Polyhydroxybutyrate production from lactate using a mixed microbial culture

ARTICLE

Polyhydroxybutyrate Production From LactateUsing a Mixed Microbial Culture

Yang Jiang, Leonie Marang, Robbert Kleerebezem, Gerard Muyzer,Mark C.M. van Loosdrecht

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BCDelft,

The Netherlands; telephone: þ31-15-278-1091; fax þ31-15-278-2355;

e-mail: [email protected]

Received 7 January 2011; revision received 3 March 2011; accepted 15 March 2011

Published online 31 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23148

ABSTRACT: In this study we investigated the use of lactateand a lactate/acetate mixture for enrichment of poly-3-hydroxybutyrate (PHB) producing mixed cultures. Themixed cultures were enriched in sequencing batch reactors(SBR) that established a feast–famine regime. The SBRs wereoperated under conditions that were previously shown toenable enrichment of a superior PHB producing strain onacetate (i.e., 12 h cycle length, 1 day SRT and 308C). Twonew mixed cultures were eventually enriched from activatedsludge. The mixed culture enriched on lactate was domi-nated by a novel gammaproteobacterium. This enrichmentcan accumulate over 90wt% PHB within 6 h, which iscurrently the best result reported for a bacterial culture interms of the final PHB content and the biomass specific PHBproduction rate. The second mixed culture enriched on amixture of acetate and lactate can produce up to 84wt%PHB in just over 8 h. The predominant bacterial species inthis culture were Plasticicumulans acidivorans and Thaueraselenatis, which have both been reported to accumulate largeamounts of PHB. The data suggest that P. acidivorans is aspecialist on acetate conversion, whereas Thauera sp. is aspecialist on lactate conversion. The main conclusion of thiswork is that the use of different substrates has a direct impacton microbial composition, but has no significant effect onthe functionality of PHB production process.

Biotechnol. Bioeng. 2011;108: 2022–2035.

� 2011 Wiley Periodicals, Inc.

KEYWORDS: polyhydroxybutyrate; mixed microbial cul-ture; lactate; microbial diversity; selective pressure

Introduction

Due to many environmental concerns on pollution causedby petroleum-based plastics, research on bioplastics hasbeen developed rapidly during the past few years. Bioplastics

are defined as plastic-like materials, which are producedfrom renewable resources and/or are biodegradable.Polyhydroxyalkanoates (PHA) are one group of bioplastics,and the only fully biodegradable bioplastic completelysynthesized by microorganisms (Chen, 2010). Manydifferent groups of bacteria are capable of producingPHA as a source of energy and carbon (Steinbuchel, 1991).The microbial PHA synthesis can be stimulated underseveral conditions, including intermittent substrate feeding,nutrient limitation and oxygen limitation (Anderson andDawes, 1990; Lee, 1996; Reis et al., 2003).

Commercial PHA production is based on the purecultures of natural PHA producers, such as Ralstoniaeutropha, or recombinant Escherichia coli (Chen, 2010; Shiet al., 1997). Due to the high costs for substrate and the needfor sterile operation, the price of PHA is much higher thanthat of traditional petroleum-based plastics (Choi and Lee,1997; Reis et al., 2003). In order to reduce the productioncost of PHA, several researchers focus on PHA productionby mixed culture biotechnology, which aims at using non-sterile processes and wastewater as substrate (Kleerebezemand van Loosdrecht, 2007). By implementing the feast–famine strategy to select for PHA producing mixed cultures,several authors have shown the great potential of thisapproach in terms of both production capacity andefficiency. Johnson et al. (2009a) reported a mixed microbialenrichment capable of accumulating up to 89wt% of PHBafter 7.6 h of continuously feeding acetate. Albuquerqueet al. (2010) enriched a mixed culture on pre-fermentedmolasses from wastewater. The maximum PHA content oftheir enrichment reached up to 75wt%.

Most of the studies on PHA production by mixed culturesare based on the use of volatile fatty acids (VFAs) as thecarbon source (Albuquerque et al., 2010; Bengtsson et al.,2010; Johnson et al., 2009a; Lemos et al., 2008; Serafim et al.,2004; Villano et al., 2010). Although VFAs are thepredominant products in pre-fermented carbohydrates(Albuquerque et al., 2007; Bengtsson et al., 2008), othertypes of fermentation products (e.g., lactate, ethanol,

Yang Jiang and Leonie Marang contributed equally to this work.

