Optimization of growth media components for ...

Optimization of growth media componentsfor polyhydroxyalkanoate (PHA) production

from organic acids by Ralstonia eutropha

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Optimization of growth media components for

polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha

Journal: Applied Microbiology and Biotechnology

Manuscript ID: AMB-10-19908.R1

Manuscript Category: Original Paper

Date Submitted by the Author:

n/a

Complete List of Authors: Yang, Young-Hun; Konkuk University, Microbial Engineering Brigham, Christopher; Massachusetts Institute of Technology, Biology Budde, Charles; Massachusetts Institute of Technology, Chemical Engineering

Boccazzi, Paolo; Massachusetts Institute of Technology, Biology Willis, Laura; Massachusetts Institute of Technology, Biomaterials Science and Engineering Hassan, Mohd Ali; Universiti Putra Malaysia, Biotechnology and Biomolecular Science Yusof, Zainal; SIRIM Berhad, Research and Technology Division Rha, ChoKyun; Massachusetts Institute of Technology, Biomaterials Science and Engineering Sinskey, Anthony; Massachusetts Institute of Technology, Biology

Keyword: polyhydroxyalkanoate, Ralstonia eutropha, organic acid, mixture model

Applied Microbiology and Biotechnology

For Peer Review

1

Optimization of growth media components for polyhydroxyalkanoate 1

(PHA) production from organic acids by Ralstonia eutropha 2

3

4

Yung-Hun Yang1, 2)

, Christopher J. Brigham1)

, Charles F. Budde3)

, Paolo Boccazzi1)

, 5

Laura B. Willis1,4)

, Mohd. Ali Hassan5)

, Zainal Abidin Mohd. Yusof 6)

, ChoKyun Rha4)

6

and Anthony J. Sinskey1,7,8)*

7

8

1) Department of Biology,

3)Department of Chemical Engineering,

4)Biomaterials 9

Science and Engineering Laboratory, 7)

Division of Health Sciences Technology, 10 8)

Engineering Systems Division, Massachusetts Institute of Technology, 77 11

Massachusetts Avenue, Cambridge, MA 02139, USA 12 2)

Current address: Department of Microbial Engineering, Konkuk University, Seoul, 13

Republic of Korea 14 5)

Department of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 15

43400 UPM Serdang, Selangor, Malaysia 16 6)

Research & Technology Division, SIRIM Berhad, P.O. Box 7035, 40911 Shah Alam, 17

Malaysia 18

19

20

21

22

* Author for correspondence (E-mail: [email protected], Tel#: 1-617-253-6721, Fax#: 23

1-617-253-8550) 24

25

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Abstract 1

We employed systematic mixture analysis to determine optimal levels of acetate, 2

propionate, and butyrate for cell growth and PHA production by Ralstonia eutropha 3

H16. Butyrate was the preferred acid for robust cell growth and high PHA production. 4

The 3-hydroxyvalerate content in the resulting PHA depended on the proportion of 5

propionate initially present in the growth medium. The proportion of acetate 6

dramatically affected the final pH of the growth medium. A model was constructed 7

using our data that predicts the effects of these acids, individually and in combination, 8

on cell dry weight (CDW), PHA content (% CDW), PHA production, 3HV in the 9

polymer, and final culture pH. Cell growth and PHA production improved 10

approximately 1.5-fold over initial conditions when the proportion of butyrate was 11

increased. Optimization of the phosphate buffer content in medium containing higher 12

amounts of butyrate improved cell growth and PHA production more than 4-fold. The 13

validated organic acid mixture analysis model can be used to optimize R. eutropha 14

culture conditions, in order to meet targets for PHA production and/or polymer HV 15

content. By modifying the growth medium made from treated industrial waste, such as 16

palm oil mill effluent (POME), more PHA can be produced. 17

18

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Keywords: polyhydroxyalkanoate, Ralstonia eutropha, organic acid, mixture model 1

