group representative of their diverse functional and
cellular roles.
Among the ten extracellular proteins identified with the highest protein
concentrations, eight were also transport related classically secreted proteins (see
Table 8.4). The majority of the transporters such as sulfate transporter, leucine ABC
chain amino acid ABC transporter substrate-binding protein, iron ABC transporter
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 185
were present in the extracellular environment and are involved in the transport of
nutrients. The synthesis of a wide range such transporters in the cell is essential for
the microorganisms to uptake the nutrients from the environment (Christie-Oleza et
al., 2012).
Bacterial biodegradation of hydrocarbons, an important process for environmental
remediation, requires the passage of hydrophobic substrates across the cell
membrane. Membrane proteins are believed to be required to facilitate the
transportation of hydrophobic compounds across the outer membrane of Gram-
negative bacteria surrounded by a hydrophilic lipopolysaccharide layer (Hearn et al.,
2008). ATP-binding cassette (ABC) transporters couple ATP hydrolysis to aid in the
transport of various molecules across cellular membranes (Michalska et al., 2012).
The involvement of ABC transporters, via their associated substrate binding protein
specificity in the uptake and metabolism of benzoic acid and other aromatics was
revealed by Giuliani et al., (2011).
186 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Table 8.4 Heat map showing the relative abundance of ten highly secreted extracellular proteins detected in the culture supernatant of the
AN – Alternating anaerobic-aerobic treatment, TS – Two stage anaerobic-aerobic treatment
* Numbers given within brackets correspond to the signal peptide cleavage site location of classically secreted proteins.
# Non-classically secreted proteins.
Protein yield
Low <----------> high
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 187
There were three classically secreted proteins identified here that matched to
glutathione transporter proteins. They are; glutathione ABC transporter substrate-
binding protein, Glutathione-binding protein precursor and glutathione-binding
protein (Table C.1 in Appendix C). One of the main roles of glutathione is protection
of cells through the regulation of the concentrations of xenobiotics within the cell by
conjugating xenobiotics and exporting them from the cells (Cole & Deeley, 2006).
PCBs are highly hydrophobic and lipophilic molecules, and therefore they are
believed to be capable of unregulated entry into the cells through the cytoplasmic
membrane which can ultimately cause intracellular toxicity (Parales & Ditty, 2017).
Therefore, bacteria capable of utilizing PCBs as their growth substrate must have
some mechanism to control the intracellular PCB concentration at sufficient level to
permit cell growth while maintaining below the intracellular toxic levels. Therefore,
the presence of glutathione related transporter proteins in the culture supernatant
might have a role in maintaining the internal PCB concentration at sub-toxic levels.
The protein with the highest concentration found in the secretome was identified as
a sulfate transporter protein (Table 8.4). Sulfur plays several key roles in bacterial
cells as it is a part of some amino acids such as cysteine, methionine and some cellular
cofactors such as biotin, coenzyme A, S-adenosylmethionine, thiamine, glutathione,
and iron-sulfur clusters (Scott et al., 2007). Sulfate is the preferred sulphur source for
most of the organisms (Aguilar-Barajas et al., 2011). The concentration of the sulfate
transporter was analyzed at different time intervals and found that increased
amounts were detected during the aerobic stages compared to lower yields in the
anaerobic stages of both AN and TS conditions (see Table C.2 in Appendix C, and
Figure 8.10). However, role of sulphate transporters in PCB hydrolysis was not clear.
188 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Figure 8.10 The concentration of the sulfate transporter protein detected in the
culture supernatant over time, under the alternating anaerobic-aerobic (AN)
conditions.
Lykidis et al. (2010) reported that the Cupriavidus necator, a Gram negative
bacterium that utilizes a variety of chloroaromatic compounds as its sole carbon and
energy source contains several ABC transporters that are able to transport aromatic
compounds. Aroclor 1260 is a synthetic chlorinated aromatic hydrocarbon mixture
used as the sole source of carbon, but there were no specific classically secreted
aromatic and xenobiotic degradation related proteins identified in the culture
supernatant. However, about 10% of uncategorized proteins were grouped together
and labelled as unclassified (Figure 8.9). In addition, about 10% of the classically
secreted proteins were grouped as ‘hypothetical’ proteins with unknown functions.
It is possible that these unclassified and unknown proteins identified here may form
new and novel secretion systems used by bacteria for growth depending on the
medium and substrates (Maffei et al., 2017).
About 10% of membrane type proteins were predicted as classically secreted
proteins (Table C.1 in Appendix C). However, it was not clear whether they are
actually secreted proteins or false positives, as they are usually not considered as
secreted proteins, but remain within the membranes of the bacterium. It has been
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 189
suggested that the SecretomeP server would identify them as secreted proteins due
to the presence of cleavable signal peptide sequences at the N-terminus (Song et al.,
2009). However, Maffei et al. (2017) recently revealed that the lipoproteins that are
fully or partially exposed to the surface occur either through known secretion
systems or by novel mechanisms. Additionally, some lipoproteins were identified as
surface-active amphiphilic metabolites or biosurfactants that play a major role in
bioremediation of hydrophobic chemical pollutants such as PCBs (Basak & Dey,
2015). The biosurfactants found during the initial biosufactant screening in Chapter
5 may have some link to the lipoproteins identified as part of the classically secreted
proteins.
The distribution of the 212 identified as classically secreted proteins at fortnightly
intervals were compared under AN and TS anaerobic-aerobic treatment conditions
(Figure 8.11). Out of 211 proteins identified in the AN conditions, 160 proteins were
found common in weeks 2, 4 and 6, with the highest number of proteins (193)
identified at Week 6 (see Figure 8.11A). 209 proteins were identified in the TS
conditions of which 161 proteins were found common in weeks 2, 4 and 6 (see Figure
8.11B). The highest number of proteins (199) with identifications were present at
week 4 under TS conditions. Even though most (140) of the proteins were found
common in all three fortnightly intervals under both AN and TS conditions, they were
present in different concentrations under each condition as indicated in the heat map
in Table C.1, Appendix C. Sulfate ABC transporter substrate-binding protein and
ethanolamine utilization protein were present only in week 6, under AN conditions.
Proteins involved in ethanolamine utilization (EUTs) have been studied and found to
be part of bacterial micro compartments (BMC) whereby BMC functions to control
metabolic reactions by confining volatile/toxic metabolite intermediates such as
alcohols and acids (Axen et al., 2014; Sturms et al., 2015). In the TS treatment,
ferrichrome ABC transporter substrate-binding protein was absent during the first
four weeks of anaerobic treatment and only present under aerobic condition at week
6. Sixteen out of twenty one common proteins found in week 2 and week 4
(anaerobic phase) of TS treatment were found to be transport related proteins.
190 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Figure 8.11 Venn diagram of the distribution of proteins identified as classically
secreted proteins among (A) alternating (AN) anaerobic-aerobic, and (B) two stage
(TS) anaerobic-aerobic conditions.
8.3.2.2.2 Non-classically secreted proteins
Cytoplasmic proteins lacking typical signal peptides or secretion motifs, but found in
the extracellular environment of bacteria are called non-classically secreted proteins
(Zhao et al., 2017). A total of 87 proteins quantified using SWATH were predicted to
be non-classically secreted proteins by the SecretomeP server (Figure 8.8). However,
the mechanisms and cellular membrane locations and features responsible for non-
classical protein secretion are not well understood (Bendtsen et al., 2005).
Similar to the analysis undertaken for classically secreted proteins, non-classically
secreted proteins were further analyzed based on their potential functions and
outcomes are presented in Figure 8.12. Out of the 87 proteins, 17% (15 proteins)
were grouped as hypothetical proteins, indicative of novel or new proteins. Six non-
classically secreted proteins were identified to be involved in motility while five
proteins were related to transporter proteins.
Only one protein matched to an enzyme called Dienelactone hydrolase in relation to
aromatic and xenobiotic degradation. This enzyme plays a crucial role in microbial
degradation of chloroaromatics via the modified chlorocatechol ortho cleavage
pathway (Schlomann et al., 1993) by catalysing the hydrolysis of dienelactone to
maleylacetate (Park et al., 2010).
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 191
Figure 8.12 Functional groupings of proteins identified as non-classically secreted
proteins.
The first published study on non-classical protein secretion in bacteria reported the
secretion of an enzyme called glutamine synthetase (GlnA) (Bendtsen et al., 2005).
The enzymes glutamine synthetase and transglutaminase were detected in the
culture supernatants. These two enzymes may have had a direct impact on the
variation of pH in the culture medium. In the AN anaerobic-aerobic conditions, weeks
1, 3 and 5 were maintained under anaerobic conditions while weeks 2, 4 and 6 were
maintained under aerobic conditions. The starting pH of the medium was set at 7.0.
