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Effect of limonene on the heterotrophic growth and
polyhydroxybutyrate production by Cupriavidus necator
H16
Authors
Guzman Lagunesa, F., Winterburna*, J.B.
a School of Chemical Engineering and Analytical Science, The Mill, The University of
Manchester, Manchester, M13 9PL, UK
*Corresponding author: [email protected]
Tel: +44(0)161 306 4891
Abstract
The inhibitory effect of limonene on polyhydroxybutyrate (PHB) production in
Cupriavidus necator H16 was studied. Firstly, results demonstrate the feasibility of
using orange juicing waste (OJW) as a substrate for PHB production. An intracellular
PHB content of 81.4 % (w/w) was attained for a total dry matter concentration of 9.58 g
L−1, when the OJW medium was used. Later, a mineral medium designed to mimic the
nutrient levels found in the complex medium derived from OJW was used to study the
effect of limonene on the production of PHB. Results showed a drop in specific growth
rate (μ) of more than 50% when the initial limonene concentration was 2% (v/v)
compared to the limonene free medium. This work highlights the importance of a
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limonene recovery stage prior to fermentation, to maintain levels below 1 % (v/v) in the
medium, adding value to the OJW and enhancing the fermentation process productivity.
Keywords:
Limonene; inhibition; Cupriavidus necator; PHB; orange peel.
1. Introduction
The wider uptake and utilisation of microbially produced biopolymers is dependent on
our ability to efficiently and economically produce these polymers, ultimately to either
give significant benefits in applications, as biomaterials, or to be available at a price
comparable to oil derived polymers. In order to achieve either of these aims a low cost,
a widely available source of carbon substrate is required, along with sufficient
biorefining and bioprocessing techniques. Approximately 1.5 million tonnes of orange
peel waste from the juicing industry are available every year as a source of fermentable
fructose for PHB production (USDA, 2016).
The production of polyhydroxyalkanoates (PHAs) has been studied during the last few
decades, as an alternative to petrochemical polymers. PHAs are a group of polyesters
that can be synthesised by a range of different microbial strains as carbon and energy
reservoir under stress conditions. Cupriavidus necator is recognised as the model
microorganism for PHA production due to its capacity of accumulate up to 80% of total
dry weight in biopolymer and the simplicity of the metabolic pathway that involves
three enzymatic reactions (Choi and Lee, 1999). PHAs not only have similar mechanical
properties to polyethylene terephthalate (PET) or polypropylene (PP) (Koller et al.,
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2010; Lee, 1996), but they are also susceptible to biodegradation and can be produced
from renewable raw materials thus reducing environmental impact and lowering
petroleum dependency. However, production costs are still a major disadvantage to a
wider use of PHAs and implementation of production strategies on a large scale
(Braunegg et al., 2004; Lee and Choi, 1998; Sudesh et al., 2000).
Many studies have been performed in order to improve productivities and reducing
production costs involving different fermentation strategies, microbial strains, upstream
and downstream processing (Chanprateep, 2010; Koller and Braunegg, 2015; Wang et
al., 2014). Moreover, the use of purified carbon sources accounts around 40% of the
total cost of production leading to several investigation groups to find alternative culture
media that can provide the conditions for the production of PHAs (Lee, 1996; Urtuvia et
al., 2014). Numerous lignocellulosic materials have been studied as potential raw
materials for the production of a medium rich in the nutrients necessary for fermentation
processes (Jain and Tiwari, 2015). These materials are often obtained as by-products
from other processes and are generally used as fuel for heat generation (Castilho et al.,
2009). The approach of such studies is to recover the fermentable sugars, to be used for
biopolymer production, through a pre-treatment step and then the solid residue can still
be used as a heat source (Kawaguchi et al., 2016; Oh et al., 2015; Tripathi et al., 2011).
One of the main lignocellulosic materials worldwide is produced by the citrus
processing industry, with orange juice being the main product (Angel Siles López et al.,
2010; Boukroufa et al., 2014). Approximately 50 million tonnes of orange fruit are
produced annually, with 3 million tonnes being used to produce fruit juice of which
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about 1.5 million tonnes of unwanted waste material (OJW) are produced every year.