2


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Introduction 1

Bioplastics are among the biomass-based products currently being developed to replace 2

petroleum-based products (Frazzetto 2003; Young 2003). A large number of bioplastics 3

have been discovered, with over 150 varieties (Williams et al. 1999), encompassing a 4

wide range of thermoplastic properties (Cornibert and Marchessault 1972). PHAs are 5

naturally occurring biopolymers produced by a diverse group of bacteria (Madison and 6

Huisman 1999; Sudesh et al. 2000) that possess characteristics similar to those of 7

chemically-synthesized polymers (Steinbüchel and Füchtenbusch 1998). 8

In general, the microbial biosynthesis of PHA results from the limitation of a nutrient, 9

such as nitrogen, sulfur, oxygen, or phosphate (Anderson and Dawes 1990) in the 10

presence of abundant carbon. These polymers are thought to serve in nature as storage 11

molecules for carbon and reducing equivalents, and in some bacterial strains, PHA can 12

account for more than 80% of CDW (Khanna and Srivastava 2005). The molecular 13

weight and monomer composition of polymer can be controlled by utilizing specific 14

PHA synthase enzymes and specific carbon substrates (Anderson and Dawes 1990; Sim 15

et al. 1997). PHAs exhibit different thermal and mechanical properties depending on the 16

types of monomers incorporated (Doi et al. 1995; Jendrossek and Handrick 2002). 17

These polymers can be rapidly hydrolyzed in soil, sea water, and lake water by 18


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microorganisms (Mergaert et al. 1992; Khanna and Srivastava 2005). Many 1

environmental factors can influence the polymer properties, which can, in turn, affect 2

the rate at which the polymer degrades (Khanna and Srivastava 2005). 3

PHAs, which have better biocompatibility than petroleum-based plastics, have been 4

used to develop medical devices, artificial organs, and therapeutic composites (Chen 5

and Wu 2005; Misra et al. 2006; Wu et al. 2009a). Despite these advantages, production 6

costs of bioplastics remain higher than those of conventional plastics and will need to be 7

lowered for bioplastics to compete in the marketplace (Choi and Lee 2000). The overall 8

cost of polymer production could be decreased by the efficient use of inexpensive 9

carbon feedstocks (Nikel et al. 2006). Several studies have investigated the production 10

of bioplastics from industrial waste streams (Ahn et al. 2000; Füchtenbusch et al. 2000; 11

Marangoni et al. 2002; Chua et al. 2003; Liu et al. 2008), which have potential as 12

inexpensive carbon sources. The fermentation of these substrates yields favorable 13

amounts of cell mass and bioplastic. However, few systematic or statistical approaches 14

have been taken to enhance the production of the desired polymer products (Nikel et al. 15

2005). 16

To develop a cost-effective strategy to produce bioplastics from oil palm waste products 17

(Hassan et al. 2002), the possibility of using anaerobically treated, clarified POME was 18


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examined. POME is an aqueous stream generated in large quantities during the milling 1

of oil palm fruits. The oil palm tree, Elaeis guineensis, is the most productive oilseed 2

plant under cultivation and is an important agricultural crop in Africa and Southeast 3

Asia (Basiron 2007; Wu et al. 2009b). In Malaysia, approximately 46 million tons of 4

POME are produced annually (Econ.mpob.gov.my/economy/Overview_2008_latest 5

130109. htm). It is estimated that for every ton of crude palm oil produced in 6

Malaysia, 2.5 tons of POME are generated. 7

POME is a colloidal suspension containing 95-96.5% water, 0.6-0.7% oil, and 4-5% 8

total solids that has high chemical oxygen demand (COD) and biological oxygen 9

demand (BOD), making it a severe pollutant that must be treated (Hassan et al. 2002; 10

Ahmad et al. 2003). POME can be fermented anaerobically by wild-type bacteria to 11

produce the volatile organic acids acetate, propionate, and butyrate in the mass ratio of 12