At each time, when the conditions changed to anaerobic, the pH slightly dropped
(Figure 8.13). However, once conditions were shifted from anaerobic to aerobic, the
pH was found to be closer to its original neutral value of 7, especially in weeks 2, 4
and 6 (Figure 8.13). Glutamine synthetase uses ammonia during the metabolism of
nitrogen by catalysing the condensation of glutamate and ammonia to form
glutamine (Takeo et al., 2013). At each anaerobic phase, the concentration of
glutamine synthetase was relatively high when compared to aerobic phase, indicating
more ammonium ion removal from the medium. The increased glutamine synthetase
concentrations may have coincided with the pH drop under anaerobic conditions as
shown in Figure 8.13. In contrast to glutamine synthetase, the enzyme
transglutaminase concentration was relatively high under each aerobic phase when
192 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
compared to anaerobic phases (see heat map in Table C.2, Appendix C). The enzyme
transglutaminase is responsible for catalysing the formation of a crosslink between a
free amine group and the γ-carboxamide group of a protein bound glutamine. The
reaction also produces ammonia (Zhang et al., 2009). Therefore, it can be assumed
that the variation of glutamine synthetase and transglutaminase concentrations
under aerobic and anaerobic conditions may have contributed to the variation of pH
levels in the medium (Figure 8.13).
Figure 8.13 Variation of glutamine synthetase concentration and pH level in the
culture supernatant under alternating anaerobic-aerobic conditions.
The distribution of the 87 non-classically secreted proteins at fortnightly intervals
were also compared under AN and TS anaerobic-aerobic treatment conditions (Figure
8.14). Similar to the classically secreted proteins, a high amount of the released
proteins under both AN and TS conditions were shared among all the fortnightly
collected samples (see Figure 8.14A and Figure 8.14B). However, and perhaps not
surprisingly, the concentrations of each protein in the culture supernatant varied at
different time intervals as shown in the Table C.1, Appendix C. When AN and TS
treatments were compared, the number of proteins shared during weeks 2, 4 and 6
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 193
was high in the culture supernatant under the TS treatment (63 proteins) compared
to the AN treatment (51 proteins). Under TS conditions, transglutaminase and
glutamine synthase were absent in anaerobic phase (in week 2 and week 4) and only
present in aerobic phase (week 6).
Figure 8.14 Distribution of non-classically secreted proteins under (A) AN anaerobic-
aerobic treatment, and (B) TS anaerobic-aerobic treatment.
Under both AN and TS conditions, a phospholipid-binding protein, dienelactone
hydrolase family protein, histone-like DNA-binding protein and flagella hook-
associated protein FlgK demonstrated relatively high concentrations throughout the
six weeks study period. An elongation factor protein concentration was relatively
high only during the initial two weeks in both, AN and TS conditions. In the TS
treatment, the only protein detected in week 2 was an efflux RND transporter
periplasmic adaptor subunit. Resistance nodulation division (RND) family
transporters belong to the bacterial efflux pumps and are widespread among Gram
negative bacteria (Nikaido & Takatsuka, 2009). They are known to catalyse and
regulate the active transport of substrates such as antibiotics and chemotherapeutic
agents and therefore may have a similar role during cell growth on PCBs.
8.4 Conclusions
So far, various studies have been conducted to identify the PCB degradation
pathways and the relevant enzymes responsible by analysing the bacterial
194 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
proteomes. However, no information is available on the types and potential role of
the enzymes secreted to the extracellular environment during microbial
bioremediation of pollutants such as PCBs. The proteomic approach to investigate
the exoproteome of the bacterial consortium grown on PCBs allowed a detailed study
on the composition and concentration of the proteins extracted from the culture
supernatant, under the AN and TS anaerobic-aerobic cultivation conditions.
During protein visualization using SDS-PAGE, it was revealed that proteins were
detected externally under both anaerobic and aerobic conditions, by the individual
facultative anaerobic bacterial culture members Achromobacter sp. NP03,
Ochrobactrum sp NP04 and Lysinibacillus sp. NP05 while growing on Aroclor 1260 as
the sole source of carbon and energy. These protein results suggested that these
bacteria were capable of secreting proteins or enzymes to the external environment
containing PCBs. In general, protein levels in the extracellular environment under
aerobic conditions were found to be lower than under anaerobic conditions.
Lysinibacillus sp. NP05 under anaerobic conditions demonstrated the highest number
of protein bands among all the samples, although the concentrations were different
for each week. In contrast and in the experiments using the consortium, the second
week of the alternating mode showed relatively high number of protein bands when
compared to the other samples, and this result correlated with the high PCB
degradation rate (49.2±2.5%) observed at week two under the alternating treatment
conditions (Figure 7.3 in Chapter 7).
Analysis of the exoproteome from the bacterial consortium reaction with Aroclor
1260 as the sole source of carbon resulted in the identification of 319 non-secreted,
212 classically secreted and 87 non-classically secreted proteins in the culture
supernatant. These proteins consisted of a heterogeneous group representative of
their diverse functional and cellular roles. Although the contribution of a high number
of cytoplasmic proteins in the extracellular environment is not clear, potentially
resulting from cell lysis or from selective straining, the presence of some proteins
involved in the intermediate steps of the PCB degradation pathways and
detoxification of toxic chemicals is a direct indication for the occurrence of PCB
Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 195
degradation by the bacterial consortium. Transporters were the largest protein group
that represented 58% of the total classically secreted proteins. However, the
mechanisms for protein or enzyme release, adaptations for cell protection from toxic
pollutants and their specific functions in PCB degradation as an extracellular protein
or enzyme remains to be further investigated.
196 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
Chapter 9: Conclusions, practical applications and recommendations for future research 197
Chapter 9: Conclusions, practical applications and recommendations for future research
9.1 Conclusions
Polychlorinated biphenyls are regarded as legacy pollutants. Even though commercial
production and use ceased in 1993, extremely stable properties have made PCBs
persistent in the environment, leading to a range of problems with ecosystem and
human health. There is a vital need to search for sustainable treatment approaches
to remediate PCB contaminated soil due to the serious shortcomings of current
physical and chemical treatment methods. Development of sustainable and effective
bioremediation techniques using microorganisms have the potential to fulfil this
need.
The effectiveness of microbial remediation of PCB contaminated soil is determined
by number of factors. The degradation rate is mainly dependent on the
environmental conditions, soil characteristics, complexity and severity of PCB
contamination, ability of native or bioaugmented microorganisms to survive and to
degrade the PCB congeners and their metabolic intermediates. As PCBs are usually
manufactured and applied as complex congener mixtures, treatment of
contaminated environments is also a complex process. As a result, bioremediation
approaches generally need a longer time span due to the slow degradation rates.
Therefore, improvement and optimization of existing bioremediation technologies
are essential in order to produce viable and cost effective treatment approaches.
The present research study focused on addressing the knowledge gaps in PCB
bioremediation that have been identified in the research literature. The study was
based on the primary hypotheses that complete degradation of complex
commercially available PCB mixtures can be achieved through a combination of
anaerobic-aerobic treatment when appropriate microbial mixtures which are capable
198 Chapter 9: Conclusions, practical applications and recommendations for future research
of increasing the aqueous solubility of PCBs and degrading PCB congeners under both
anaerobic and aerobic conditions are incorporated.
Accordingly, potential PCB degrading microorganisms were isolated from the natural
environment and their PCB degradation potentials were determined as individuals
and as a consortium under different environmental conditions. The ability of bacteria
to make hydrophobic PCBs soluble in aqueous media and to facilitate the uptake of
PCBs into the cells by releasing extracellular proteins and/or secreted enzymes were
also tested. The findings presented in this thesis details the new knowledge created
to bridge a number of current knowledge gaps in the microbial based bioremediation
of PCBs.
The schematic representation of the summary of major research findings described
in Chapters 4-8, is given in Figure 9.1 and the main conclusions derived from the study
are discussed below in Sections 9.1.1 to 9.1.5.
According to Figure 9.1, PCBs were added into the minimal salts based bacterial
growth medium as the sole source of carbon. Biosurfactants that were released and
detected in the extracellular environment showed a direct impact on increasing the
solubility of hydrophobic PCBs in the aqueous medium by the PCB degrading
microorganisms, as discussed in detailed in Chapter 5. The major proteins that were
found to be related to PCB degradation and detoxification and discussed in Chapter
8 are only shown in the Figure 9.1. The lipoproteins detected in the culture
supernatant may have increased PCB solubilization together with the biosurfactants.