Abbreviations: DO, dissolved oxygen; F/F ratio, feast-to-famine ratio; HRT, hydraulic

retention time; PHA, polyhydroxyalkanoate; PHB, polyhydroxybutyrate; SBR, sequen-

cing batch reactor; SRT, solid retention time; TSS, total suspended solid; VFAs, volatile

fatty acids.

Correspondence to: Robbert Kleerebezem

Additional Supporting Information may be found in the online version of this article.

2022 Biotechnology and Bioengineering, Vol. 108, No. 9, September, 2011 � 2011 Wiley Periodicals, Inc.

succinate, and glycerol) can be found in wastewater at highconcentrations as well (Temudo et al., 2007). Dabrock et al.(1992), Gao et al. (2009), and Hofvendahl and Hahn-Hagerdal (2000) have shown that many factors can triggerlactate production in the fermentation. The influence ofsubstrates other than VFAs, on PHA production by mixedcultures has hardly been investigated. So far, only Beccariet al. (2002) studied the effect of ethanol on PHA storage;however, their work is based on activated sludge withoutacclimation to ethanol, rather than on an enrichmentspecific for PHA production. A handful of the studies byDionisi et al. (2001, 2004, 2005, 2006, 2007) use lactate asone of the carbon sources to select a PHA producing mixedmicrobial culture. Yet only one of these studies investigatedthe short-term impact of organic acid on PHA production(Dionisi et al., 2004). Moreover, the maximal PHA storagecapacity of the enrichments in these studies was relativelylow (about 50wt%).

The substrate composition may have a direct influence onthe microbial community structure of the biomass, whichwill subsequently affect the PHA producing capacity. P.acidivorans, the dominant bacterial species in an enrichmentthat can accumulate up to 89 wt% PHB on acetate (Jianget al., 2010b; Johnson et al., 2009a), shows high specificuptake rates for a wide range of VFAs. Its specific uptake ratefor lactate is significantly lower. Therefore, it is interesting toevaluate whether a highly efficient PHA producing culturecan be enriched with lactate as substrate. Two SBRs wereoperated under equal conditions as used by Johnson et al.(2009a) for a highly effective acetate based PHB producingculture: 308C, 12 h cycle length, pH 7 and 1 day solidretention time (SRT). Both bioreactors were inoculated withactivated sludge, but cultivated either with lactate or amixture of acetate and lactate as substrate. The maximumPHA producing capacity and microbial communitystructure were studied. A metabolic model developed byJohnson et al. (2009b) was adjusted to characterize thekinetic and stoichiometric properties of the cultures.

Materials and Methods

Culture Enrichment in Sequencing Batch Reactors(SBR)

SBRs were used to enrich the PHB-producing mixedmicrobial culture. The basic SBR set-up was the same asdescribed by Johnson et al. (2009b). A double-jacket glassbioreactor with a working volume of 2 L (Applikon,Schiedam, the Netherlands) was used for the enrichmentand maintenance of the PHA-producing mixed cultures.The temperature of the reactors was set to 308C. The pH ofthe reactor medium was maintained at pH 7 by the additionof 1M HCl and 1M NaOH. The air flow rate to the reactorwas maintained at 0.2 LN/min using a mass flow controller(Brooks Instrument, Ede, the Netherlands). The off-gas wasrecirculated at about 1.2 LN/min through the reactor.Therefore, the total gas flow rate through the reactor was

1.4 LN/min. The temperature, pumps, stirrer, airflow, andpH were controlled by a biocontroller (Biostat B plus,Sartorius Systems, Bohemia, NY). Operation of the SBR wasbased on 12 h batch cycles consisting of start phase, feedingphase, reaction phase, and biomass effluent phase. Nosettling phase was included in the batch cycle. The SRT wastherefore equal to the HRT, and both were set to 1 day.