3:1:1, respectively (Yee et al. 2003). The feasibility of utilizing this treated POME as a 13

carbon source for the production of PHAs by Ralstonia eutropha has been demonstrated 14

(Hassan et al. 2002). In order to confirm and extend these preliminary findings, we have 15

used a mixture analysis model to optimize the concentrations of volatile organic acids in 16

minimal medium. We have determined the effects that altering the ratio of these organic 17

acids has on microbial cell growth, PHA production, and monomer composition of the 18


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polymer. We report here the steps we have taken toward the goals of enhancing 1

bioplastic productivity and lowering production costs. 2

3

Materials and Methods 4

Bacterial strains and growth media 5

R. eutropha H16 (Wilde 1962) was precultured in 3 mL of dextrose free Tryptic Soy 6

Broth (TSB) for 24 h at 30°C. The cells were harvested and washed twice with sterile 7

water, then used to inoculate 5 mL of PHB minimal medium, prepared as previously 8

described (York et al. 2003) with a final NH4Cl concentration of 0.1 g/L. Cell growth 9

was monitored by measuring optical density (OD) at 600 nm, starting from an initial 10

OD600 of 0.05. For media containing high phosphate buffer (10X), we used 67 mM of 11

Na2HPO4, 62.5 mM of NaH2PO4, 26 mM of K2SO4, and 10 mM of NaOH. Cells for 12

high phosphate media growth experiments were grown initially in 5 mL PHB minimal 13

medium in a test tube for 48 h on a roller drum or shaker. For larger volume mixed acid 14

experiments with high phosphate media, 1 mL of cells from an overnight culture grown 15

in TSB was used to inoculate 50 mL of high phosphate minimal medium in 250 mL 16

flasks and then grown for 48 h at 30°C. For 1 L cultures with mixed acids, 1 mL of 17

cells from an overnight culture grown in TSB was used to inoculate 50 mL of TSB in a 18


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250 mL flask, which was then incubated for 24 h at 30°C. These cells were harvested, 1

washed with sterile water twice, used to inoculate 1 L of high phosphate minimal 2

medium, and then grown for 72 h at 30°C before harvesting. The cells were harvested, 3

washed twice with cold water, and lyophilized for 48 h. Note that when describing 4

acid concentrations, all values refer to the % (w/v) of the organic acid sodium salt added 5

to the media. All media used in this study were supplemented with 10 µg/mL 6

gentamicin. 7

8

Design of experiments and mixture analysis 9

To develop a strategy for optimizing cell growth and PHA production, a mixture 10

analysis model of three acids in the minimal medium was developed and populated 11

using a standard mixture analysis methodology (Nowruzi et al. 2008) and the Minitab 12

V14 program (http://www.minitab.com). For the design of mixture analysis experiments 13

to populate the model, we used a simplex lattice method augmented by the design of 14

axial points (0.17:0.17:0.67, 0.17:0.67:0.17, and 0.67:0.17:0.17 for acetate: propionate: 15

butyrate). The degree of lattice for this mixture analysis is three; therefore, the 16

experimental design contains the set of all 13 combinations, where each value of 17

organic acid is 0.00, 0.33, 0.67 and 1.00 (Table 1). The experimental data for response 18


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trace plots are also shown in Table 1. All experiments were performed using 50 mL 1

cultures with 0.3% total organic acid content. To plot mixture contours, a mixture 2

regression using the model fitting method was applied with full quadratic component 3

terms initially included. In the data analysis, the coefficients with p-value below 0.1 4

were used as parameters (Online Resource 1). 5

6

Analytical methods 7

PHA quantity and composition were determined by gas chromatography using a slight 8

modification of a method described previously (Brandl et al. 1988). Approximately 10 9

mg of freeze-dried cells from each experiment were weighed and placed in Teflon-10

stoppered glass vials. For methanolysis, 1 mL CHCl3 and 1 mL CH3OH/H2SO4 (85:15 11

v/v) were added to the samples, which were then incubated at 105°C for 2 h, cooled to 12

room temperature, and incubated on ice for 10 min. After adding 0.5 mL of cold water, 13

the samples were vortexed for 1 min, then centrifuged at 2000 × g. The organic phase 14

was extracted by pipette, and moved to clean borosilicate glass tubes containing Na2SO4. 15