According to the existing literature (see chapters 8.1), PCB degradation is believed to
be an intracellular process and therefore the enzymes responsible for anaerobic
degradation and aerobic oxidation pathways shown inside the cell in the Figure 9.1
would not be expected in the extracellular environment. However, some of the
previously identified enzymes such as dienelactone hydrolase and 3-oxoadipate co A
transferase that are responsible for some of the intermediate steps in PCB
degradation pathways were found outside in the culture supernatant as highlighted
in Figure 9.1 (also see Sections 8.3.2.1 and 8.3.2.2). Relatively high levels of different
oxidoreductases were also found in the culture supernatant as both secretory and
Chapter 9: Conclusions, practical applications and recommendations for future research 199
non-secretory proteins, which are usually engaged in detoxification of toxic
hydrocarbons.
It is postulated that bacteria capable of utilizing PCBs as their growth substrate need
some mechanism to control the intracellular PCB concentration at sufficient level to
permit cell growth while maintaining and controlling the intracellular toxic levels.
Therefore, as shown in Figure 9.1, the presence of some classically secreted
glutathione related transporter proteins in the culture supernatant might have a key
role in maintaining the internal PCB concentration at sub-toxic levels as one of the
main functions of glutathione is protection of cells through the regulation of internal
xenobiotic concentrations by conjugating and exporting them from the cells. Nearly
half of the secretory proteins were related to cellular transport (see Figures 8.9 and
9.1) and they were found to be involved in various cellular functions such as uptake
of nutrients, regulation of cytoplasmic metabolites concentration, and prevention of
the effects of toxins by catalyzing their active efflux.
200 Chapter 9: Conclusions, practical applications and recommendations for future research
Figure 9.1 A schematic representation of the summary of major findings of the research study.
Chapter 9: Conclusions, practical applications and recommendations for future research 201
9.1.1 Isolation, screening and identification of potential PCB degrading
microorganisms
As discussed in Chapter 4, the selective enrichment screening was done to isolate and
identify the potential PCB degrading microorganisms from the natural environment.
The main conclusions derived are as follows:
• From 11 microorganisms initially tested, two obligate aerobic and four
facultative anaerobic bacterial strains that can utilize commercial PCB mixture,
Aroclor 1260 as their sole source of carbon were isolated from the natural
environment.
• They were identified as Chryseobacterium sp. NP01, Delftia sp. NP02,
Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp. NP05 and
Pseudomonas sp. NP06 using 16S rRNA gene sequence based molecular
identification.
• This finding is also the first evidence of reporting the PCB degradation ability of
Chryseobacterium and Delftia species.
9.1.2 Screening of bacterial isolates for their ability to produce biosurfactants to
make hydrophobic PCBs soluble in aqueous media
As discussed in Chapter 5, six bacterial cultures isolated during Chapter 4 were further
tested for their ability to produce biosurfactants as one of the limitations in biological
breakdown is poor bioavailability due to the extreme hydrophobic nature of PCBs. The
main conclusions from chapter 5 are as follows:
• Microorganisms capable of degrading PCBs discovered in this study, were also
found to have the potential to produce some surface active substances to facilitate
the enhancement of solubility of the hydrophobic PCBs.
• The PCB solubility results was positively correlated with the biosurfactant
production.
202 Chapter 9: Conclusions, practical applications and recommendations for future research
• Chryseobacterium sp. NP01 and Lysinibacillus sp. NP05 demonstrated the highest
biosurfactant production, PCB solubility and chloride ion accumulation when
254 Appendix B: PCB data related to bacterial consortium study
Appendix C: Extracellular protein analysis 255
Appendix C: Extracellular protein analysis
256 Appendix C: Extracellular protein analysis
Table C.1 Relative abundance of extracellular proteins of bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 found in the culture supernatant under alternating (AN) and two stage (TS) anaerobic-aerobic conditions as a heat map.
297 WP_054550133.1 SRPBCC domain-containing protein Yes#
Appendix C: Extracellular protein analysis 269
No. Accession number Protein
AN
(Weeks)
TS
(Weeks) Secreted*
2 4 6 2 4 6
298 OLU07233.1 TIGR01244 family protein Yes#
299 WP_088153633.1 DUF2950 domain-containing protein Yes#
Notes:
AN – Alternating anaerobic-aerobic treatment, TS – Two stage anaerobic-aerobic treatment
* Numbers given within brackets correspond to the cleavage site location within the amino acid sequence of classically secreted proteins.
# Non-classically secreted proteins.
Protein yield
Negative Low <----------> high
270 Appendix C: Extracellular protein analysis
Table C.2 Relative abundance of extracellular proteins of bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05
found in the culture supernatant under alternating (AN) anaerobic-aerobic conditions as a heat map. Classically secreted proteins were predicted using the
SignalP 3.0 Server and the non-classically secreted proteins were predicted using the SecretomeP 2.0 Server. Week 1, 3, 5 were kept under anaerobic and
Supplementary Material 1: Abstracts of conference papers relevant to the thesis 293
Supplementary Material 1: Abstracts of
conference papers relevant to the thesis
294 Supplementary Material 1: Abstracts of conference papers relevant to the thesis
Supplementary Material 2: Publications relevant to the thesis 295
Supplementary Material 2: Publications
relevant to the thesis
Science of the Total Environment 651 (2019) 2197–2207
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Science of the Total Environment
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Solubilization and degradation of polychlorinated biphenyls (PCBs) bynaturally occurring facultative anaerobic bacteria
Gathanayana Pathiraja a, Prasanna Egodawatta a, Ashantha Goonetilleke b, Valentino S. Junior Te'o a,⁎a School of Earth, Environmental and Biological Sciences, Queensland University of Technology (QUT), Brisbane 4001, Queensland, Australiab School of Civil Engineering and Built Environment, Queensland University of Technology (QUT), Brisbane 4001, Queensland, Australia
H I G H L I G H T S G R A P H I C A L A B S T R A C T
• The best strains degraded PCBs underboth anaerobic and aerobic conditions.
Article history:Received 20 August 2018Received in revised form 9 October 2018Accepted 9 October 2018Available online 11 October 2018
Editor: Jay Gan
A combination of solubilization and degradation is essential for the bioremediation of environments contami-natedwith complex polychlorinated biphenyls (PCB)mixtures. However, the application of facultative anaerobicmicroorganisms that can both solubilize and breakdown hydrophobic PCBs in aqueousmedia under both anaer-obic and aerobic conditions, has not been reported widely. In this comprehensive study, four bacteria discoveredfrom soil and sediments and identified as Achromobacter sp. NP03,Ochrobactrum sp. NP04, Lysinibacillus sp. NP05and Pseudomonas sp. NP06, were investigated for their PCB degradation efficiencies. Aroclor 1260 (50 mg/L), acommercial and highly chlorinated PCB mixture was exposed to the different bacterial strains under aerobic,anaerobic and two stage anaerobic–aerobic conditions. The results confirmed that all four facultative anaerobicmicroorganisms were capable of degrading PCBs under both anaerobic and aerobic conditions. The highest chlo-rine removal (9.16± 0.8mg/L), PCB solubility (14.7± 0.93mg/L) and growth rates as OD600 (2.63 ± 0.22)wereobtained for Lysinibacillus sp. NP05 under two stage anaerobic-aerobic conditions. The presence of biosurfactantsin the culturemedium suggested their role in solubility of PCBs. Overall, the positive results obtained suggest thathigh PCB hydrolysis can be achieved using suitable facultative anaerobic microorganisms under two stageanaerobic-aerobic conditions. Such facultative microbial strains capable of solubilization as well as degradationof PCBs under both anaerobic and aerobic conditions provide an efficient and effective alternative to commonlyused bioaugmentation methods utilizing specific obligate aerobic and anaerobic microorganisms, separately.
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health impacts. Polychlorinated biphenyls (PCBs) are one such toxicchemical group consisting of 209 different chlorinated organic com-pounds. PCBs are persistent in the environment due to low reactivity,high chemical stability and extreme hydrophobicity (Beyer and Biziuk,2009). Conversely, high lipophilicity makes PCBs soluble in fats.Such characteristics results in bioaccumulation, bioconcentration andbiomagnification along the food chains leading to numerous healthimplications in humans and animals (ATSDR, 2000). Due to theirpersistence in the environment, sites contaminated with PCBs such aselectricity distribution stations, service areas and dumpsites as well assediments in the nearby waterbodies are still posing significant threatsto human and ecosystem health.