The two new mixed cultures were individually enrichedon lactate or a mixture of lactate and acetate (1:1 Cmolbased). Both systems were inoculated with activated sludgefrom a nutrient (i.e., organic carbon and nitrogen) removalsewage treatment plant (Kralingseveer, The Netherlands).The medium composition used in this study was the same asdescribed by Johnson et al. (2009a), except the carbonsource. In 1 L medium for one normal SBR cycle, the carbonsource was either 16.2mM Na(D,L-)Lac for the lactate SBRor 12.5mM NaAc�3H2O and 8.33mM Na(D,L-)Lac for themixed substrate SBR. The other chemical compounds in themedium were: 6.74mM NH4Cl, 2.49mM KH2PO4,0.55mM MgSO4�7H2O, 0.72mM KCl, 1.5mL/L traceelements solution according to Vishniac and Santer(1957) and 5mg/L allylthiourea (to prevent nitification).The SBR previously enriched on sole acetate (Johnson et al.,2009a) was maintained from previous studies for compar-ison. The total suspended solid (TSS) concentration at theend of the cycle was in the range of 0.5–0.6 g/L in all SBRsduring stable operation. The stable operational performancewas defined as a stable ammonia concentration and TSS atthe end of the cycle and a constant length of the feast phaseand pH profile for at least five cycles. The biomass from thisstable operational stage was collected for cycle measure-ments, fed-batch experiments, and microbial communitystructure analysis.

PHA Storage Maximization in Fed-Batch Reactor

A fed-batch reactor was used to evaluate the maximum PHAstorage capacity of the biomass collected from the SBRs. Theexperiments were conducted in a similar setup as the SBRs,but in a fed-batch mode. At the beginning of eachexperiment, the reactor was filled with 1 L biomass fromthe enrichment SBR and 1 L of organic carbon- andammonium-free medium was added. In this way, microbialgrowth in the fed-batch reactor is limited by the amount ofammonium remaining from the previous SBR cycle. ThePHA accumulation experiment was started by feeding apulse of 60mmol of the substrate the culture was grown on.Further substrate was supplied to the reactor via pH controlusing a 1.5M solution of the substrate stock solution (lacticacid and/or acetic acid) instead of the 1MHCl solution usedin the SBR. A few drops of antifoam (Sigma–Aldrich,Zwijndrecht, the Netherlands) were added when necessary.After about 10 h, the experiments were stopped.

Analytical Methods

For both SBR cycle measurements and fed-batch experi-ments, the progress of the experiments was monitored via

Jiang et al.: Polyhydroxybutyrate Production From Lactate 2023

Biotechnology and Bioengineering

online (DO, temperature, pH, acid and base dosage, off-gas O2 and CO2) and offline (substrate, ammonia, TSS,PHA)measurements. A detailed description of the analyticalprocedures for online and offline measurements can befound elsewhere (Johnson et al., 2009b).

Biological Oxygen Monitoring

The activity of the enrichments on different substrates (i.e.,lactate and acetate) was evaluated by measuring the biomassspecific oxygen uptake rate with a biological oxygen monitor(BOM). The incubation chamber was filled with 40mLoxygen saturated biomass from the SBR. The endogenousrespiration of the biomass collected at the end of the cyclewas checked to be negligible. The incubation chamber(308C) was closed and stirred with a magnetic stirrer. Whenthe temperature and DO were stabilized, 0.5mL of a 30mMsubstrate solution (Na(D,L-)Lac or NaAc�3H2O) was dosedinto the incubation chamber. The temperature and DO inthe chamber were measured using a dissolved oxygen meter(Jenway 9300, Stone, UK). The specific oxygen uptake ratewas calculated from the oxygen depletion rate and thebiomass concentration (measured as TSS).

Microbial Composition Analysis

Microbial composition analysis started with DNA isolationand 16S rRNA gene amplification. The obtained 16S rRNAgene sequences have been stored in GenBank underaccession numbers: HQ171181–HQ171185. They werecompared to sequences stored in GenBank using thenBLAST algorithm. FISH was used to validate the resultsobtained by DGGE analysis and to identify the cells underthe microscope. A detailed description of the microbialcomposition analysis procedures can be found elsewhere(Jiang et al., 2010a; Johnson et al., 2009a). The primers andprobes used in this work are listed in Table I. In order toidentify the bacterial species involved in PHA synthesis, Nileblue A was used to stain the PHA granules. The procedure of

post Nile blue A staining is described in Johnson et al.(2009a).