These samples were then filtered and injected into a gas chromatograph (Agilent, CA, 16

USA) equipped with a DB-Wax capillary column (Agilent, 30 m x 0.32 mm x 0.5 µm). 17

The volatile acids were monitored by high-performance liquid chromatography (HPLC) 18


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using an Aminex HPX-87H column (Bio-Rad, CA, USA) at 50°C with a diode array 1

detector at 210 nm. The mobile phase was 5 mM sulfuric acid, with a flow rate of 0.6 2

mL/min. 3

4

Results 5

Optimizations of mixed acid concentration, buffer concentration, and C/N ratio. 6

Initial experiments were performed to determine the experimental range of organic acid 7

concentrations to be explored for the mixture analysis. Specifically, we examined the 8

growth of R. eutropha H16 in minimal media containing individual organic acids or 9

combinations of mixed acids as sole carbon sources, various buffer concentrations, and 10

various carbon to nitrogen (C/N) ratios. We tested utilization of the individual acids, 11

and found R. eutropha exhibited maximal growth in defined minimal medium 12

containing 0.5% acetate, 0.4% propionate, or 0.5% butyrate as the sole carbon source 13

(Fig. 1). When R. eutropha H16 was grown on the 3:1:1 (acetate: propionate: butyrate) 14

maximum growth was observed with a 0.3% total acid concentration (Fig. 2a). 15

The growth of R. eutropha in the standard minimal medium was accompanied by an 16

increase in the final pH to values as high as 9.5. Therefore, experiments were performed 17


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to study cell growth on the mixed acid carbon source using different concentrations of 1

phosphate buffer added to control pH. A range of phosphate buffer concentrations from 2

10 mM to 500 mM was examined, and media with up to 130 mM phosphate buffer 3

exhibited an increase in final culture optical density that was proportional to the amount 4

of buffer added (Fig. 2b). The buffer concentration of 130 mM phosphate (i.e., high 5

phosphate media, compared to our initial media) was therefore employed in all further 6

mixed acid culture experiments. Various C/N ratios were examined by adding different 7

levels of mixed acids to the standard minimal medium. We found that 40 C/N allowed 8

for the most robust cell growth using mixed acids as the carbon source (Fig. 2c), while 9

the % PHA of CDW was highest at 80 C/N (Fig. 2d). Although 80 C/N provided the 10

highest PHA content, the low cell densities of these cultures made them difficult to 11

analyze. We therefore used 40 C/N in all further mixed acid experiments. 12

The initial pH of the culture medium also affected cell growth, especially with 13

propionate and acetate as carbon sources. With increases in the initial pH of the growth 14

medium, R. eutropha showed a lower final OD600 on acetate as the main carbon source. 15

However, when cells were grown on propionate, a different trend emerged: the final 16

optical density increased as the initial pH was increased up to 7.3, then went down with 17

further increases in pH (Fig. 3a). The increase in pH of the cultures was greater when 18


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acetate was used as the carbon source than when the other organic acids were used (Fig. 1

3b). The reason why acetate consumption increased the pH of the growth medium was 2

not clear, and no significant production of by-product was detected by HPLC analysis 3

(data not shown). A similar pH increase was observed in R. eutropha cultures in another 4

study (Wang and Yu 2001), but no explanation for this phenomenon was given. 5

6

Modelling the effects on Cell Growth, pH, Amount of PHA, and 3HV Content 7

To populate the mixture analysis model of the effect of the three acids on cell growth, 8

