Jacobs Journal of Microbiology and Pathology · compounds describe synthetic compounds, and compound ... Pseudomonas putida in batch fermen-tation. Effect of inoculation volume: Different

Cite this article : Perumallapalli S jyothi, Syda vali shaik, M.V.V. Chandana Lakshmi et al. Comparison study of Biodegradation of Phenanthrene by Thalassospira frigidphilosprofundus and Pseudomonas putida. J J Microbiol Pathol. 2019; 6(1): 023.

Research ArticleComparison study of Biodegradation of Phenanthrene by Thalassospira frigidphilos-

profundus and Pseudomonas putida

Perumallapalli S jyothi1, Syda vali shaik2 , M.V.V. Chandana Lakshmi 3*

1Department of Chemical Engineering, AUCOE (A), Andhra University, Visakhapatnam-03, Andhra Pradesh, India.E-mail:[email protected] of research and development .Unique Biotech Limited. Hyderbad.India3Department of Chemical Engineering, AUCOE (A), Andhra University, Visakhapatnam-03, Andhra Pradesh, India.

*Corresponding author: M.V.V. Chandana Lakshmi, Department of Chemical Engineering, AUCOE (A), Andhra University, Visakhapatnam-03, Andhra Pradesh, India. E-MAIL: [email protected]

Received Date: 12-28-2018 Accepted Date: 01-11-2019

Published Date: 01-21-2019

Copyright: © 2019 Perumallapalli S jyothi

ABSTRACTThe Polycyclic aromatic hydrocarbons (PAHs) are toxic and carcinogenic, it creates so many serious problems to the environment, and it shows necessity for degrading these PAHs. Using microbial transformation, it is possible to decrease the concentration of these PAHs. The aim of the present study is to analyze the biodegradation of Polycyclic Aromatic Hydrocarbons (PAHs) compounds by the bacterial strains Pseudomonas putida and Thalassospira frigidphi-losprofundus, examine and find the strain having high potential for maximum degradation of PAHs. In this work, one PAHs namely Phenanthrene were studied as these show highest concentration at contaminated sites. These bacterial strains were tested under parameters of pH(6-8), inoculum size (1-3ml), temperature(200C-400C), and incubation period (1-11days) and the optimum growth conditions obtained for Pseudomonas putida were pH 7,inocu-lum size 1ml, 300C, 7days and for Thalassospira frigidphilosprofundus were pH 7, inoculum size 1ml, 200C, 7days respectively. By varying the concentrations of PAH compounds from 100mg/l - 400mg/l, degradation results of PAHs by Pseudomonas putida were Phenanthrene (35%,38%,33% and 0.7%), Thus the degradation phenomenon of PAHs gave Thalassospira frigidphilosprofunds has better degradation potential than Pseudomonas putida.

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INTRODUCTION

Polycyclic aromatic hydrocarbons:

All substances originated into the environment ei-ther by biogenic or anthropogenic sources. Anthropogenic compounds describe synthetic compounds, and compound classes as well as elements and naturally occurring chemi-cal entities which are mobilized by man’s activities. These substances are released into the environment in amounts that are unnatural due to human activity. Anthropogenic inorganic and organic pollutants are dispersed throughout the atmosphere, hydrosphere and lithosphere, and they have tendency to transform into another compounds which may be toxic, less toxic and not toxic to flora and fauna. [1]

Polycyclic aromatic hydrocarbons (PAHs) are pol-lutants, often carcinogenic, mutagenic, or toxic, found in most terrestrial ecosystems that arise from industrial op-erations and from natural events such as forest fires. Major components of petroleum, they are continuously released into natural environments, posing a serious risk to human health [2]. Polyaromatics, nitrogen and halogens containing organic compounds are recalcitrant compounds which is difficult to degrade by microorganism. Such type of com-pounds have higher bioaccumulation and bio magnification potency when enters into the biotic entities. Understanding the dynamic process of bioaccumulation and bio magnifica-tion is very important in protecting human beings and oth-er organisms [1]

Materials and methodsMicroorganisms and Growth media:

In this study, a psychrophilic bacteria Thalassospira frigidphilosprofundus NCIM 3SC21 and Pseudomonas putida NCIM 2650 were collected from National Collection of In-dustrial Micro-organisms, Pune, India. The Microorganism Thalassospira frigidphilospro-fundus (NCIM 3SC21) was maintained in marine broth (MB) medium

Thalassospira frigidphilosprofundus Standardization of physical parameters:

A number of physical parameters were evaluat-ed for their effect on growth conditions by Thalassospira frigidphilosprofundus, Pseudomonas putida in batch fermen-tation.