Microorganisms play an important role in the removal of toxicchemical compounds from the environment. Biological conversion ofhighly and moderately chlorinated PCB congeners into less chlorinatedcongeners has been reported to take place through dechlorinationunder anaerobic conditions (Praveckova et al., 2015; Agullo et al.,2017). In comparison, lower and moderately chlorinated congenerscan be degraded by oxidative bacteria under aerobic conditions throughupper and lower biphenyl degradation pathways (Field and Sierra-Alvarez, 2008). Therefore, to achieve complete degradation, one of themost promising bioremediation strategies is to combine the anaerobicdechlorination and aerobic oxidation (Passatore et al., 2014). Althoughnumerous sediment and soil based studies have been conducted eitherunder anaerobic or aerobic conditions separately, using potentialPCB degrading bacteria (Adrian et al., 2009; Payne et al., 2011; Wangand He, 2013; Murinova et al., 2014), studies based on combinedanaerobic-aerobic conditions are limited (Evans et al., 1996; Masteret al., 2002; Long et al., 2015). Studies by Evans et al. (1996) andMaster et al. (2002) used two separate groups of bacteria capable ofreductive dechlorination and aerobic oxidation, respectively, to degradePCBs in contaminated soil slurry. No studies appear to have beenundertaken so far on PCB degradation using facultative anaerobic mi-croorganisms under two-stage anaerobic-aerobic conditions. However,there is a recent study based on facultative anaerobic bacteria mediatedin situ delignification and enhanced gas release under microaerophilicconditions in soil containing lignocellulose (Rashid et al., 2017).
Past research literature on biochemical pathways and intracellularlocalization of enzymes responsible for PCB degradation suggest thatPCBs have to be solubilized first for easier passage through the cellwall and into the cytoplasm prior to being metabolized. Therefore, anincrease in the rate of solubilization could accelerate the entrance ofPCBs into the cells and their subsequent degradation (Ohtsubo et al.,2004). Most of the research studies undertaken so far on PCB solubilityhave been based on the addition of chemical or biological surfactantswith limited investigation into the actual application ofmicroorganismsproducing surfactants (Singer et al., 2000; Fava and Di Gioia, 2001;Occulti et al., 2008; Viisimaa et al., 2013). Chemical surfactants havethe advantage of being economical, but are often toxic to biologicalsystems (Abraham et al., 2002). In comparison, biosurfactants generallyexhibit higher interfacial tension reduction activities compared tochemical surfactants, and are less toxic and readily biodegradable(Viisimaa et al., 2013). However, the main disadvantage in the use ofcommercially available biosurfactants is the high cost (Aparna et al.,2012).
Therefore, the use of suitable biosurfactant producing microbialstrains, which are also capable of degrading PCBs under both, anaerobicand aerobic conditions, would be an attractive alternative to the use ofeither chemical or biological surfactants or PCB degrading aerobicand anaerobic bacterial groups, separately. Indeed, the application ofbiosurfactant-producing and pollutant-degrading microorganismsoffers the dual advantage of a continuous supply of biodegradablesurfactants and the ability to degrade pollutants (Megharaj et al., 2011).
The aim of this study was to isolate potential naturally occurringfacultative anaerobic bacteria from soil and sediment environmentsand to investigate their capability for degrading Aroclor 1260, a complex
and widely used commercial PCB mixture, under comparative anaero-bic, aerobic and two stage anaerobic-aerobic conditions while solubiliz-ing a hydrophobic PCBmixture. The outcomes of the study are expectedto contribute to the development of more efficient and effective bacte-rial mediated bioremediation treatment of PCB contaminated soils andsediments.
2. Materials and methods
2.1. PCB source
Aroclor 1260 was selected as the commercial grade PCB source forthis study and obtained as a GC/FID grade technical mixture fromAccuStandard Inc. (New Haven, CT, USA). Aroclor 1260 represents acomplex PCB mixture consisting of about 75 different penta to nonachloro biphenylswith an average of 6.3 chlorines per biphenylmolecule(Bedard et al., 2007). Aroclor 1260was prepared as a 50mg/mL stock inGCMS grade acetone, before use.
2.2. Screening, enrichment and identification of possible PCB degradingmicroorganisms
2.2.1. Sampling of soil and sedimentsIn order to isolate possible facultative anaerobic PCB degrading bac-
teria, six soil samples around the Brisbane City area and six sedimentsamples from Brisbane River (27.4745° S, 153.0293° E) and CoombabahLake, Gold Coast (27.54° S, 153.22° E), Australia were collected intosterile glass bottles and transported on ice to the laboratory. Soil andsediment samples were separately homogenised and 50 g from eachcomposite soil and sediment mixtures were added to duplicate250mLErlenmeyer flasks. After contaminationwith Aroclor 1260 to ob-tain 50 mg/kg PCB concentration, flasks were incubated stationaryunder aerobic conditions at room temperature (23 °C ± 1 °C) for onemonth. Similarly, 50 g of each composite soil and sediment mixturewere added to duplicate 50 mL polypropylene vials with screw caps,contaminated with Aroclor 1260 to obtain 50 mg/L concentration andincubated stationary inside the anaerobic chamber (COY lab products)main compartment at room temperature (23 °C ± 1 °C) for onemonth. The atmosphere inside the anaerobic chamber was maintainedconstant at 4.9% H2, 10.7% CO2 and 84.4% N2.
2.2.2. Selective enrichment of possible PCB degrading bacteriaIsolation ofmicroorganisms capable of utilizing PCBswas carried out
through a series of selective enrichments using DSMZ medium 465a(Atlas, 2005) as the base minimal salt medium (MSM) with the follow-ing modifications. After autoclaving the medium, instead of 0.5 g/Lhydroxybiphenyl in ethanol, Aroclor 1260 in GCMS grade acetone wasadded as the sole carbon and energy source to give 50 mg/L final PCBconcentration. Four 250 mL Erlenmeyer flasks containing 100 mL ofsterile MSM medium were prepared. Two flasks were inoculated with10 g of composite soil, and the other two were inoculated with 10 gof sediment previously contaminated with Aroclor 1260. Two flasks(onewith soil and the other with sediment) were incubated aerobicallyin a platform shakermaintained at 150 rpmand 28 °C. The other two re-maining flasks were incubated anaerobically under static conditions inan incubator kept inside the anaerobic chamber with the temperaturekept at 28 °C. Four serial transfers (10% of the enrichment medium)were carried out from each flask at weekly intervals into fresh sterileMSM containing 50mg/LAroclor 1260. From the final flask, supernatantwas plated on nutrient agar (CM003, Oxoid) and incubated overnight at28 °C. Morphologically different colonies were isolated and streaked onfresh nutrient agar plates.
2.2.3. Characterization of potential PCB degrading bacteriaBacterial isolates obtained from selective enrichment were inocu-
lated in duplicate on minimal salt agar plates containing 50 mg/L of
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Aroclor 1260 as sole source of carbon and incubated at 28 °C in parallelunder anaerobic and aerobic conditions to determine their growth tol-erance in the presence/absence of atmospheric oxygen. Minimal saltagar with no added Aroclor 1260, but an equal volume of acetone thatwas used to add Aroclor 1260 into the minimal salt agar in the growthexperiment, was used as the negative control to determine whetherthere was any contribution from leftover acetone on bacterial growth.In comparison, nutrient agar medium alone was used as the positivecontrol. Isolates with the ability to grow on PCBs under both, anaerobicand aerobic conditions were selected for further characterization basedon colony morphology on nutrient agar plates and cell morphologyusing Gram staining. Additionally, the bacterial isolates with highPCB solubility and degradation potential were further characterizedbased on their ability to utilize different carbon and nitrogen sourcesusing Biolog PM1 and PM3B plates containing 95 separate sole carbonand nitrogen sources. Full-length 16S rRNA gene sequences were PCRamplified from purified genomic DNA (gDNA) isolated from puremicrobial isolates in order to ascertain their identification as shown inFig. 1.
2.2.3.1. Genomic DNA extraction and polymerase chain reactions (PCR).Bacterial gDNAs were extracted using Isolate II genomic DNA kit(Bioline) and different gDNAs were used as templates for PCRamplification of 16S rRNA genes. Full length universal 16S rRNAprimers, 27F and 1492R (Integrated DNA Technologies, Inc.), wereused and amplifications were performed according to the MyTaq HSRed DNA polymerase protocol (Bioline). The following PCR cyclingconditions were used: 1 × cycle: 95 °C/10 min, 30 × cycles: 95 °C/30 s,50 °C/30 s, 72 °C/2 min, 1 × cycle: 72 °C/7 min, and held at 4 °C. Thequality of the PCR products was checked by electrophoresis at 100 Vfor 1 h using a 1% agarose gel. The ~1.5 kb RNA gene PCR productswere extracted and purified using the Isolate II PCR and gel extractionkit protocol (Bioline).