Data Treatment and Modeling

The SBR and fed-batch experiments were evaluatedaccording to the approach proposed by Johnson et al.(2009b). The metabolic model developed in this study wasadjusted from the model described in the same paper, whichwas based on acetate as sole substrate Johnson et al. (2009b).In this study, the model was extended for the use of lactate,and the use of a mixture of acetate and lactate. The reactionsinvolved in the metabolic model are summarized in Table II.The structure of the kinetic description remainedunchanged compared to the model developed in Johnsonet al. (2009b), but the maximum stoichiometric yields forlactate were recalculated. The maximum stoichiometricyields were derived from the balances of the conservedmoieties (NADH, ATP, AcCoA) in these reactions, asdescribed by Johnson et al. (2009b). As can be seen fromSupplementary Appendix A, all of them are represented as afunction of the efficiency of oxidative phosphorylation (d, P/O ratio). To simplify the model, the P/O ratio is assumed tobe 2.0 in all cultures, thereby neglecting potential smalldifference caused by the different microorganisms. In the

Table I. Oligonucleotide probes for FISH analysis and primers for PCR-DGGE analysis in this study.

Code Function Sequence (50–30) Specificity Refs.

EUB338 Probe gctgcctcccgtaggagt Bacteria Amann et al. (1990)

EUB338II Probe gcagccacccgtaggtgt Bacteria Daims et al. (1999)

EUB338III Probe gctgccacccgtaggtgt Bacteria Daims et al. (1999)

Beta42a Probe gccttcccacttcgttt Betaproteobacteria Manz et al. (1992)

Gama42a Probe gccttcccacatcgttta Gammaproteobacteria Manz et al. (1992)

UCB823 Probe cctccccaccgtccagtt P. acidivorans Johnson et al. (2009a)

ULB450 Probe cttccatactctaggtac Novel Gammaproteobacteria This study

341F-GC Primer cctacgggaggcagcagb Bacteria Schafer and Muyzer (2001)

907R Primer ccgtcaattcmtttgagttt Bacteria Schafer and Muyzer (2001)

aIn order to minimize erroneous hybridizations, the unlabeled probe Gamma42a was used to compete with the Beta42a probe.b341F-GC primer contains GC-clamp (50-cgcccgccgcgccccgcgcccgtcccgccgcccccgcccg-30) at the 50-end of the primer.

Table II. Reactions considered in the metabolic model on a carbon-mole

base (adapted from Johnson et al., 2009b). P/O ratio is the efficiency of the

oxidative phosphorylation and was assumed to be 2.0 for all enrichments in

this study.

Reaction Stoichiometry

1 Acetate uptake, activation 1HAcþ 1ATP! 1AcCoA

2 Lactate uptake, activation 1HLacþ 2/3ATP! 2/3AcCoAþ1/3CO2þ 2/3NADH2

3 Catabolism 1AcCoA! 1CO2þ 2NADH2

4 PHB production 1AcCoAþ 0.25NADH2! 1PHB

5 Anabolism 1.267AcCoAþ 0.2NH3þ 2.16ATP!1Xþ 0.267CO2þ 0.434NADH2

6 PHB consumption 1PHBþ 0.25ATP! 1AcCoA

þ 0.25NADH2

7 Oxidative phosphorylation 1NADH2þ 0.5O2! dATP

2024 Biotechnology and Bioengineering, Vol. 108, No. 9, September, 2011

case of using a mixture of acetate and lactate as a substrate, asummation of two separate models for sole acetate and solelactate was exerted. The metabolic model was constructedbased on the stoichiometry and kinetic equations shown inSupplementary Appendices A and B. Both data treatmentand modeling were conducted using the software MicrosoftExcel.

Results

SBR for Mixed Culture Enrichment

A total of three SBRs were operated during an extensiveperiod to study the potential use of lactate as substrate forPHB production. One SBR was fed with acetate (SBR 1) andwas operated under stable condition for more than 4 years.The two newly started SBRs were fed with lactate (SBR 2)and with a mixture of lactate and acetate (SBR 3). Thetypical feast–famine profile was established only a few cyclesafter inoculation with activated sludge. The feast–famineregime was readily identifiable from the DO profile: directlyafter feeding, the DO rapidly decreased rapidly (feast phase),and after substrate depletion the DO increased sharply,indicating the start of the famine phase. Based on the sharpincrease of the DO after substrate depletion, the length of thefeast phase can readily be estimated from the onlinerecorded DO profiles. Figure 1 shows the development of thelength of the feast phase in the SBRs operated with lactateand with the acetate/lactate mixture.