Effect of inoculation volume:

Different levels of inoculum 1-3(%v/v) were added to both marine broth and nutrient broth media to study the effect of inoculum level on growth conditions by Thalasso-spira frigidphilosprofundus, Pseudomonas putida in batch fermentation.

Effect of pH:

The pH of the medium was optimized by adjusting the pH range 6-8 by adding buffers.

Effect of temperature:

Culture flasks containing the medium were incu-bated at different temperatures ranging from 200-400 C to study their effect on conditions of microorganisms

Effect of carbon source:

The carbon source like glucose was added at 1-3 g/l levels to study the effect on growth conditions of microor-ganisms.

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Carbon source utilization:

1. Microorganisms were taken and inoculated in ma-rine broth which was incubated for 24 hours at 200C.

2. Then the cell mass was centrifuged at 4 ºC at 7000 rpm for 10 minutes.

3. After that, supernatant was discarded and mass was resuspended in 1 ml of marine broth. (Haimou et al 2004)

4. 100μl of suspended cell mass was transferred to marine broth tubes with 100 mg/l, 200 mg/l, 300 mg/l, 400 mg/l, 500 mg/l of Phenanthrene. 1 % of glucose without any PAH (Phenanthrene) in marine broth was kept as con-trol for growth.

5. After that in order to monitor the growth, OD at 595 nm were taken at regular time interval varying from 0 to 7 days.

Degradation study:

1. The microorganisms were inoculated in Marine broth media without any carbon source.

2. Then the OD was taken at 595 nm and adjusted to 0.1.

3. 500μl was taken from 0.1 OD adjusted culture and transferred to marine broth media with 100 mg/l of PAH.

4. These were incubated at 20ºC/ 160 rpm for 7 days.

5. After 7 days of incubation above media were used for extraction and quantification of Phenanthrene Repeat the steps 7,8,9 for 200, 300, 400 mg/l also

Quantification of PAHs

1. After 7 days of shaking, residual PAH was extracted from culture (5ml).

2. For extraction equal volume dichloromethane was added to the degradation set up.

3. The tubes were then vortex for 10 min and kept for another few minutes to separate aqueous and organic phases. Upper aqueous layer was extracted and dried over sodium sulfate.

4. There after organic phase was pipette out and kept for drying overnight.

5. The residual was re-suspended in equal volume of n-hexane.

6. The extracted phenanthrene was 10 times diluted in n-hexane.

7. The residual Phenanthrene concentration was de-termined from standard curve of respective PAH.

8. Absorbance of Phenanthrene was taken at 252 and 221nm respectively.

Standard Curves of PAHs

1. Preparation of stock solution: 5 mg/ml of concen-tration, PAH (Phenanthrene and Naphthalene, anthracene, Fluorine) solution was prepared and dissolved in Hexane.

2. The above stock solution of volume 1-10 ml were taken and again dissolved in 10 ml of Hexane.

3. Duplicates were prepared to avoid the error.

4. Absorbance at 200-400 nm was noted using UV-Vis Spectrophotometer.

5. A standard graph was plotted against O.D. Vs. Con-centration.

Results and discussions

There are a number of reports on degra-dation of PAHs using Pseudomonas putida. This part of the document shows the degradation of PAHs by Thalassospira frigidphilosprofundus when tested under a wide range of conditions for the physical parameters like inoculum size, pH, temperature and nutritional parameter like carbon. Cultures maintained at these optimized conditions were

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grown under selective nutrient supplementations. The re-search is shows on the to know the Carbon source limits of organisms (Pseudomonas putida, Thalassospira frigidphilos-profundus) and to check degradation potential of organisms and biodegradation was estimated.