2.2.3.2. Ligation, transformation and cloning of PCR products for sequencing.The gel purified ~1.5 kb 16S RNA gene fragments were ligated intothe pGEM-T Easy Vector (Promega) and transformed into high efficiencyE. coli JM 109 competent cells. The transformed cells were screened forblue andwhite colonies by plating on Luria-Bertani (LB) agar plates con-taining ampicillin (100 μg/mL), Isopropyl β-D-1-thiogalactopyranoside
Fig. 1. Process overview for full length (1.5 kb) 16S rRNA based genomic D
(IPTG, 0.5mM) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside(X-Gal, 80 μg/mL). Recombinant colonies were selected and checked forthe presence of 16S gene inserts by colony PCR using M13 forward andreverse primers (Integrated DNA Technologies, Inc.) that anneal to out-side of the pGEM-T vector (Promega) multiple cloning site.
2.2.3.3. Recombinant plasmid isolation and sequencing. Recombinantclones of each bacterial culture were grown in liquid LB broth contain-ing ampicillin (100 μg/mL), before plasmid isolation and purificationswere performed using the QIAprep (QIAGEN) Spin Miniprep Kit. Twoseparate sequence reactions using M13F and M13R primers werecarried out for each recombinant plasmid, according to the standardBigDye Terminator (BDT) v3.1 cycle sequencing kit (Thermo FisherScientific) protocol. DNA sequencing was performed using 3500Genetic Analyzer (Applied Biosystems, Hitachi) and the programFinchTV 1.4.0 (Geospiza Inc.) was used for editing of raw nucleotideDNA sequence data. The DNA sequences were polished by the removalof the plasmid vector sequences and merging of the two overlappingsequences to obtain a full length 1.5 kb contiguous sequence wasdone using the Emboss Needle tool of the European BioinformaticsInstitute (EMBL-EBI). The resulting full length 16S rRNA genesequences were then submitted to determine the closest matchingsequences by comparing with the bacterial and archaeal 16S RNAdatabases using the Sequence Match tool of the Ribosomal DatabaseProject (RDP) (Cole et al., 2014) and Basic Local Alignment SearchTool (BLAST) of NCBI, USA. The searches were limited to ‘Type’ bacterialstrains.
2.3. PCB degradation by individual bacterial cultures under aerobic, anaer-obic and two-stage anaerobic-aerobic conditions
Erlenmeyer flasks containing 75 mL of sterile MSM (same mediumused for selective enrichment) were prepared and 50 mg/mL Aroclor1260 stock solution in GCMS grade acetone was added to each flask assole source of carbon to give 50 mg/L Aroclor 1260 concentration.Contents of theflaskswere vigorously shaken for 3min to allow acetoneto evaporate. For each bacterial culture, three separate sets of experi-ments were carried out in parallel under aerobic, anaerobic and two-stage anaerobic-aerobic conditions. In all anaerobic and two stageexperiments, flasks containing media were prepared and incubated in
NA isolation, cloning and sequencing based molecular identification.
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an anaerobic chamber (COY laboratory products, Inc.) under strict an-aerobic conditions. The liquid minimal salt medium in the flasks werekept equilibrated for oneweek inside the anaerobic chambermain com-partment before theywere inoculatedwith the seed cultures. Palladiumcatalysts located inside the chamberwere used to scrub any residual ox-ygen present in the chamber and the samples were transferred underanaerobic conditions without changes to the internal atmosphere inthe chamber through a heavy duty vacuum airlock compartment. Theenvironment inside the anaerobic chamber was maintained constantunder 4.9% H2, 10.7% CO2 and 84.4% N2 (BOC Australia). Under these an-aerobic conditions, hydrogen gas present inside the anaerobic chamberand controlled at 4.9% (mol/mol) concentration as thepotential electrondonor in the present study.
Aliquots of bacterial seed cultures (7.5 mL) grown overnight in LBbroth were centrifuged at 5000 ×g for 10 min. The resulting cell pelletswere washed twice in MSM, resuspended in 1 mL of the samemedium,and then transferred into the anaerobic chamber and used as theinoculum for each flask. Before inoculation of bacterial cultures, flaskswere maintained at pH 7. Flasks used in anaerobic experiments werekept at 28 °C in an incubator kept inside the anaerobic chamber, withoccasional gentle shaking by hand over the six weeks period. In aerobicexperiments, after inoculationwith seed cultures,flaskswere incubatedat 28 °C and 150 rpm under aerobic conditions for six weeks. The two-stage experiment was started and continued at 28 °C under similaranaerobic conditions (with occasional shaking by hand) for the firstfour weeks, and the flasks were removed from the anaerobic chamberand transferred to aerobic conditions at 28 °C and 150 rpm during thelast two weeks. The experiments were conducted in triplicate whilethe controls were conducted in duplicate. Minimal salt media spikedwith 50 mg/L Aroclor 1260 with no added bacteria seed cultures wereused as abiotic controls. Minimal salt media spiked with equal volumesof acetone that were used to dissolve 50 mg/L Aroclor 1260 were inoc-ulated with bacterial seed cultures similar to the experiment and usedas media controls.
2.3.1. Sample collection and analysisSamples (4mL) were withdrawn from each flask at time zero and in
weekly intervals to determine pH, cell growth and PCB solubility. Inorder to have representative samples, liquid aliquots were removedfrom themiddle of the culturemediumwhile flaskswere kept under ag-itation. Bacterial cell densities were calculated as colony forming units(CFU) using standard plate count. The DU730 Beckman Coulter UV/VISspectrophotometer was used to measure the cell growth at 600 nmoptical density and calibrated Hanna HI 2221 pH meter was used tomeasure the pH according to the APHA method 4500-H (APHA, 2012).At the end of six weeks, chloride ion concentration in each flask wasmeasured as described below in order to determine the amount ofchlorines released from the PCB mixture, by the direct action of themicrobes. If bacteria were able to dechlorinate the PCB molecules, thereleased chloride ions were expected to accumulate in the culturemedium. To see whether there is any significant contribution to thechloride level in the culture medium due to the lysis of bacterialcells, the chloride measurements were performed before and afterthe sonication of the samples. Samples were first centrifuged at5000 ×g for 10 min and the resultant supernatant was filteredthrough 0.2 μm sterile filter discs to remove the bacterial cells. Thecell free culture supernatant was used to measure the chloride ionconcentration using the Dionex ICS-2100 ion chromatographyaccording to USEPA method 300.0 (USEPA, 1993). For the calibrationcurve preparation, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm and100 ppm standard sodium chloride solutions were used. The abioticcontrols andmedia controls (see Section 2.2) were used in parallel inorder to subtract the background chloride levels coming from theminimal salt medium, leaking of any chloride due to bacterial celllysis and any traces left from the LB medium used to cultivate thebacterial seed cultures.
2.3.2. PCB extraction and analysisLiquid aliquots (1mL) sampled in weekly intervals were transferred
to 8 mL glass vials fitted with Teflon-lined screw caps. PCBs were ex-tracted with 2.5 mL of GC grade n-hexane (USEPA, 2007) by vigoroushorizontal shaking on a platform shaker for 4 h at 250 rpm underroom temperature, followed by centrifugation at 5000 rpm for 10 min(Adrian et al., 2009). The solvent phase was used for subsequentPCB analysis. Before extraction, 25 μL of 2, 4, 5, 6-tetrachloro-m-xylene(10 μg/mL in hexane) was added to each sample as the surrogate stan-dard to determine the extraction efficiency (USEPA, 2007). Surrogaterecovery was 98.98% ±19.11% (n = 308).
PCB extracts and standards were spiked with 2,2′,4,4′,5,5′-hexabromobiphenyl as an internal standard (USEPA, 2007). Total solu-ble PCB levels were determined as per the USEPA method 8082A(USEPA, 2007). Thermo Scientific Trace 1310 (in splitless mode, at260 °C inlet temperature, 80 mL/min split flow and 1.2 min splitlesstime) equipped with SSL injector with splitless liner with glass wool,Thermo TG-5SilMS analytical column (30m× 0.25mm ID × 0.25 μm)and, TriPlus RSH auto sampler were used for PCB analysis. Heliumwas used as the carrier gas at 1.2 mL/min constant flowrate. Thecolumn was kept at 40 °C for 2 min, and then the temperature wasraised to 300 °C at 15 °C/min and kept for 5 min. The ThermoScientific Trace Finder EFS software was used for the screening andquantitation of total soluble PCBs and PCB homolog groups usingthe Thermo Scientific triple stage quadrupole Mass spectrometer(TSQ8000 EVO). Retention times for each PCB homolog group wastaken from the existing literature (Walker and Feyerherm, 2013)and full scan acquisition and timed selective reaction monitoring(SRM) modes were used to distinguish the PCB homolog groups.Relative amounts of dissolved PCB levels and homolog groups weredetermined by nine-point calibration using Aroclor 1260 standardsolutions and area integration.
2.4. Screening for biosurfactant production
At the end of the six week experiment, 10 mL culture supernatantfrom each flask of the aerobic batch experiment were centrifuged at5000 ×g for 10 min and filtered through 0.2 μm filters to separate thebacterial cells. The cell free supernatants were used for the subsequentbiosurfactant screening tests.