This waste includes the skin, pulp and seeds and accounts for about 50% (w/w) of the
total amount of oranges processed for juice, making it a very wasteful process and
representing a disposal challenge for the industries involved (USDA, 2016; Boukroufa
et al., 2014; Choi et al., 2013). Different strategies for the valorisation this waste as a
whole have been tested, from burning it as heat and power source to the use as hard
metals sieve in water treatment (Balu et al., 2012; Bampidis and Robinson, 2006;
Santos et al., 2015). The use of OJW as starting material for different biotechnological
processes is currently being assessed; however, the complexity of the composition, and
the presence of toxic substances, the process has not been successfully established
(Pourbafrani et al., 2007). The use of an autohydrolysate of orange peel (Rivas et al.,
2008) as complex medium rich in fructose for the production of PHAs by C. necator
H16 represents a new approach for the use of this carbon-rich material that has obtained
promising results. However, our preliminary studies revealed that even when fructose
concentration was close to the optimum found for a mineral medium (Aramvash et al.,
2015), a significant drop in the specific growth rate is triggered by increasing the
concentration of OJW solids at the beginning of the media preparation process. This
suggested that some inhibitory substance was accumulating in the medium.
OJW can contain up to 1.6 % (w/w) of orange essential oil (OEO), with important
applications in several industries, including food, cosmetics and pharmaceutical. This
essential oil accumulates in small oil sacs of 0.4 to 0.6 mm in diameter and is located at
irregular depths in the flavedo at the outer peel of the fruit (Angel Siles López et al.,
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2010) and in addition to its characteristic smell it also has shown inhibitory effects on
the growth of several pathogenic strains (Zahi et al., 2015) (Muthaiyan et al., 2012;
Subramenium et al., 2015). Approximately 90% of the OEO consists of limonene, a
naturally occurring monoterpene; consequently, studies on the antimicrobial effect of
orange essential oil have focused on the limonene titration. According to literature,
concentrations as low as 0.05% can inhibit cell growth for bioethanol production (Choi
et al., 2013; Joshi et al., 2015). Furthermore, different approaches focused on the
holistic implementation of citrus wastes have highlighted the importance of recovery of
the OEO prior its biotechnological processing, enhancing the productivities of the
microbiological stage, and adding value to the starting material (Lohrasbi et al., 2010;
Ruiz and Flotats, 2014).
In this work, the biomass and PHB accumulation by C. necator H16 using an OJW
autohydrolysed medium were evaluated. The effect of limonene on the strain’s growth
kinetics was as well assessed with the objective to determine its tolerance to the main
component of OEO.
2. Materials and Methods
2.1.Microbial strain
Freeze dried C. necator H16, from the DSMZ-German Collection of Microorganisms
and Cell Cultures, DSM No. 428, was purchased and activated according to supplier
instructions. Master and working stock were created using MicroBankTM cryovial
system (Pro-Lab Diagnostics, UK) and kept at the −80 °C. Short term storage plates 5
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consisting of nutrient agar (Sigma-Aldrich, UK) were prepared every time a batch of
experiments was started.
2.2. Media preparation
2.2.1. Orange juicing waste medium
The feasibility of using OJW as starting material for the production of PHAs was
assessed. OJW was obtained from a local juicing bar. Material was stored as received at
−20° C until used. The process proposed by Rivas et al. (2008) (Rivas et al., 2008) to
produce sugar rich medium from OJW was followed. After defrosting, the OJW was
submitted to a drying stage at 60° C for 48h and then ground using a standard food
processor. An extra run was performed using fresh material, whole and ground, to
measure the effect of the drying stage over the carbohydrates extraction process. Two
initial ratios of OJW solids to distilled water were used, 1:8 and 1:12 (w:w). A
hydrolysis stage was then performed using an autoclave where the temperature was
maintained at 121° C for 20 minutes. In order to determine the effect of the grinding
stage on the sugar concentration in the medium, both options, ground OJW and whole
OJW were tested during the extraction step. Solids were spun down using a centrifuge
Sigma 6-16S (Sigma, Germany) at 7000 rpm and supernatant was separated by
decantation. A 10M NaOH solution was used to adjust the initial pH of the media to a
value of 7.0 ± 0.2. Finally, solutions were sterilised by filtration using 0.2 μm
polyethersulfone (PES) membrane filtration units (Thermo Fisher Scientific Inc., UK)
and transferred to shake flasks for the fermentation experiments.