Optimization of physical parameters:

The physical parameters, which affect the growth conditions of microorganisms, were optimized at shake flask level and the results were discussed below. The present investigation was carried out to optimize the cho-sen parameters.

Optimum conditions for Pseudomonas putida:

Effect of inoculation volume:The organism with different inoculum sizes name-

ly 0.5, 1, 1.5 was tested for 0 to 7 days of incubation and the optimum growth conditions was observed at 7th day with the inoculum size of 1ml.[7]

Table 1: Effect of inoculum size Incubation in days

I n o c u -lum size

0 1 3 5 7

0.5 ml 0.26 0.29 0.6 0.76 0.92

1 ml 0.26 0.37 0.71 1.21 1.68

1.5 ml 0.26 0.71 1.24 1.07 0.84

Figure 1: Effect of inoculum size

Effect of pH:The effect of pH on growth conditions of Pseudomo-

nas putida was studied in the pH range of 6, 7, and 8 under submerged fermentation. The optimum pH for growth from Pseudomonas putida was found to be 7. [7]Table 2: Effect of pH

Incubation in days

pH 0 1 3 5 7

6 0.31 0.38 0.64 0.72 0.86

7 0.32 0.47 0.76 0.89 1.12

8 0.29 0.34 0.51 0.67 0.81

Figure 2: Effect of pH

Effect of temperature:

The effect of temperature on growth conditions of Pseudomonas putida was studied in the temperature range of 200, 300, 40 o C under submerged fermentation. The op-timum temperature for growth from Pseudomonas putida was found to be 30oC. [3]

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Table 3: Effect of temperatureIncubation in days

Tempera-ture

0 1 3 5 7

20°C 0.26 0.26 0.41 0.64 0.73

30°C0.26 0.41 0.68 0.97 1.46

40°C 0.26 0.35 0.61 0.82 0.97

Figure 3: Effect of temperature

Effect of carbon source:

of Pseudomonas putida The effect of carbon source on growth conditions

was studied in the carbon source range of 1g/l, 2g/l, 3g/l under submerged fermentation. The optimum carbon source for growth from Pseudomonasputida was found to be 3g/l.[4]

Table 4: Effect of carbon sourceIncubation in days

C a r b o n g/l

0 1 3 5 7

1 0.28 0.37 0.71 0.78 0.87

2 0.34 0.39 0.75 0.86 0.92

3 0.36 0.41 0.84 0.94 1.23

Figure 4: Effect of carbon source

Optimum conditions for Thalassospira frigidphilos-profundus:

Effect of inoculation volume:

The organism with different inoculum sizes (0.5, 1, 1.5) was tested for 0 to 7 days of incubation and the opti-mum growth conditions were observed at 7th day with the inoculum size of 1ml.[5]

Table 5: Effect of inoculation volumeIncubation in days

I n o c u -lum size

0 1 3 5 7

0.5 ml 0.21 0.27 0.56 0.79 1.02

1 ml 0.21 0.34 0.84 1.42 1.75

1.5 ml 0.21 0.63 1.47 0.94 0.81

Figure 5: Effect of inoculum size

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Effect of pH:

The effect of pH on growth conditions of Thalas-sospira frigidphilosprofundus was studied in the pH range of 6, 7, and 8 under submerged fermentation. The results obtained were as follows and The optimum pH for growth from Thalassospira frigidphilosprofundus was found to be 7.[6]

Table 6: Effect of pHIncubation in days

pH 0 1 3 5 7

6 0.24 0.34 0.47 0.63 0.94

7 0.28 0.39 0.64 0.97 1.43

8 0.27 0.32 0.41 0.56 0.79

Figure 6: Effect of pH

Effect of temperature:

Thalassospira frigidphilosprofundusThe effect of temperature on growth conditions of

was studied in the temperature range of 200, 300, 40 o C under submerged fer-mentation. The optimum temperature for growth from Tha-lassospira frigidphilosprofundus was found to be 20oC. [6]