2.4.1. Drop collapse testThe drop collapse test was used as a primary screening to deter-
mine the ability of bacterial strains for their biosurfactant productioncapacity based on past literature (Bodour et al., 2003; Alvarez et al.,2015; Panjiar et al., 2015; Joy et al., 2017). 20 μL cell free culturesupernatant was mixed with 5 μL of 0.1% methylene blue solutionand placed on parafilm paper as a drop using a pipette. The purposeof adding methylene blue was for easy visualization of the droplet.Diameter of the drop was measured after 1 min using 1 mm gritpaper placed underneath the parafilm paper. 1% (w/v) sodium dode-cyl sulphate (SDS) solution was used as the positive control whilephosphate buffered saline (PBS) solution and abiotic controls wereused as negative controls. The results where the diameter of thedroplet was at least 1 mL larger than the one made by the negativecontrol were considered as positive for biosurfactant production.When there is no biosurfactant present, the droplet remains stableas the polar water molecules are repelled from the hydrophobicsurface. When the interfacial tension between the liquid droplet andthe hydrophobic surface is reduced, the drop spreads or collapses(Alvarez et al., 2015).
2.4.2. Emulsification index (EI24)3 mL of cell free culture supernatant and 3 mL of mineral oil were
taken into a graduated test tube and vigorously shaken for 2 min toform an emulsion. The mixture was allowed to stand still for 24 h and
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the height of emulsion layer was measured (Nayak et al., 2009). Theemulsification index was calculated using Eq. (1) (Panjiar et al., 2015).
EI₂₄ %ð Þ ¼ Height of emulsion formed after 24 hrs=Total height of solution� 100
ð1Þ
Emulsions formed following the reactions with different culturesupernatants were compared to the controls. The positive control usedwas a 1% (w/v) solution of synthetic surfactant sodiumdodecyl sulphatein deionised water, whereas the abiotic and medium only controlsamples were used as the negative controls.
2.4.3. HaemolysisTo detect the haemolytic activity indicative of biosurfactant
production, 50 μL of the cell free culture supernatant was spotted onthe middle of Tryptone soya agar plates containing 5% sheep blood(Thermo Fisher Scientific) and the plates were incubated at 28 °C for48 h (Alvarez et al., 2015). Plates were visually examined forhaemolysis. The level of clearance of red blood cellswas considered pro-portional to the concentration of biosurfactant. A yellow transparentzone indicated complete lysis of red blood cells and was regarded asbeta (β) or complete haemolysis. The appearance of dark green zonesbeneath the place where the supernatant was spotted was consideredas alpha (α) or partial haemolysis of blood cells. Alpha and betahaemolysis were considered as positive for biosurfactant production.No change in the blood agar plates indicated gamma (γ) or nohaemolysis (Joy et al., 2017).
3. Results and discussion
3.1. Identification of PCB utilizing facultative anaerobic culture members
After selective enrichment screening, three Gram negative (NP 03,04 and 06) and one Gram-positive (NP05) rod-like shape bacterialstrains capable of utilizing PCBs as sole source of carbon under bothanaerobic and aerobic conditions were isolated. During 16S rRNA genesequencing based identification, the closest matching sequences wereobtained from National Center for Biotechnology Information (NCBI)and Ribosomal Database Project (RDP) databases and are summarisedin Table 1. The RDP database was used as a curated database as itprovides the aligned and annotated rRNA gene sequence data (Coleet al., 2014; Wang et al., 2007), while NCBI was used as a non-curateddatabase. There were no differences between the two databases forculture numbers NP04 and NP05 as they obtained the same closestrelatives from both databases. However, similarity of culture numbersNP03 and NP06 were limited to the generic level between the twodatabases. There is no universal definition existing for species levelidentification using 16S rRNA gene sequencing and use of acceptablecriteria for establishing a species match varies widely in differentstudies (Janda and Abbott, 2007). Therefore, if similarity was not
Table 1Comparison of closest relatives of isolated bacteria based on NCBI and RDP databases.
a Similarity score - percent sequence identity over all pairwise comparable positions when r
100%, then identification was limited up to the generic level as there isno guarantee of having 1% divergence to obtain accurate identification.As none of the cultures showed 100% similarity to the existing data-bases, the four bacterial cultures were named according to their genusfollowed by the strain number. 16S rRNA sequences of the four cultures(NP03, NP04, NP05 and NP06) were deposited in NCBI GenBank data-base under the accession numbers KY711179, KY711180, KY711181and KY711182, respectively.
There was no visible growth in the media controls that containedequal volume of acetone used to dissolve Aroclor 1260. This findingconfirmed that the bacteria were not able to utilize acetone as their car-bon source even if there was residual acetone left in the medium afterevaporation. Bacteria such as Achromobacter sp. and Ochrobactrum sp.have been found in PCB contaminated sediments, but only under aero-bic conditions (Dudasova et al., 2014; Murinova and Dercova, 2014).However, in this study, it was found that the four facultative anaerobicbacterial strains Achromobacter sp. NP03, Ochrobactrum sp. NP04,Lysinibacillus sp. NP05 and Pseudomonas sp. NP06 were able to growon minimal salt agar containing Aroclor 1260 under aerobic and anaer-obic conditions indicating their ability to utilize PCBs as their sole sourceof carbon under both environmental conditions.
The facultative anaerobic bacterial strains with high PCB solubilityand degradation potential were further characterized based on theirability to utilize different carbon and nitrogen sources using BiologPM1 and PM3B plates. Achromobacter sp. NP03, Ochrobactrum sp.NP04 and Lysinibacillus sp. NP05 were able to utilize L-Proline as solesource of carbon and nitrogen. In addition, L-lactic acid and methyl py-ruvate were utilized as sole source of carbon (Table S1, SupplementaryInformation) and L-Glutamic acid, Ala-His, Ala-Leu and Gly-Gln wereused as nitrogen sources (Table S2, Supplementary Information) athigh rates by Achromobacter sp. NP03, Ochrobactrum sp. NP04 andLysinibacillus sp. NP05.
3.2. PCB degradation under aerobic, anaerobic and two stage anaerobic-aerobic conditions
3.2.1. Growth profiles measured at OD600 while grown on Aroclor 1260 ascarbon source
Fig. 2 shows the growth profiles of four facultative anaerobic strainsunder aerobic, anaerobic and two stage anaerobic-aerobic conditions.As evident in Fig. 2a, all four strains reached saturation by week oneunder aerobic conditions (OD600 of 0.51, 0.7, 0.62 and 0.46 for NP03,04, 05 and 06, respectively) and did not improve much further withtime. According to the literature, these results suggest the possibilityof all four strains consuming lower chlorinated PCB congeners withinthe firstweek andwere unable to degrade highly chlorinated congenersunder aerobic conditions (Field and Sierra-Alvarez, 2008). This conceptcan be supported by the fact that Aroclor 1260, the PCB source used inthis study consists of less than 10% (by weight) of lower chlorinatedhomolog groups (containing four or fewer chlorines per biphenyl
Fig. 2. Growth of four facultative anaerobic bacterial strains under (a) aerobic,(b) anaerobic, and (c) two stage anaerobic-aerobic conditions. Error bars represent thestandard deviation of mean values (n = 3).
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molecule) compared to the highly chlorinated homologs (Mayes et al.,1998; ATSDR, 2000). To provide further support for these observations,Pieper and Seeger (2008) reported that aerobic bacteria are capable ofdegrading biphenyls as the sole source of carbon and energy, andusually involves the biodegradation of PCBs with less than four chlorineatoms. Therefore, the inability of bacterial cultures to degrade highlychlorinated congeners and the limited availability of lower chlorinatedcongeners in the medium may have negatively contributed to the lim-ited growth rate under aerobic conditions.
In comparison, during anaerobic conditions, all four strains of NP03,NP04, NP05 and NP06, showed higher cell densities with OD600 of 1.22,1.31, 1.04 and 0.82, respectively, reaching maximum growth by week 4(Fig. 2b). Significantly high growth rates under anaerobic conditionswithout the presence of carbon sources other than PCBs is an indicationof biphenyl ring cleavage in addition to dechlorination. According toexisting literature, anaerobic dechlorination is a reductive process thatuses PCBs as electron acceptors, but the carbon rings are usually notcleaved (Wiegel andWu, 2000;Hughes et al., 2009). In caseswhere bac-teria are not capable of breaking down the carbon ring structure, they
will require additional carbon sources in order tomaintain their growthduring dechlorination of PCBs (Wu et al., 2000; Bedard et al., 2006;Adrian et al., 2009; Wang and He, 2013). During this study, 8.7 × 106,5.0 × 106, 4.9 × 106 and 4.5 × 106 cells/mL initial cell densitiesat week zero were increased by 200 folds to 2.0 × 108, 1.3 × 108,1.3 × 108 and 1.0 × 108 cells/mL at week four for NP03, NP04, NP05and NP06, respectively. This fact confirmed the capability of these mi-croorganisms to utilize PCBs as their carbon source other than electronacceptors under anaerobic conditions without the requirement foradditional carbon sources.