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2.2.2. Mineral medium
The effect of limonene over the cell growth of C.necator H16 was studied adding
different concentrations of the terpene to the mineral media developed by Aramvash et
al. (2015) for the production of PHB. A basal mineral salt medium was prepared with
the following composition: KH2PO4 1.75 g L−1; MgSO4.7H2O 1.2 g L−1; NH4CL 2 g
L−1; citric acid 1.7 g L−1; trace elements solution 10 ml L−1. The trace elements
solution was composed of ZnSO4.7H2O 2.25 mg L−1; FeSO4.7H2O 10 mg L−1;
CaCl.2H2O 2 mg L−1; Na2B4O7.7H2O 0.23 mg L−1; (NH4)6Mo7O24 0.1 mg L−1;
CuSO4.5H2O 1 mg L−1; MnSO4.5H2O 0.6 mg L−1; HCl (35%) 10 mL L−1. Fructose was
used as the carbon source at a concentration of 25 g L−1. Salts, trace elements and
fructose solutions were prepared separately. All solutions were autoclaved at 121 °C
during 20 mins; once they reached room temperature, the tree solutions were mixed.
The initial pH of the medium was adjusted to 6.8. Limonene (Thermo Fisher Scientific,
UK) was filtered using a 0.2 μm PET membrane filter to assure sterility; the
corresponding quantity was then added to the mineral media to reach the concentration
required. Concentrations of 0, 0.5, 1, 1.5 and 2 % (v/v) of limonene were tested for this
work.
2.3.Cultivation conditions and inoculum preparation
For every experiment, a single colony from the short term storage stock was taken,
keeping aseptic conditions, and inoculated into 10 mL of nutrient broth No. 2 (Sigma-
Aldrich, UK) contained in 50 mL falcon tubes. Tubes were placed on an orbital shaker
where conditions were maintained at 30° C and 200 rpm. After 24 h of cultivation, an
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adaptation stage was performed taking 2 mL of the broth to inoculate 20 ml of, either,
OJW-based medium or limonene-free mineral media contained in 50 ml falcon tubes
and cultivated for 48 h under the same conditions. Finally, for the limonene effect
experiments, 10 ml of the limonene-free medium were used to inoculate 100 ml of the
mineral media with limonene added, using 500 ml Erlenmeyer flasks. When working
with the OJW-based medium 10 ml of the adaptation stage broth were used to inoculate
100 ml of identical medium in 500 ml shake flasks. All experiments were run in
triplicate; results are presented as the mean, with error bars showing ± 1 standard
deviation.
2.4.Analytical methods
2.4.1. Partial OJW characterization
Total carbohydrates, crude protein, crude fibre and water content measurements were
carried out to the OJW in order to characterise the material. The phenol-sulphuric acid
method described by Nielsen was used for total carbohydrate determination (Nielsen,
2010). Standard procedures 954.01, 962.09 described in the Official Methods of
Analysis for the AOAC (AOAC, 1990) were followed for the determination of crude
protein and fibre. A protein factor of 6.25 was used to calculate the protein content.
Water content was determined by measuring the weight difference between fresh
material and the material after dried. Samples of fresh OJW were located into a drying
oven at 60° C during a period of 48 h.