SBR 2 was operated for more than 4 months and twodifferent stable periods could be identified during thisenrichment process (Fig. 1A). Initially, the length of the feastphase stabilized at around 70min (stage I, Fig. 1A). After1 month of stable operation, the SBR 2 destabilized forunknown reasons and the length of the feast phase increaseddramatically to about 180min. Just after a few cycles, theSBR 2 re-stabilized automatically without any additionaloperation and the length of the feast phase decreased to lessthan 40min afterwards (stage II, Fig. 1A). In contrast, thelength of the feast phase in SBR 3 remained constant, oncestable operation was obtained (Fig. 1B). Due to the fact thatacetate and lactate were not consumed at the same rate inSBR 3, the end of the feast phase in this reactor wascharacterized by a stepwise increase of the DO (Fig. 2D).Acetate was firstly depleted after 35min, leading to a firstsharp increase of the DO. Lactate was depleted after around60min, resulting in the second increase of the DO. SBR 3was operated for nearly 3 months. The feast phase lengthunder the stable operational condition in the acetate fed SBR1 was around 40min (data not shown).

For a more detailed evaluation of the performance of theSBRs, cycle measurements were conducted once the SBRswere considered stable. The patterns of substrate consump-tion and products formation during these cycle measure-ments are displayed in Figure 2. In the feast phase, carbonsubstrates were rapidly consumed and primarily used for

PHB production (Y feast;ObsPHB;S , Table III). PHB was the only

polymer produced independent of the substrate used in thisstudy. The PHB content at the end of the feast phase wasabout 50wt% of biomass dry weight in all cycle measure-ments (Table III). At the beginning of the famine phase, arapid increase of the DO was observed due to the depletionof the external substrate. During the famine phase, thestored PHB was degraded and used for biomass synthesisand respiration.

Characterization of the Microbial Community Structure

To understand the microbial community structure in theSBRs, DGGE analysis of PCR-amplified 16S rRNA genefragments was performed. Biomass for this analysis wascollected when stable operation was established in the SBRs.Since two stable stages were observed in SBR 2, two differentbiomass samples enriched on lactate were analyzed.Figure 3A represents a typical DGGE gel, in which the lanenumber corresponds to the number of the SBRs. Thedifferent banding pattern in each lane indicated that themicrobial community in each experimental run was distinct.It was observed that the number of dominant bands in eachsample was directly related to the composition of the carbon

Figure 1. Development of the feast phase length (^) over time for SBR 2 (A) and

SBR 3 (B). Since a stepwise increase of the DO was observed in SBR 3, plus signs (þ)

indicate the length of the first part of the feast phase. The white squares (&) and gray

triangles (~) on the x-axis indicate when respectively the cycle and accumulation

experiments were performed.



source. Only one major band was observed when a singlecarbon source was used, whereas the biomass enriched onthe mixture of acetate and lactate was dominated by twotypes of bacterial species as indicated by two different bandsin the gel.

In order to identify the bacterial populations, five bandswere excised for sequence analysis (B1–B5, Fig. 3A). Allbands gave explicit sequences, which were compared to thesequences stored in GenBank (NCBI). A neighbor-joiningtree (Fig. 4) was generated from the obtained sequences,providing the phylogenetic affiliation of these bacteria. Thesequence derived from band B1 and B4 showed 100%similarity to the sequence of P. acidivorans, which has beenreported as a new genus within the gammaproteobacteriawith a superior PHB producing capacity on acetate (Jianget al., 2010b). From the first stage of the enrichment onlactate and from the biomass enriched on a mixture ofacetate and lactate, the sequence of band B2 and B5 wasfound to be affiliated to Thauera selenatis (100% sequencesimilarity). Thauera species have been reported as PHAproducers and are commonly present in wastewatertreatment plants (Dionisi et al., 2005; Lemos et al., 2008;Silva et al., 2010). Consistent to the decreased length of thefeast phase in SBR 2, the dominant bacterial species in thisenrichment shifted as well. The sequence of band B3indicated that the dominant bacterium was an uncultured

bacterium belonging to the gammaproteobacteria. Theclosest known sequence (99% similarity) to this bandoriginated from an uncultured bacterium detected inconventional activated sludge from petroleum refineriesin Brazil (Silva et al., 2010). The PHA producing capacitywas not evaluated in that paper. The closest known culturedrelative of the uncultured gammaproteobacterium was P.acidivorans (93% similarity).