Table 7: Effect of temperatureIncubation in days

Tempera-ture

0 1 3 5 7

20°C 0.21 0.42 0.76 1.24 1.75

30°C 0.21 0.24 0.54 0.76 0.91

40°C 0.21 0.25 0.32 0.35 0.39

Fig. 7 Effect of temperature

Effect of carbon source:

The effect of carbon source on growth conditions of Thalassospira frigidphilosprofundus was studied in the carbon source range of 1g/l, 2g/l, 3g/l under submerged fermentation. The optimum carbon source for growth from Thalassospira frigidphilosprofundus was found to be 3g/l.[5]

Table 8: Effect of carbon sourceIncubation in days

Carbon% 0 1 3 5 7

1 0.21 0.31 0.42 0.59 0.63

2 0.26 0.35 0.56 0.86 0.98

3 0.3 0.43 0.68 1.02 1.46

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Figure 8: Effect of carbon source

Carbon source utilization:

The microorganisms were taken and inoc-ulated in marine broth which was incubated for 24 hours at 200C and after the cell mass was centrifuged at 4 ºC at 7000 rpm for 10 minutes and obtained supernatant was discard-ed and mass was re suspended in 1 ml of marine broth. This 1 ml mass was transferred to marine broth tubes with 100 mg/l, 200 mg/l, 300 mg/l, 400 mg/l, 500 mg/l of Phenan-threne as a sole carbon source. And take1 % of glucose with-out any PAH (Phenanthrene and Naphthalene, anthracene fluorine) in marine broth was kept as control for growth.

After that in order to monitor the growth, the OD at 595 nm were taken at regular time interval i.e. 0 day, 1 day ,3 day, 5 day and 7 day

Phenanthrene at 100mg/l:

Phenanthrene is added 100mg/l in both the media of or-ganisms. OD595 at an interval of every 2 days and the results is like this the maximum growth of both species (Thalasso-spira frigidphilosprofundus, Pseudomonas putida) are 1.77 and 1.42 respectively in 7thday. The growth of both species is having the nearly similar growth, but Thalassospira frigid-philosprofundus species are having slightly higher growth compare with Pseudomonas putida.

Figure 9: Growth using Phenanthrene at 100mg/l

Incubation Days

Microorganisms 0 1 3 5 7 9 11

Thalassospira frigidphilosprofundus 0.37 0.62 0.84 1.16 1.77 1.74 1.34

Pseudomonas putida 0.32 0.46 0.81 0.931 1.42 1.4 1.27

Table: 9 Growth using Phenanthrene at 100mg/l

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Phenanthrene is added 200mg/l in both the me-dia of organisms. OD595 at an interval of every 2 days and the results is like this the maximum growth of both species (Thalassospira frigidphilosprofundus, Pseudomonas putida)are 0.94 and 0.98 respectively 3rd day. The growths of Tha-lassospira frigidphilosprofundus are having slightly growth increased are 1.36 in 5th day as comparison of Pseudomonas putida.

Figure 10: Growth using Phenanthrene at 200mg/l

Phenanthrene at 300mg/l

Phenanthrene is added 300mg/l in both the media of organisms. OD595 at an interval of every 2 days and the results is like this the growth of Thalassospira frigid-philosprofundus are having slightly growth increased day by day. The Pseudomonas putida also slightly growth increased up to 5th day but after this growth is slightly decreased as compared to Thalassospira frigidphilosprofundus

Table 11: Growth using Phenanthrene at 300mg/l

Incubation Days

Microorgan-isms

0 1 3 5 7 9 11

Thalassospi-ra frigidphi-losprofundus

0.67 0.93 1.42 2.14 2.81 2.75 2.18

pseudomonas 0.67 0.78 1.15 1.92 2.16 2.21 1.98

Incubation Days

Microorganisms 0 1 3 5 7 9 11

Thalassospira frigidphilosprofundus 0.54 0.76 0.94 1.36 2.47 2.31 1.62

Pseudomonas putida 0.51 0.66 0.98 1.25 1.76 1.64 1.31

Table 10: Growth using Phenanthrene at 200mg/l

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Figure 11: Growth using Phenanthrene at 300 mg/l

Phenanthrene at 400mg/l:

pseudomonasThe species has no growth in the con-centration of 400 mg/l Phenanthrene similarly Thalasso-spira frigidphilosprofundus have the same condition in this concentration but it has the growth that means it has the tolerance up to this level also.