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2.4.2. Biomass measurements
Samples were taken periodically throughout the experiments and the cell density was
evaluated by optical density measurements at a wavelength (λ) of 600 nm (OD600),
using a spectrophotometer UVmini-1240 (Shimadzu, USA). The dry matter content of
fermentation media was measured by transferring approximately 2 mL of cell
containing broth into a pre-weighed 2 mL micro test tube (Eppendorf, DE), cells were
then spun down at 13,000 rpm for 10 minutes using an Eppendorf MiniSpin centrifuge
(Fisher Scientific, UK). The resulting supernatant was decanted and frozen to be used in
residual nutrient determinations. The remaining cell pellet was washed twice using
distilled water and then dried at 60C until constant weight was reached, 48 hours after.
Residual biomass concentration was calculated by the subtraction of the PHB
concentration from the total dry matter.
2.4.3. PHB determination
Gas chromatography (GC) was employed for PHB quantification according to the
method developed by Riis and Mai (1988) (Riis and Mai, 1988). A gas chromatography
system model 7820A (Agilent Technologies, USA) coupled with an autosampler
Combi/Pal from Varian was used for this study. A Poraplot Q-HT 10×32 mm column
was used and the detection system selected was a flame ionization detector (FID) set at
200° C. The injection volume and temperature were 1 μL and 230° C respectively.
Temperature program started at 120°C to be gradually increased during 3 minutes until
230° C, temperature was then held until finish the analysis. Helium was used as the
carrier gas. A calibration curve was prepared using purified PHB as a standard (Sigma-
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Aldrich, UK) at different known concentrations. Peak areas of the samples were then
correlated to concentration using the calibration curve obtained.
2.4.4. Carbohydrate measurement
The concentration of fructose, glucose and sucrose, in the supernatant collected from
TDM samples, was determined using a Dionex Ultimate 3000 HPLC equipment. The
refractive index intensity of the samples was measured using a RefractoMax 521
(ThermoFisher Scientific, UK) detector, set at 50 C, peak area and concentration were
correlated using a calibration curve constructed by running standards of known
concentration. An Aminex HPX-87C Column was used to achieve the separation at a
temperature of 50 C. The mobile phase used was 5 mM sulphuric acid at a flow rate of
0.6 mL min−1. Samples were diluted 10 times to assure a good column performance
using HPLC grade water and filtered using nylon syringe filters 0.45 μm pore size prior
analysis.
2.4.5. Total nitrogen measurement
Total nitrogen quantification was performed using a Shimadzu TOC-VC equipment
coupled with both an ASI-V autosampler unit and a TNM-1 total nitrogen detector. A
calibration curve was created by the equipment, from a master solution of NH4Cl at a
concentration of 50 mg L−1. An aliquot of 750 μL of free solids supernatant was taken
to a final volume of 15 mL, required for the machine, and filtered through nylon syringe
filters 0.45 μm pore before injection.
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3. Results and discussion
3.1.OJW as starting material for PHA production
Results for the water content of the material show a solids content around 20 ± 0.6 %
(w/w) for the OJW tested, this is similar to that observed by Pourbafrani et al.(2010),
20± 0.8 % of solids content, when working with citrus waste coming from a juice
factory (Pourbafrani et al., 2010). The difference in the water content removed from the
ground OJW and the whole “as juiced” material was around only 6.5 % (w/w) after 48 h
of drying, leading to the decision of grinding the material after the drying stage. The
composition of the OJW material was found to be similar to those reported in other
studies on the valorisation of this by-product. Results for the total carbohydrates and
crude fibre assays were 18.4 and 66. 31 %, respectively. This corresponds to those
reported by Rivas et al. [29] a total soluble sugars content of 16.9 % and a crude protein
of 63.05 %. The protein determination by Kjendhal digestion yielded a content of 7.22
%, slightly above the 6.50 % reported by Rivas et al.; this variation can be expected
when analysing natural materials of different origin.