Apart from the DGGE analysis, FISH was performed toanalyze the presence of the bacterial species in the culture.Two specific probes (UCB823 and ULB450) were used toindicate P. acidivorans and the novel gammaproteobacter-ium. Considering only one dominant band was observedfrom SBR 2-I biomass on the DGGE gel, and because we hadno specific probe available, Thauera sp. was indicated by theBeta42a probe (together with an unlabeled Gamma42aprobe). The general FISH probe mixture (EUB338 I–III) wasadditionally used to visualize all bacterial species in thesample.

The FISH results from the SBRs are consistent with theDGGE result. P. acidivorans was found to be the dominantbacterial species in SBR 1. In the two stages of SBR 2 (I andII), the predominant bacterial species were respectivelyThauera sp. and the novel gammaproteobacterium. In SBR3, both P. acidivorans and Thauera sp. were observed to beabundant. Since the PHB producing capacity of the novel

Figure 2. Results of the cycle experiments on SBR 1 (A), SBR 2-I (B), SBR 2-II (C), and SBR 3 (D). The solid blue line represents the DO (%), the orange circles ( ) represent the

PHB content in the cells (wt%), and the red squares ( ) and pink diamonds ( ) represent respectively the acetate and the lactate concentration in the reactor liquid (Cmmol/L).


gammaproteobacterium had never been examined, post-staining with Nile blue A was performed on the biomasscollected from SBR 2-II at the end of an accumulationexperiment (Fig. 3G). The result indicates that the novelgammaproteobacterium enriched in SBR 2-II was the mainorganism responsible for such a high PHB producingcapacity.

Short-Term Substrate Shift Experiments

In order to understand the reason why different types ofbacteria dominate the SBRs fed with different substrates, theactivities of the enrichments in this study on varioussubstrates were evaluated by BOM. The results demon-strated that the biomass from the different reactors had aclear substrate specificity (Fig. 5). The biomass selected onacetate (P. acidivorans dominated, SBR 1) showed a veryhigh activity on acetate, but a remarkably lower activity onlactate. In contrast, the enrichments on lactate (Thauera sp.

from SBR 2-I and the novel gammaproteobacterium fromSBR 2-II) showed a high activity on lactate, but aconsiderable lower activity on acetate. The biomass fromSBR 3 (a mixture of P. acidivorans and Thauera sp.),enriched on a mixture of acetate and lactate, displayed acompromised response to both substrates. The activity ofthis enrichment on acetate was slightly lower than that of thebiomass from SBR 1. When incubated with lactate, thebiomass from SBR 3 still showed about half of the activityobtained from the biomass in SBR 2-I and SBR 2-II.

PHB Production in Fed-Batch Reactor

To evaluate the PHB production rate and capacity of thedifferent enrichments, fed-batch accumulation experimentswere performed. After around 10 h operation under fed-batch mode, a PHB content of more than 80wt% wasreached in all four enrichments (Table III). The highest PHBcontent was reached in SBR 2-II. This enrichment, with the

Table III. Overview of observed variables, and model derived yields and biomass specific rates in the SBRs and fed-batch reactors.

SBR 1 acetate SBR 2-I lactate SBR 2-II lactate SBR 3a acetateþ lactate

Observed

Dominant bacteria P. acidivorans Thauera sp. g-Proteobacteria P. acidivorans and Thauera sp.