PHA production, and 3HV content, R. eutropha was grown with 13 culture 9

compositions of mixed acids, as described in Materials and Methods (Table 1). Each 10

culture condition was grown for 48 h and tested in duplicate. For each culture, we 11

measured the cell growth, medium pH, amount of PHA per 50 mL culture, PHA content 12

in the cells (% of CDW), residual CDW (CDW minus PHA), and the 3HV content of 13

PHA. 14

Contour plots of each variable, generated by the modelling software, were used to 15

predict growth and PHA production over the range of compositions tested (Fig. 4). To 16

examine the accuracy of these models, three additional mixture compositions, which 17


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had not been included in the first set of 13 conditions, were selected and tested. The 1

three conditions used for the model validation tests were (acetate: propionate: butyrate): 2

0.1:0.1:0.8, 0.1:0.4:0.5, and 0.6:0.3:0.1. The results of these cultures validated the 3

predictive power of the model for CDW, PHA content, total amount of PHA per volume 4

of culture, and 3HV%. The results indicated that the model contour plots accurately 5

reflected each of the empirical trends (Fig. 5), and that the model could be used to 6

reliably predict the results of growth on various acid mixtures. From the results outlined 7

in Fig. 4, an increase in the initial concentration of butyrate augmented cell growth and 8

PHA production. Interestingly, the optimal acid composition for total PHA production 9

predicted by the model is not pure butyrate, but rather 0.2:0.0:0.8. The reason that a 10

small fraction of acetate in the medium is beneficial is not currently understood. 11

Butyrate concentration correlated positively with cell weight and amount of PHA, while 12

propionate concentration correlated positively with 3HV content in the final PHA. 13

We determined that the effect of butyrate on cell growth and PHA production was not 14

due simply to an increase in total carbon available in the media. When we normalized 15

carbon levels to 124 mM of total carbon and examined differences in growth between 16

conditions of 0.3% (3:1:6), 0.34% (3.6:1:1) and 0.33% (3:1.5:1), the culture with the 17

highest concentration of butyrate reached the highest OD600 (Online Resource 2). 18


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The amount of 3HV in the final PHA was proportional to the amount of propionate 1

added to the culture, as previously reported (Bhubalan et al. 2008). With a constant 2

amount of propionate in the media, and different acetate:butyrate ratios, combinations 3

with higher acetate led to PHAs containing more 3HV. For example, polymer from the 4

0.67:0.33:0 (acetate: propionate: butyrate) cultures contained 7.7% 3HV, while the 5

polymer from the 0:0.33:0.67 (acetate: propionate: butyrate) culture contained 4.6% 6

3HV. However, cultures containing higher acetate yielded less PHA and total biomass 7

(Table 1). These results, which are based on model predictions and empirical 8

experiments, can be applied to adjust conditions to optimize cell growth and polymer 9

production. The model can also be used to maximize polymer production with a given 10

monomer composition (i.e. culture conditions can be identified that will maximize 11

polymer accumulation with a minimum 3HV content). 12

13

PHA Production 14

We have found that increasing the ratio of butyrate to the other organic acids increased 15

total biomass and PHA content of R. eutropha, consistent with previous reports 16

(Chakraborty et al. 2009). Based on our model predictions and experiments, the 17


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reported acid composition from treated POME (3:1:1 acetate: propionate: butyrate) is 1

not an optimal ratio for maximizing cell growth and PHA production (Hassan et al. 2

2002). Our model shows that supplementing the original 3:1:1 mixture to change the 3

ratios of the volatile acids could yield several alternative media compositions that are 4

more favourable for cell growth and production of PHAs (Online Resource 3). To test 5

some of the predictions listed in Online Resource 3, we compared 3:1:1 (acetate: 6

propionate: butyrate, Fig. 6a) to 3:1:6 (acetate: propionate: butyrate, Fig. 6b) conditions 7

using larger (1 L) culture volumes. The concentration of each volatile acid was 8

monitored by HPLC. CDW and PHA content increased continuously until 41 h (Fig. 9

6a and b). When the results in Fig. 6a and 6b are compared, it can be seen that the 10

maximum PHA obtained from 3:1:6 (41 h, CDW 1.33 g/L, PHA 0.86 g/L) is 11

significantly higher than that obtained from 3:1:1 (41 h, CDW 1.10 g/L, PHA 0.64 g/L). 12