The concentrations of carbohydrates measured in the supernatants obtained for the
different conditions tested are showed in table 1.The fructose concentration in the
aqueous extracts was improved by almost 15 % comparing the whole material to the
ground OJW, after the drying step. The maximum concentration of fructose obtained
was 24.74 g L−1 when ground OJW was used. The drying strategy simplifies the
handling of OJW, reducing the risk of microbial growth and, as the results show,
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concentrating the target compounds in the solid fraction. The results also confirmed the
observations made in previous reports that have studied the effect of the particle size on
hydrolysis and extraction processes for citrus by-products, namely that the grinding
stage enhances carbohydrate recovery by increasing the surface area in contact with the
aqueous fraction (Agbor et al., 2011; Choi et al., 2013; Lopresto et al., 2014). The
process proposed in this contribution only focused on the effect of the grinding stage,
not taking into account the resulting particle size. Nevertheless, previous studies
focused on lignocellulosic materials show that reduction of particle size below 0.400
mm has little impact on the rates and yields of hydrolysis process (Agbor et al., 2011).
Three carbohydrate peaks were identified by HPLC analysis, glucose, fructose and
sucrose; consumption over time was determined from the difference in the peak areas.
Initial solids loading during media preparation led to a corresponding difference in the
fructose extracted from the peels. The autohydrolysis treatment proved effective for
fructose recovery, where Rivas et al. (2008) (Rivas et al., 2008) reported maximum
concentrations of 16 g L−1 of fructose, this study obtained 23 g L−1. Treatment with an
initial ratio of orange peel of 1:12 (w) lead to an initial fructose concentration of 14 g
L−1 and complete consumption was achieved after 72 h of fermentation. The depletion
of fructose for treatments with ratio 1:8, initial fructose concentration of 23 g L−1, was
not achieved for the frame time of the study, indicating that other nutrients were
limiting.
Figure 1 shows the cell growth curves as well as the PHB concentration time course for
the extraction treatments studied. The specific growth rate value for the media with an
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initial solids load of 1:12 (w:v), (figure 1.b) reached the highest value for the different
treatments studied, 0.18 h−1, with an intracellular PHB percentage above 80%. These
values are similar to those obtained for C. necator H16 when grown in a phosphate
buffered medium, using organic acids as carbon source reaching a maximum PHA
content of 83.7 % (Yang et al., 2010). Other efforts have focused on the implementation
of glycerol as carbon source for PHA production, as this by-product of the biodiesel
process is available in great quantities. In 2012, Tanadchangsaeng and Yu growing C.
necator H16 in a mineral media added with 20 g L−1 of glycerol achieving a μmax of
0.11h−1 and 70% of PHB accumulation (Tanadchangsaeng and Yu, 2012). The
treatment with an initial ratio of solids of 1:8 (figure 1.a) exhibited slower growth for a
higher concentration of fructose, this can be related to some inhibitors present in the
broth as result of the extraction process conditions (Mohan et al., 2015; Talebnia et al.,
2007). A recent study showed that methanol contained in the crude glycerol can inhibit
the cell growth of C. necator DSM4058, while a μmax of 0.47 h−1 was obtained when 50
g L−1 of glycerol were used as carbon source in inhibition free conditions (Salakkam
and Webb, 2015).
A maximum dry matter concentration of 9.58 g L−1 with a percentage of PHB of 76 %
was achieved with a medium prepared from whole (non-ground) orange peel. However,
an increased intracellular PHB content, 80%, was reached for the same concentration of
initial solids, 1:8 (w:w), but adding the grinding stage. With yields on consumed
fructose (YX/S) of 0.41 and 0.39, whole and ground peel respectively. These results
indicate that the medium prepared with whole OJW generates provides slightly better
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conditions for cell propagation, whereas ground OJW gives an improved PHB
production. Final biomass and intracellular polymer concentrations are comparable to
those attained by Aramvash et al. (2015), using a mineral medium with an initial
fructose concentration of 35 g L−1, reaching 7.48 g L−1 of PHB with a maximum of 90
% of PHB accumulation. Other strains have been tested on different wastes, Halomonas
campisialis is capable of using 84 % of the sugars in an orange peel based medium,
leading to a final PHB content of 42 % (w/w) corresponding to 0.33 g L−1 after 48 h of
fermentation, lower than those reported here (Kulkarni et al., 2015).
Residual biomass results show that after the first 30 hours the total matter reaches a
plateau stage and stays constant for a period of 40 h when it starts to increase again.