In the two-stage anaerobic-aerobic cultivations as shown in Fig. 2c,all four strains showed similar growth patterns to the anaerobic condi-tions (Fig. 2b) up to week four during anaerobic conditions. However,after switching from anaerobic to aerobic conditions between weekfour to six, Lysinibacillus sp. NP05 started increasing in growth fromOD600 of 1.04± 0.07 to 2.63± 0.22, which is nearly three times its orig-inal cell density. Similarly, Ochrobactrum sp. NP03, Achromobacter sp.NP04 and Pseudomonas sp. NP06 also indicated slight increase in celldensities (1.39 ± 0.08, 1.48 ± 0.23 and 0.99 ± 0.06, respectively)when changing conditions from anaerobic to aerobic as indicated inFig. 2c. Based on these results, it can be postulated that all fourorganisms are capable of performing dechlorination under anaerobicconditions in a similar way at different rates and Lysinibacillus sp.NP05 subsequently hydrolyses the carbon ring structure extensivelyunder aerobic conditions compared to the other three organisms.
3.2.2. Variation of PCB solubility in aqueous mediumPCB concentrations were measured as total soluble PCBs in order to
investigate the change of solubility levels in themedium resulting frompotential bacterial activity under aerobic, anaerobic and two stage con-ditions. Solubility of PCBs in themediumwas an indirectmeasure of themicroorganisms attacking the PCBs and converting them from insolubleto soluble forms. Theoutcomes of this experiment are shown in Fig. 3. Atthe initial concentration of 50mg/L, it was observed that the PCBs addedto the flasks appeared to be not completely soluble, but most remainedat the bottom as small clumps prior to the addition of the bacterialcultures. The total soluble PCB levels in the abiotic controls with no bac-teria addedwasmeasured and found to remain very low throughout thecultivation period, with values found to be 0.15 ± 0.02 mg/L, 0.57 ±0.07 mg/L and 0.5 ± 0.23 mg/L under aerobic, anaerobic and twostage anaerobic-aerobic conditions, respectively. Such solubility rangesare consistentwith previous reports (ATSDR, 2000; Bedard et al., 2007).
Under aerobic conditions, Lysinibacillus sp. NP05 showed the highestcapacity to solubilize PCBs compared to Ochrobactrum sp. NP03,Achromobacter sp. NP04 and Pseudomonas sp. NP06 (Fig. 3a). Thisincrease in activity by Lysinibacillus sp. NP05 started after week oneand reached optimal activity by week five before it started to decrease.However, Lysinibacillus sp. NP05 did not increase in cell growth, butshowed similar cell densities to the rest of the three strains as shownin Fig. 2a. The increased solubility over the aerobic incubation periodwith no corresponding increase in cell growth shown by Lysinibacillussp. NP05 could possibly be due to two reasons. Firstly, their ability tosecrete some surface-active compounds that ultimately lead to in-creased solubility of the hydrophobic PCB mixture (Singer et al., 2000;Cameotra and Bollag, 2003). Secondly, the bacterium was unable tocarry out the dechorination of highly chlorinated congeners under aer-obic conditions as reported by Furukawa (2000) even though it mayhave the capacity to solubilize them.
In contrast to the aerobic conditions and as shown in Fig. 3b, PCBsolubility of all four cultures increased significantly under anaerobicconditions. A similar trendwas also observed in thefirst fourweeks dur-ing the anaerobic stage of the two stage anaerobic-aerobic conditions(see Fig. 3c). It was noted that soon after the addition of bacterialcultures, the insoluble PCB pellets started to disappear in the flasks,presumably as the PCBs became soluble due to the action of the micro-organisms. There are two possible explanations for the mode of action
Fig. 3. PCB solubility under (a) aerobic, (b) anaerobic, and (c) two stage anaerobic-aerobicconditions. Error bars represent the standard deviation of mean values from triplicates.Initial is the total soluble PCBs prior to the addition of microbes and week 0 isimmediately after addition of microbes.
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by themicroorganisms that resulted in the increase in solubility of PCBs.Firstly, the dechlorination of PCBs could transform low water solublehighly chlorinated congeners intomorewater soluble lower chlorinatedcongeners as reported by Yin et al. (2011). If highly chlorinated conge-ners were dechlorinated into lower chlorinated congeners, there shouldbe more chlorides released to the medium. Therefore, measurementof chloride ions accumulated in the medium is a direct indicationof dechlorination, which is discussed in Section 3.2.3. Secondly, theenhancement of the solubility of hydrophobic PCBs was due to the pro-duction of bioemulsifiers or surface-active molecules (biosurfactants)by the microorganisms (Federici et al., 2012).
Findings of the two stage experiments are shown in Fig. 3c. Duringthe two stage study, the first four weeks were dedicated to anaerobicconditions before switching over to aerobic conditions in the last twoweeks. All four strains demonstrated similar trends in the first fourweeks under anaerobic conditions (Fig. 3b vs Fig. 3c). However, in thesecond stage, when the conditions shifted from anaerobic to aerobic,
the solubility of PCBs declined in Achromobacter sp. NP03,Ochrobactrumsp. NP04 and Pseudomonas sp. NP06, whereas in Lysinibacillus sp. NP05,there was a significant increase in PCB solubility. Parallel to theincreased PCB solubility, the growth of Lysinibacillus sp. NP05 also in-creased significantly during this period as shown in Fig. 2c aerobicphase. This confirmed the ability of Lysinibacillus to make hydrophobicPCB mixture soluble in the aqueous medium under both, anaerobicand aerobic conditions. Furthermore, it also confirmed the ability to de-chlorinate the highly chlorinated congeners during the anaerobic phaseand to degrade the resulting lower chlorinated congeners during theaerobic phase. This is an added advantage in bioremediation applica-tions as the organism can survive and degrade the PCB compoundsunder varying anaerobic and aerobic conditions.
3.2.3. Chloride ion build up and variation of pH in the aqueous mediumThe release of chlorides and their concentrations in the liquid me-
dium were measured as explained in Section 2.2.1. These measure-ments were taken as an indication of the dechlorination process thatoccurred following the addition of the four bacteria strains. The chlorideion concentrations in the abiotic controlswere alsomeasured and foundto be relatively constant throughout the experiment and there was noconsiderable difference between the initial and final chloride levelsunder aerobic, anaerobic and two stage anaerobic-aerobic conditions.The chloride ion concentrations in the controls were in the range of37.0 ± 0.9 mg/L. These background levels were concluded to comefrom chloride containing compounds in the basal minimal salt mediumcomprising of MgCl2·6H2O, CoCl2·6H2O, MnCl2·4H2O, NiCl2·6H2O andCuCl2·2H2O. The background chloride values coming from the abioticcontrols and media controls were first subtracted from the experimen-tal values, before the final values were plotted as shown in Fig. 4.
According to Fig. 4, accumulation of chloride ions in aerobic andanaerobic conditions provided clear evidence that the four facultativemicrobes discovered have the ability to dechlorinate the Aroclor 1260mixture under both, aerobic and anaerobic conditions. Significantlyhigh chloride ion concentrations in the combined anaerobic-aerobictreatment of all four cultures when compared to separate aerobic andanaerobic treatments further confirmed the effectiveness of combininganaerobic and aerobic degradation rather than isolated aerobic oranaerobic applications (Tartakovsky et al., 2001; Long et al., 2015). In-creasing levels of chloride ions in the culture medium were reportedby Yin et al. (2011) as a direct indication of dechlorination of PCB mol-ecules. Under anaerobic and two stage conditions, Lysinibacillus sp.NP05 demonstrated the highest chloride ion levels when compared tothe other three cultures and they were 5.2 ± 0.7 mg/L and 9.16 ±0.8 mg/L, respectively. Pseudomonas sp. NP06 had the lowest chloridelevels under all three conditions. As Aroclor 1260 theoretically contains60% chlorine by weight, the maximum chlorine level expected in50 mg/L Aroclor 1260 concentration is 30 mg/L. Therefore, the chlorideyield of 9.16 ± 0.8 mg/L observed in Lysinibacillus sp. NP05 under twostage anaerobic-aerobic treatment is an indication of the removal ofone third of total chlorine present in the Aroclor 1260 mixture.