In addition, measuring the consumption of volatile acids showed that butyrate was 13

depleted first, regardless of its initial concentration in the medium (Fig. 6a and b). This 14

observation was confirmed by using a culture medium containing a uniform percentage 15

of each volatile acid (1:1:1 acetate: propionate: butyrate) (Online Resource 4). The rapid 16

depletion of butyrate is consistent with a report which suggested that R. eutropha can 17

direct 67% of butyrate into the TCA cycle, whereas only 33% of acetate can enter the 18


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TCA cycle (Chakraborty et al. 2009). Our experiments clearly show that butyrate 1

increased PHA accumulation more effectively than acetate. This may be related to the 2

observation that butyrate can be incorporated into PHA without decomposition of the 3

molecule (Doi et al. 1988). 4

5

Discussion 6

Many efforts have been undertaken to seek renewable sources of carbon for the 7

production of PHA (Tan et al. 1997; Loo et al. 2005; Nikel et al. 2005). Industrial and 8

agricultural effluents are promising because these aqueous streams contain various 9

sugars, organic acids, and other organic compounds that can be recovered and used as 10

substrates for fermentation. Despite the advantages of turning waste streams into carbon 11

feedstocks, several problems remain to be solved regarding the use of industrial wastes 12

as carbon sources for PHA production. First and foremost, the carbon composition of an 13

untreated waste stream is unlikely to be optimal for biological polymer production. 14

Untreated waste streams may contain harmful components, which could inhibit bacterial 15

growth and, in turn, limit PHA production. Furthermore, the composition of these 16

streams may not be easy to control and reproduce (Zaldivar et al. 2001). One strategy 17

that can therefore be employed is to treat a waste stream in order to generate a more 18


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desirable feedstock, as has been demonstrated by using anaerobic fermentation of 1

POME to produce organic acids (Hassan et al. 1996). 2

In this study, we applied systematic optimization modelling of mixed acid ratios to 3

establish the effect of each volatile fatty acid and to maximize cell growth and PHA 4

production. We first reformulated our minimal medium, raising the phosphate buffer 5

concentration so as to increase buffering capacity and changing the C/N ratio. We then 6

populated a model using data from cultures with various mixed acid compositions, and 7

confirmed the predicted results with additional mixed acid cultures. With the validated 8

model we can predict growth and PHA production of R. eutropha on any combination 9

of acetate, propionate, and butyrate. We also observed that the initial pH of the growth 10

medium resulted in differences in cell growth: depending on the type and concentration 11

of each organic acid, different final pH values were observed, suggesting that 12

fermentation proceeds by different routes and different metabolites are produced. These 13

results suggest several strategies to make treated POME a better substrate for cell 14

growth and PHA production. One way to stimulate biomass production, starting with a 15

3:1:1 (acetate: propionate: butyrate) mixture, is to increase the amount of butyrate, 16

which is consumed by R. eutropha faster than the other volatile acids tested in this work. 17

More butyrate would increase CDW as well as PHA content. To increase the proportion 18


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of 3HV in the resulting PHA, cells can be grown with more propionate and higher 1

initial pH. Previous work has shown that introducing 3HV into a PHB chain reduces the 2

melting temperature and increases the toughness of the resulting plastic, making it a 3

more useful material (Anderson and Dawes, 1990). Copolymers of 3HB and 3HV 4

synthesized by R. eutropha are also known to have lower molecular weights, compared 5

to the PHB homopolymer (Kunioka and Doi 1990; Scandola et al. 1992). The mixed 6

acid model allows one to formulate a medium for production of PHA with a desired 7