This growth coincides with a loss of intracellular PHB. This cessation of cell growth is
usually related to the nitrogen source reaching the limiting concentration, triggering
PHA accumulation at the same time (Koller et al., 2010; Rodríguez-Contreras et al.,
2015). An initial reduction of the total nitrogen concentration was observed during the
first stages of the fermentation with a corresponding accumulation of biomass, which
stayed constant after 40 % of the initial total nitrogen had been consumed, for all the
conditions tested. This indicates that only a part of the total nitrogen is accessible and
available to the microorganism (Haas et. al., 2015). This could be caused by the
Maillard reaction between sugars, proteins and peptides during autoclaving, or protein
conversion into a protease-resistant form. Bioavailable nitrogen was nonetheless the
limiting nutrient and the triggering factor for bacteria to switch its metabolism to PHB
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production, as the synthesis of the polymer started when the nitrogen consumption, and
hence cellular growth, stopped. Table 2 presents a summary of all results obtained.
3.2.Effect of limonene on C. necator growth and PHB production
The variation of cell growth, fructose uptake, residual nitrogen and polymer production
over the course of the experiments is shown in figure 2 for the different limonene
concentrations tested. The experiments were carried as previously described and
proceeded as expected, with biomass and PHA production occurring within the first
72h. Higher initial levels of limonene led to lower titre readings for biomass and
polymer accumulation.
The biomass concentrations were followed through time for all limonene conditions
tested. For each limonene concentration different kinetic behaviour was exhibited by C.
necator H16. As the initial limonene concentration was increased, stronger growth
inhibition effect can be observed. The lag phase observed during the first hours of
fermentation increased considerably with the concentration of limonene, lasting around
40 h when the limonene concentration was 2 % (v/v) (figure 2.e). The final
concentrations of biomass and PHB were strongly affected by the presence of the
terpene, losing about 30% of intracellular PHB content when the initial concentration of
limonene rose from 1 to 1.5%.
Many studies on the antimicrobial effect of orange essential oil have centred on the
effect of limonene, as this terpene represents 90% (w/w) of OES (Di Pasqua et al.,
2006; Vuuren and Viljoen, 2007). In the present study, results for the cell growth and
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PHB production of C. necator H16, indicate that the addition of limonene at
concentrations as low as 0.5% (v/v) has a negative effect, leading to a value in specific
growth rate 9% lower than those observed for a limonene free medium, dropping from
0.19 to 0.17 h−1. This agrees with previous studies testing the inhibitory effect of orange
essential oils and limonene against several organisms, which showed that the minimal
inhibitory concentration of limonene was in the range of 1-4% for Gram-negative
strains, while for the Gram-positive Staphylococcus aureus and Brochotrix
thermospacta a maximum sublethal concentration of 1.68 mg L−1 was determined (Ruiz
and Flotats, 2014).
The final concentrations of dry matter, as well as PHB titre, were affected by the
presence of limonene. Experiments run in the absence of limonene, figure 2.a, reached a
maximum dry matter concentration of 10.18 g L−1 after 60 hours of fermentation while
adding 0.5% (v/v) of limonene, figure 2.b, caused a drop in the dry matter concentration
to 8.28 g L−1 after 72 h. It is interesting to notice that higher limonene concentrations
shortened the time taken to reach the trigger point for the synthesis of the biopolymer
when compared to lower concentrations of the terpene, whilst it was possible to detect a
slight amount of PHB earlier on in the fermentation for the higher concentrations tested
indicating that the presence of limonene acts as an external stress on the cell population.
It was also possible to observe for all the limonene concentrations a decrease in residual
biomass during the second half of the fermentation. These results are in agreement with
those reported by Aramvash et al. (2015) using 35 g L−1 of fructose leading to 7.48 g
L−1 of PHB at maximum concentration, for a medium optimised for PHB production.
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The lag phase present during the first hours of fermentation increased considerably with
the concentration of limonene, lasting around 40 h for the maximum concentration
tested. The final concentration of biomass, as reported for other strains (Marques et al.,
2014; Mohammad Pourbafrani, 2007), and PHB was strongly affected by the presence
of limonene.