Length feast phase (min) 38 82 34 35 80

PHB max. feast (wt%) 52 48 53 51

PHB max. acc. (wt%) 88 81 92 84

Time PHB max. (h) 9.2 11.3 10.0 8.3

Time PHB >80wt% (h) 4.2 11.3 2.7 7.3

SBR cycle

Feast

Y feast;ObsPHB;S (Cmol/Cmol) 0.67 0.61 0.66 0.67 0.60

Y feast;ObsX;S (Cmol/Cmol) 0.00 0.10 0.06 0.00 0.10

Y feast;ObsCO2 ;S

(Cmol/Cmol) 0.34 0.30 0.29 0.34 0.30

Famine

Y famine;ObsX;PHB (Cmol/Cmol) 0.67 0.67 0.55 0.67 0.67

Y famine;ObsX;PHB (Cmol/Cmol) 0.33 0.34 0.46 0.33 0.33

Parameters

qmaxs (Cmol/Cmol/h) �4.38 �1.77 �3.93 �4.38 �1.77

mmax (Cmol/Cmol/h) 0.00 0.17 0.23 0.00 0.17

k ((Cmol/Cmol)1/3/h) �0.16 �0.18 �0.13 �0.16 �0.18

fP. acidivorans (Cmol/Cmol) — — — 0.47

mATP (mol/Cmol/h) 0.00 0.00 0.06 0.00 0.00

Fed-batch experiment

Accumulation

Yacc:;ObsPHB;S (Cmol/Cmol) 0.61 0.64 0.63 0.61 0.68

Yacc:;ObsX;S (Cmol/Cmol) 0.04 0.07 0.05 0.04 0.05

Yacc:;ObsCO2 ;S

(Cmol/Cmol) 0.35 0.29 0.32 0.35 0.28

Parameters

qmaxPHB (Cmol/Cmol/h) 1.74 0.96 2.07 1.74 0.96

mmax (Cmol/Cmol/h) 0.09 0.10 0.16 0.09 0.10

mATP (mol/Cmol/h) 0.00 0.00 0.61 0.00 0.00

P 1.31 1.44 3.22 1.38 1.44

fP. acidivorans (Cmol/Cmol) — — — 0.47

fmaxPHB (Cmol/Cmol) 8.30 5.10 13.50 8.30 5.10

aSBR 3 was modeled as the sum of two separate cultures of which one is solely using acetate (left column) and the other solely lactate (right column).



novel gammaproteobacterium as the dominant bacterialstrain, could accumulate more than 90wt% PHB (the finalPHB concentration can reach approximately 4.3 g/L, datanot shown). To demonstrate the complete data set obtainedby the accumulation experiments, the results for one of theexperiments on SBR 2-II biomass are shown in Figure 6.The PHB content increases quickly in the first 90min,corresponding to the rapid consumption of the substrate.After about 350min, the PHB content hardly increases, eventhough substrate consumption continues. This phenom-enon has been observed in all cases in this study (data notshown). This observation suggests that some unknowncarbon storage compounds are produced by the enrich-ments, although the solid evidence is still missing. Besidesthe maximum PHB content, the PHB production efficiencywas also estimated by evaluating the time needed for

different enrichments to accumulate more than 80wt%PHB. The highest PHB production efficiency was againobserved in the SBR 2-II enrichment, which accumulated80wt% PHB in less than 3 h (Table III).

Modeling and Kinetic Analysis

To obtain an accurate evaluation of biomass specificreaction rates and a better understanding about the kineticsof PHB production on different substrates, a metabolicmodel was developed for both cycle and accumulationexperiments. The model developed for the lactate grownbiomass was derived from the metabolic model described byJohnson et al. (2009b), which was developed for acetate asthe substrate. The only structural difference between both

Figure 3. DGGE gel and FISH microscopic photographs. A: DGGE gel of PCR-amplified 16S rRNA gene fragments from the mixed cultures enriched in the three SBRs. The lane

numbers refer to the corresponding SBR numbers. SmartLadder (Eurogentec, Maastricht, the Netherlands) was loaded in two lanes, labeled with M. The bands labeled with B1-5

were excised and re-amplified for microbial identification. B: Fluorescence microscopy images of the mixed cultures enriched on acetate (SBR 1) stained with Cy3-labeled probe for

P. acidivorans (UCB823, red), FLUOS-labeled probe for betaproreobacteria (Beta42a, green) and Cy5-labeled probe for Eubacteria (EUBmix, blue). The pink color indicates both Cy3-

labeled probe and Cy5-labeled probe hybridized. C: Fluorescence microscopy images of the mixed cultures enriched on lactate (SBR 2-I) stained with Cy3-labeled probe for novel

Gammaproteobacteria (ULB450, red), FLUOS-labeled probe for betaproreobacteria (Beta42a, green) and Cy5-labeled probe for Eubacteria (EUBmix, blue). The light blue color

indicates both FLUOS-labeled probe and Cy5-labeled probe hybridized. D: Fluorescence microscopy images of the mixed cultures enriched on lactate (SBR 2-II) stained with Cy3-

labeled probe for novel Gammaproteobacteria (ULB450, red), FLUOS-labeled probe for betaproreobacteria (Beta42a, green) and Cy5-labeled probe for Eubacteria (EUBmix, blue).