3HV content. 8

Furthermore, the validated mixture optimization model can also guide the development 9

of alternative procedures for upstream treatment of POME, in order to improve the 10

proportions of certain acids in the final composition. For example, the composition of 11

organic acids generated from POME has been shown to be affected by controlling the 12

pH of the treatment conditions (Hassan et al. 1996). 13

In this work, a model designed to improve PHA production from mixed organic acids 14

by R. eutropha H16 was constructed and validated. The results discussed here 15

represent a significant step towards the production of low-cost bioplastic using organic 16

waste streams as a carbon feedstock. 17

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Acknowledgements 1

The authors thank Mr. John Quimby and Ms. Karen Pepper for their contributions to 2

this manuscript. This work was supported by a grant from the government of Malaysia 3

and the Malaysian Office of Science, Technology and Innovation (MOSTI). The result 4

of this grant, the Malaysia-MIT Biotechnology Partnership Program (MMBPP), is a 5

collaboration between MIT, SIRIM Berhad, Universiti Putra Malaysia, and Universiti 6

Sains Malaysia. The authors would like to thank the members of this program for their 7

collegial collaborations. 8


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(POME) using membrane technology. Desalination 157:87-95 3

Ahn WS, Park SJ, Lee SY (2000) Production of Poly(3-hydroxybutyrate) by fed-batch 4

culture of recombinant Escherichia coli with a highly concentrated whey solution. Appl 5

Environ Microbiol 66:3624-3627 6

Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial 7

uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450-472 8

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56:17-34 1

2

3


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Table. 1 1

Experimental design points chosen through a simplex lattice methodology for the 2

mixture analysis model and their experimental results. 3

ID # Acetate Propionate Butyrate

CDW

(mg/ 50mL)

PHA content

(wt%)

3HV%

1 1.00 0.00 0.00 38.6±1.3 66.5±0.5 0.0±0.0

2 0.67 0.33 0.00 46.9±0.1 71.1±1.9 7.7±1.7

3 0.67 0.00 0.33 57.5±0.5 76.8±0.5 0.0±0.0

4 0.33 0.67 0.00 51.7 ±2.7 72.9±1.4 17.1±3.2

5 0.33 0.33 0.33 61.3±2.2 77.6±0.8 8.5±1.3

6 0.33 0.00 0.67 71.7±1.5 83.0±3.3 0.0±0.0

7 0.00 1.00 0.00 43.0±3.9 70.0±3.9 35.1±2.7

8 0.00 0.67 0.33 60.2±1.0 76.8±2.2 15.4±2.8

9 0.00 0.33 0.67 66.0±1.9 83.7±0.2 4.6±0.7

10 0.00 0.00 1.00 66.1±4.9 82.2±0.3 0.0±0.0

11 0.67 0.17 0.17 49.1±0.6 77.7±3.7 4.7±0.8

12 0.17 0.67 0.17 53.2±1.5 74.6±0.9 18.4±3.6

13 0.17 0.17 0.67 70.0±0.1 78.1±2.6 2.0±0.3

4

5

6


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Figure legends 1

2

Fig. 1 Growth of R. eutropha in minimal medium with acetate, propionate, or 3

butyrate as the sole carbon source. Cultures were inoculated to an initial 4

OD600 of 0.05 and the reported values were measured after 48 h of growth. 5

6

Fig. 2 Optimizations of total mixed acid concentration (A), buffer concentration 7

(B), C/N ratio (C) and PHA production (D). Measurements were made 8

after 48 h of growth. For (A), media was buffered with 70 mM phosphate 9

buffer. In all experiments a ratio of 3:1:1 (acetate:propionate:butyrate) was 10

used. For (B-D), 0.5% total mixed acids were used. 11

12

Fig. 3 Effect of initial pH on cell growth. OD600 (A) and final pH (B) were 13

measured after 48 h of growth as functions of initial pH. Cultures 14

contained 0.5% (w/v) acetate (filled circles), 0.4% (w/v) propionate (open 15

circles), or 0.5% (w/v) butyrate (closed triangles). 16

17

Fig. 4 Optimization of mixed acid composition. The effects on CDW (A), final 18

pH (B), amount of PHA produced (C), PHA content (wt%) (D), 3HV content 19

(wt%) (E) and residual CDW (F) of different mixed acid compositions. 20

21

22

Fig. 5 Confirmation of mixed acid model by comparison of experimental values 23

(solid bars) and predicted values (white bars) at 0.1:0.1:0.8 (acetate: 24


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propionate: butyrate), 0.1:0.4:0.5 and, 0.6:0.3:0.1 using high phosphate 1