PHB content inside the cells dropped from 68 % to 59 % (w/w) when 0.5% (v/v) of
limonene was added to the mineral media. Some growth was still observed, however,
with cell growth in the presence of 2 % (v/v) of terpene, reaching 2.6 g L−1 of dry mass
with a content of 22 % of PHB after 98h of fermentation. This differs to previous
reports on the ethanol producing yeast Saccharomyces cerevisiae, where no cell growth
was observed in the presence of 0.5 % of limonene and cell viability reached 0 after 3
hours of cultivation (Pourbafrani et al., 2007). More recently, Tao et al. (2014)
described the antifungal effect of limonene over a pathogenic strain that infects citrus
plants, Penicililum digitatum, at concentrations around 0.24 %, reporting a mycelial
growth inhibition of 43 %. C. necator showed stronger resistance when compared with
above mentioned strains losing 25% of its growth with concentrations up to 1% (v/v) of
limonene. A greater effect was triggered when the concentration of limonene was
increased to 1.5% (v/v) with C. necator H16 losing 50% of its specific growth rate.
Total nitrogen measurements never reached complete depletion. However, a constant
residual concentration of 220 mg L−1 of nitrogen was reached for the limonene free
control experiment after 40 hours, and similar behaviour can be observed for limonene
concentrations of 0.5 and 1 % (v/v). The nitrogen uptake was significantly reduced
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when the concentration of limonene was increased from 1% to 1.5% (v/v), the residual
total nitrogen being 60% higher when compared to lower concentrations. Figure 3
shows the percentage of total nitrogen uptake for the different concentrations of
limonene.
Biomass and product yields, as well as specific growth rate and maximum PHB content,
were calculated for the different limonene concentrations tested and the results are
shown in figure 4. The trend for specific growth rate and PHB content shows sigmoidal
behaviour with a major drop occurring between 1 and 1.5 % (v/v) of initial limonene.
The effect was less pronounced for the consumed substrate yields on dry matter and
product; however total consumptions were considerably lower for concentrations of
limonene above 1%, as previous data showed.
The values of μ showed a decrease of more than 50% when the concentration of
limonene was 2 % (v/v); however, compared with the reports for some pathogenic
bacterial strains, C. necator was able to show some growth in such conditions (Espina et
al., 2011; Marques et al., 2014). Results indicate that C. necator is able to grow in
media prepared from orange peel media if the concentration if limonene is as low as
1%, with a 10% reduction in specific growth rate. When Salakkam and Webb (2015)
studied the effect of methanol on a glycerol consuming strain of C. necator, results
showed that, similar to limonene, the presence of the alcohol inhibited growth at all of
the concentrations tested. Even when both compounds exhibit similar sigmoidal profile
as inhibitors of cellular growth, limonene triggered a stronger inhibition at lower
concentrations. This similar behaviour could be due to the mechanism by which
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methanol and limonene inhibit cell growth. Alcohol and terpenes are molecules that
affect the structure of the plasmatic membrane of the cell and its functions. Lipid
composition and protein conformation is changed, leading to leakage of intracellular
material in the case of limonene, and lowering the proton motive force in case of
methanol (Mohammad Pourbafrani, 2007; Zahi et al., 2015; Salakkam and Webb,
2015). However, direct comparison between different systems needs to be considered
carefully as the affinity of the different compounds for the microorganism’s membrane
may play a role.
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4. Conclusions
The technical feasibility of value added biopolymer production using OJW as a starting
material has been demonstrated and a process established to recover fructose from
OJW, to a maximum concentration of 24.74 g L−1. From this OJW medium a maximum
PHB concentration of 7.34 g L−1 was obtained with no nutriment supplementation.
The effect of the initial limonene concentration on PHB productivity and cell growth
was assessed, using a mineral media to replicate the OJW medium, with the addition of
different limonene concentrations. The investigation into limonene tolerance indicates
the existence of a threshold inhibitory concentration of 1 %(v/v).
Acknowledgements
We thank Dr. Saul Alonso Tuero for his insightful advice during the writing process.