Figure 12: Growth using for Phenanthrene at 400 mg/l

Degradation study: The culture inoculated in fresh Marine broth media without any carbon source. Then the OD was taken at 595 nm and adjusted to 0.1.On that medium I was taken 500ul from 0.1 OD and it is transferred to marine broth media with 100 mg/l of PAH and These were incubated at 20ºC/ 160 rpm for 7 days. After 7 days of incubation above me-dia were used for extraction and quantification of Phenan-

Incubation Days

Microorganisms 0 1 3 5 7 9 11

Thalassospira frigidphilosprofundus 0.71 0.83 0.89 0.9 0.92 0.84 0.76

Pseudomonas putida 0.72 0.75 0.77 0.77 0.78 0.78 0.78

Table 12: Growth using for Phenanthrene at 400mg/l

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threne Repeat the steps 7,8,9 for 200, 300, 400 mg/l also.After 7 days of shaking, residual PAH was extracted

from culture (5ml). For extraction equal volume dichloro-methane was added to the degradation set up. These tubes were then vortex for 10 min and kept for another few min-utes to separate aqueous and organic phases. Upper aque-ous layer was extracted and dried over sodium sulfate. There after organic phase was pipette out and kept for dry-ing overnight and the residual was re-suspended in equal volume of n-hexane. The extracted phenanthrene was 10 times diluted in n-hexane. The residual Phenanthrene con-centration was determined from standard curve of respec-tive PAH. Absorbance of Phenanthrene was taken at 252 and 221nm respectively.

Figure 13: Incubated at 200C/180 rpm for 7 days

Figure 14: Aqueous and organic phases

Extracted upper aqueous layerFigure 15:

Degradation of Phenanthrene:The degradation of Phenanthrene by both collected

species the maximum degradation occur by Thalassospira frigidphilosprofundus species compare with Pseudomonas putida species it has maximum degradation occur in 100 ppm and 200 ppm concentration (52.75 and 51.37 respec-tively) it degrades the half of the initial concentration simi-larly Pseudomonas putida species also showing good degra-dation potential near same concentration of Phenanthrene that is 35.250 and 37.625 respectively, but compare to Tha-lassospira frigidphilosprofundus it has less potential that is nearly less than 15% of its total concentration. From the two collected species Thalassospira frigidphilosprofundus is much potential to Phenanthrene degradation.

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Figure 16: Percentage of Phenanthrene degradation

Conclusion

In the present study, Pseudomonas putida, Thalas-sospira frigidphilosprofundus was chosen for PAHs deg-radation. The physical parameters like inoculum size, pH, temperature and nutritional parameters like carbon source each played an important role in PAHs degradation

Preliminary optimized conditions of physical pa-rameters for the biodegradation of PAHs by Pseudomonas putida were 1 (% v/v) inoculum size, 7 pH, 30ºC tempera-ture and for nutritional parameters 3g/l.

The research is focused on the to know the carbon source utilization of organisms (Pseudomonas putida, Tha-lassospira frigidphilosprofundus) and to check degradation potential of organisms. After completion of experimental part the results are unexpected that Thalassospira frigidphi-losprofundus is showing more capable of degrading PAHs over Pseudomonas putida.

Biodegradation of PAHs was studied by varying the concentration from 100 mg/l to 400 mg/l. Pseudomonas putida has the tolerance limit up to 300mg/l and Thalasso-spira frigidphilosprofundus has the tolerance limit of up to 400 mg/l . Percentage of degradation for Phenanthrene for a concentration range of 100mg/l -400 mg/l using Pseudo-monas putida is Thalassospira frigidphilosprofundus

35%,38%,33% and 0.7% respectively and are 53%, 51%, 45%,

14%.

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Table 13: Percentage of Phenanthrene degradation

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