buffer. 2

3

Fig. 6 Production of PHAs from 3:1:1 (A) and 3:1:6 (B) organic acids (acetate: 4

propionate: butyrate) in high phosphate buffer media. Acetate (●), 5

propionate (○), butyrate (▼), CDW (△) and PHA (■) concentrations were 6

monitored. Depletion of NH4+ occurred within 12 h. 7


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1

2

Fig. 1 3


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1

2

Fig. 2 3

4

5

6

7

8

9

10

11


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1

2

Fig. 3 3


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1

2

3

Fig. 4 4


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1

Fig. 5 2


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1

Fig. 6 2

3


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Online Resource 1 2

Parameters for mixture contour in these experiments 3

CDW pH PHAs PHA % 3 HV %

Coef4) P5) Coef P Coef P coef P coef P

A1) 39.8 - 8.93 - 26.2 - 66.17 - 0.03 -

P2) 47.0 - 8.03 - 34.6 - 75.63 - 37.86 -

B3) 70.7 - 8.29 - 58.1 - 83.68 - -0.02 -

AP 32.3 0.000 0.79 0.032 27.2 0.005 - - -18.24 0.000

AB 37.3 0.000 0.55 0.098 44.4 0.001 28.70 0.003 - -

BP 25.5 0.001 0.79 0.029 26.7 0.005 - - -32.17 0.001

AP(A-P) -35.8 0.003 - - -33.9 0.024 - - 12.52 0.001

AB(A-B) -23.6 0.010 - - -32.2 0.025 - - - -

BP(B-P) - - - - - - - - - -

AAPB - - -12.26 0.043 - - - - 66.48 0.020

APPB -348.5 0.002 - - -391.1 0.009 - - 247.09 0.000

APBB - - - - - - - - -78.71 0.007

AP(A-P)sq - - - - - - - - - -

AB(A-B)sq - - - - - - - - - -

BP(B-P)sq - - - - - - - - 160 0.107

R-sq 99.88% 98.05% 99.67% 92.63% 95.20%

R-sq(pred) 98..04% 94.97% 96.42% 80.59% 84.73%

1) A: Acetate 4

2) P: Propionate 5

3) B: Butyrate 6

4) Coef.: coefficient 7

5) P: Statistical probability 8

9


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2

3

Online Resource 2 Comparison of growth between compositions with same number of 4

carbons (3:1:6, 3.6:1:1 and 3:1.5:1(acetate: propionate: butyrate)) and 5

control (3:1:1(acetate: propionate: butyrate)) for 48hr at 30°C 6


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Online Resource 3 1

Predicted amount of cell mass, PHA and wt % of 3HV. 2

3

Ratio 1)

CDW (mg) 2)

PHAs (mg) % CDW (%) 3 HV wt%

3:1:1 52.3 38.9 75.0 6.5

3:1:4 65.4 53.7 81.5 2.4

3:1:6 68.7 57.4 82.8 1.3

1:1:1 58.6 45.6 78.3 9.9

1) Acetate: Propionate: Butyrate 4

2) CDW predicted when cultures were grown using the given carbon source ratio in 50 5

mL mimimal media in 250 mL flasks at 30°C for 48 hrs. 6

7

8


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Online Resource 4 Consumption of volatile acids in 1:1:1 (acetate: propionate: 4

butyrate) composition. Acetate (●), propionate (○), and butyrate (▼) were measured 5

6


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