This study was funded by a grant from the Mexican Council for Science and
Technology, CONACyT: 351189.
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List of Figures
Fig. 1. Fructose, dry matter, PHB and total nitrogen concentrations obtained for the
growth of C. necator H16 using different OJW media. Where: fructose, ; total
nitrogen, ; total dry matter, ; triangles are the concentration of PHB, ▲; and
residual biomass,▼. a) for an initial load of orange peel 1:8 (w:w); b) for the
1:12 (w:w) initial ratio OJW treatment.
Fig. 2. Variation cell growth, PHB production and, fructose and nitrogen uptakes, for
different initial concentrations of limonene. a) Limonene free; b) 0.5 % (v/v); c)
1 % (v/v) ; d) 1.5 % (v/v); e) 2 % (v/v). Where: fructose, ; total nitrogen, ; total
dry matter, ; concentration of PHB, ▲; and residual biomass, ▼.
Fig. 3. Percentage of total nitrogen uptake for the different initial percentages of
limonene in volume. Where: Limonene free (); 0.5 % (); 1 % (▲);1.5 % (▼); and for
2 % ().
Fig. 4. Variation of specific growth rate (), dry matter and PHB yields over substrate
( and ▲ respectively), and intracellular percentage of polymer (▼) as function
of the initial concentration of limonene.
List of Tables
Table 1 Carbohydrate concentrations obtained for different extraction treatments
studied
Table 2 Kinetic parameters obtained for the microbial growth of C. necator H16 using
the different OJW media obtained.
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Table 1. Carbohydrate concentrations obtained for different extraction treatments studied.
%
WaterInitial solids ratio
[Initial fructose]
(g L−1)
[Initial glucose]
(g L−1)
[Initial sucrose]
(g L−1)
Ground OJW80
1:12 11.91 10.58
Ground OJW 1:8 24.74 13.62 9.90
Whole OJW73.5
1:12 12.09 10.97
Whole OJW 1:8 22.08 14.65 5.92
Manual peeling peels 60 1:12 14.13 10.74
Fresh OJW whole
N/D
1:8 4.51 4.78 3.06
Fresh OJW ground6.96 6.13 3.34
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Table 2. Kinetic parameters obtained for the microbial growth of C. necator H16 using the different OJW media obtained.
Initial solids
Ratio
(w:v)
[Fructose]0
(g L−1)
*[Dry matter]f
(g L−1)
*[PHB]f
(g L−1)Yx/s Yp/s
μmax
(h−1)% PHB
Ground OJW 1:12 14.94 6.21 5.03 0.40 0.43 0.179 80.9
Ground OJW 1:8 23.14 9.01 7.34 0.46 0.39 0.122 81.5
Whole OJW 1:8 22.22 9.58 7.31 0.52 0.41 0.118 76.3
*Concentrations of dry matter and PHB were measured after 72 h. Yields were calculated against fructose consumed.
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Fig. 1. Fructose, dry matter, PHB and total nitrogen concentrations obtained for the
growth of C. necator H16 using different OJW media. Where: fructose,; total nitrogen,
; total dry matter, ; triangles are the concentration of PHB, ▲; and residual
biomass,▼.
a) For an initial load of orange peel 1:8 (w:w).
b) For the 1:12 (w:w) initial ratio OJW treatment.
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Fig. 2. Variation cell growth, PHB production and, fructose and nitrogen uptakes, for different initial concentrations of limonene . Where:
fructose, ; total nitrogen, ; total dry matter, ; concentration of PHB, ▲; and residual biomass, ▼.
a) Limonene free b) 0.5 % (v/v)
c) 1 % (v/v)
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Fig. 3. Percentage of total nitrogen uptake for the different initial percentages of
limonene in volume. Limonene free (); 0.5 % (); 1 % (▲);1.5 % (▼); and for 2 %
().
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Fig. 4. Variation of specific growth rate (), dry matter and PHB yields on substrate (
and ▲ respectively), and intracellular percentage of polymer (▼) as function of the
initial concentration of limonene.
35