The pH of the culture supernatants were also monitored togetherwith chloride ion build up and are shown in Fig. 5. The pH values ofthe abiotic controls remained relatively constant throughout the exper-iment (7.07 ± 0.14) under anaerobic, aerobic and two stage conditions.At the end of the aerobic and anaerobic experiments, the pHdid not sig-nificantly change and were 7.49 ± 0.07 and 6.95 ± 0.03, respectively.However, significant pH reduction was observed at the end of all thetwo stage anaerobic-aerobic experiments with values of 5.15 ± 0.03,4.98 ± 0.06, 4.97 ± 0.01 and 6.14 ± 0.03 obtained for Achromobactersp. NP03,Ochrobactrum sp. NP04, Lysinibacillus sp. NP05, and Pseudomo-nas sp. NP06, respectively (Fig. 5). When variations of the pH valueswere compared with the accumulation of the chloride ion levels in themedium, the negative correlation is clearly visible. Based on these re-sults, the lowering of pH under two stage anaerobic-aerobic conditionsappeared to have correlated well with the increase in chloride ions. The
Fig. 4. Chloride ion accumulation in the culturemedia after six weeks. The values shown here are after subtracting the background chloride levels coming from abiotic controls andmediacontrols. Error bars represent the standard deviation of mean values from triplicates.
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high levels of chlorides under combined anaerobic-aerobic conditions isattributed to the dechlorination of highly chlorinated congeners underanaerobic conditions first, followed by further degradation of resultinglower chlorinated congeners after switching to aerobic conditions.
3.3. Biosurfactant production
An extracellular biosurfactant producing Lysinibacillus sp. waspreviously reported with the ability to solubilize different aliphaticand aromatic hydrocarbons such as hexane, benzene, toluene, dieseland kerosene (Panjiar et al., 2015). In addition, a bacterium isolatedfrom refinery wastewater was identified as Ochrobactrum sp. with thepotential to produce exopolysaccharide bioemulsifier and was able todegrade diesel oil (Ramasamy et al., 2014), n-octane, mineral light andheavy oils, crude oil (Calvo et al., 2008). However, there is no research
Fig. 5. Variation of pH and chloride ion concentrations after six weeks under aerobic, anaerobicrepresent the standard deviation of mean values from triplicates.
literature available that confirms the ability of these bacterial speciesto produce biosurfactants, which makes extremely hydrophobic PCBssoluble in aqueous medium while concomitantly degrading PCBs.Therefore, this study can be considered a first to report the potentialof Lysinibacillus, Achromobacter and Ochrobactrum species to producebiosurfactants that ultimately increased the solubility of PCBs.
In this study, the drop collapse and emulsification index tests wereused as quantitative methods (Ramasamy et al., 2014), while thehaemolytic assay was used as the qualitative method (Thavasi et al.,2011) to test for biosurfactant production. Results of these three testsare summarised in Table 2.
Among the four bacterial cultures, three showed positive results forthe drop collapse test. The drop spreads or collapses proportional to theconcentration of biosurfactants in the supernatant, due to the reductionof interfacial tension between the liquid droplet and the hydrophobic
and two stage anaerobic-aerobic conditions the initial pH was adjusted to 7.0. Error bars
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surface (Alvarez et al., 2015). A maximum drop diameter of 5.3 ±0.3 mmwas observed for the culture supernatant of Achromobacter sp.NP03. Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 also demon-strated to have relatively high diameters of 5.0 mm, when comparedto 3.3 ± 0.3mmdiameter in the negative control (see Table 2). The rel-atively high droplet diameters of the supernatants confirms the releaseof biosurfactants by these bacterial cultures into the culture medium.
As summarised in Table 2, the highest emulsification index of 50%was observed in culture supernatants of Lysinibacillus sp. NP05. Thishigh result was followed by Achromobacter sp. NP03 (33.3%) andOchrobactrum sp. NP04 (16.7%). Emulsion formation was not observedin the culture supernatant of Pseudomonas sp. NP06. The production ofbiosurfactants was evident by the formation of the emulsion layer byLysinibacillus, Achromobacter and Ochrobactrum strains. The ability ofbiosurfactant production by these potential PCB degradingmicroorgan-isms to increase the solubility of hydrophobic PCBs without addition ofchemical or biological surfactant is an added advantage in bioremedia-tion applications.
Similar to positive results from the emulsification index test,Lysinibacillus sp. NP05 performed well during the haemolytic assay. Itshowed strong yellow transparent zones in Tryptone soya agar contain-ing sheep blood after incubation at 28 °C for 48 h indicative of completehaemolysis of red blood cells. In contrast, Achromobacter sp. NP03and Ochrobactrum sp. NP04 showed partial haemolysis having semi-
Fig. 6. Growth profile, PCB hydrolysis and pH variation of Lysinibacillus sp. NP05 under two stagfrom triplicates.
transparent zones surrounded by dark green areas. Pseudomonas sp.NP06 showed negative to minor haemolysis and this also coincideswith the low chloride accumulation observed.
Based on the three biosurfactant tests, the descending order of pref-erence for the potential of biosurfactant production is Lysinibacillus sp.NP05 N Achromobacter sp. NP03 N Ochrobactrum sp. NP04 N Pseudomo-nas sp. NP06. When the results of biosurfactant screening testswere compared with PCB solubility and chloride ion accumulation,Lysinibacillus sp. NP05 that exhibited the highest biosurfactant produc-tion potential, also demonstrated the highest total PCB solubility andchloride ion accumulation. These results provide a clear and positivecorrelation between biosurfactant production and PCB solubilizationand subsequent degradation. Not surprisingly, Pseudomonas sp. NP06that demonstrated the lowest biosurfactant production capability alsohad the lowest chloride ion levels in the culture supernatant.
According to the results presented in this study, it can be argued thatthe rate of microbial degradation of PCBs is decided not only by theirability of break down the PCB molecules, but also bymaking the hydro-phobic PCBs soluble in the aqueous medium. Therefore, it is importantto include suitable and different culture members with the ability toproduce biosurfactants in order to facilitate the bioavailability of PCBs.Such a strategy will be highly effective in field and scale up bioremedi-ation applications.
Overall, the data from the growth characteristics (Fig. 2), PCBsolubility (Fig. 3), chloride build up (Fig. 4), pH variation (Fig. 5)and biosurfactant production (Table 2) support the identificationof Lysinibacillus sp. NP05 as the best performer out of four facultativeanaerobic strains tested followed by Achromobacter sp. NP03,Ochrobactrum sp. NP04 and Pseudomonas sp. NP06. As shown inFig. 6, increase in growth rate parallel to the increased PCB solubilityunder combined anaerobic-aerobic treatment by Lysinibacillus sp.NP05 is an indication of the consumption of Aroclor 1260 as its carbonand energy source other than solubilization. Moreover, decrease in pHover the two weeks of aerobic phase as per Fig. 6 would coincide withthe occurrence of advanced PCB degradation steps to further break-down the chlorinated intermediates.
4. Conclusions
Based on an exhaustive review of past research literature, this re-search can be considered as the first comparative study which assessed
e anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values
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the capability and growth characteristics of facultative anaerobicbacteria in degrading PCBs under anaerobic, aerobic and two-stageanaerobic-aerobic cultivation conditions. The study found four bacterialstrains identified as Achromobacter sp. NP03, Ochrobactrum sp. NP04,Lysinibacillus sp. NP05 and Pseudomonas sp. NP06, to have the capabilityfor degrading commercial PCB mixture, Aroclor 1260 as the sole sourceof carbon under both, anaerobic and aerobic conditions. Among the fourstrains tested, Lysinibacillus sp. NP05 performed the best based on theresults from the comparative experiments on cell growth, PCB solubil-ity, chloride build up and biosurfactant production.
The results suggested that microorganisms capable of degradingPCBs also have the potential to produce surface active substances tofacilitate hydrophobic PCBs which are soluble in aqueous media andconsequently enhanced the bioavailability and degradation of PCBs.The two stage anaerobic-aerobic conditions produced the best overallresults when assessed on cell growth, PCB solubility and chloride buildup. In field scale soil remediation applications, facultative microorgan-isms have the potential to be better candidates as they can surviveand degrade PCBs under both anaerobic and aerobic conditions, whileachieving relatively higher degradation rates. Furthermore, based onthese results there is an opportunity to produce and apply tailor-madebacterial consortia for future process designs and applications resultingin shorter time frames, while effectively hydrolyzing PCBs.
Acknowledgement
The authors would like to acknowledge the Queensland University ofTechnology (QUT) for the Australian Government Research TrainingProgram (RTP) scholarship provided to the first author for undertakingthis doctoral study. We also gratefully thank the QUT Central AnalyticalResearch Facility (CARF) operated by the Institute of Future Environments(IFE) where the analytical data reported in this paper were obtained.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2018.10.127.
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