The pink color indicates both Cy3-labeled probe and Cy5-labeled probe hybridized. E: Fluorescence microscopy images of the mixed cultures enriched on the mixture of acetate and

lactate (SBR 3) stained with Cy3-labeled probe for P. acidivorans (UCB823, red), FLUOS-labeled probe for betaproreobacteria (Beta42a, green) and Cy5-labeled probe for Eubacteria

(EUBmix, blue). The pink color indicates both Cy3-labeled probe and Cy5-labeled probe hybridized. The light blue color indicates both FLUOS-labeled probe and Cy5-labeled probe

hybridized. F: Fluorescence microscopy image of SBR 2-II biomass collected at the end of an accumulation experiment, which was stained with Cy3-labeled probe for novel

Gammaproteobacteria (ULB450, red), FLUOS-labeled probe for betaproreobacteria (Beta42a, green) and Cy5-labeled probe for Eubacteria (EUBmix, blue). The pink color indicates

both Cy3-labeled probe and Cy5-labeled probe hybridized. G: Overlay of a phase contrast image and a fluorescence microscopy image of the same field as in (F), post-stained with

Nile blue A to indicate the presence of PHA, indicating that the uncultured bacteria was the main organism responsible for the high PHB producing capacity.


models is the maximum stoichiometric yields(Supplementary Appendix A). The experimental data fromsole substrate experiments (Fig. 7A–F) were used forcalibrating the model. In contrast, the experimental datafrom the mixture of acetate and lactate (SBR 3) was used tovalidate the model by combining the individual modelsfor acetate and lactate, with the following assumptions:

� As the predominant bacteria in SBR 3, P. acidivorans andThauera sp. are specialist on acetate and lactaterespectively (Fig. 5), the consumption of lactate byP. acidivorans and the consumption of acetate by Thauerasp. are neglected.

� The model parameters for biomass grown in SBR 3 weredirectly obtained from SBR 1 and SBR 2-I. P. acidivorans

Figure 4. Neighbor-joining tree based on 16S rRNA sequences, showing the phylogenetic affiliation of band B1-5 from the DGGE gel in Figure 3A. The Greek symbols indicate

different subclasses of the Proteobacteria, that is, alphaproteobacteria, betaproteobacteria, and gammaproteobacteria. The bar indicates a 10% sequence difference.

Figure 5. The biomass specific oxygen uptake rate (mmol/g/h) measured by

BOM for the cultures enriched on acetate (SBR 1), lactate (SBR 2I-II) and a mixture of

acetate and lactate (SBR 3). For all enrichments, the activity on both acetate and

lactate was determined. The experiments were performed in duplicate; error bars

indicate the standard deviation.

Figure 6. Results of an accumulation experiment on SBR 2-II. The pink diamonds

( ) represent the cumulative uptake of lactate (Cmmol), the orange circles ( )

represent the PHB content in the cells (wt%), and the black triangles (~) represent

the active biomass (Cmmol) in the reactor. The amount of ammonium (mmol) in the

reactor is depicted by the green asterisks ( ). The solid blue line and the dashed brown

line represent respectively the cumulative oxygen uptake and the cumulative carbon

dioxide evolution. Lines only indicate the trend here.



and Thauera sp. were the dominant bacterial species inSBR 3 and were individually enriched on acetate andlactate in SBR 1 and SBR 2-I. It is therefore assumed thatthe kinetic properties of the two bacterial species areunchanged.

� It is impossible to obtain the individual biomassconcentration of P. acidivorans and Thauera sp. fromTSS measurement. A new parameter ( fP. acidivorans) wasintroduced to estimate the relative abundance ofP. acidivorans and Thauera sp. in the total biomass.

Figure 7. Results of the cycle and accumulation experiments on SBR 1 (A and B), SBR 2-I (C and D), SBR 2-II (E and F) and SBR 3 (G and H). The solid lines represent modeled

data, the symbols represent the corresponding measured data. ( ) Acetate; ( ) Lactate; (~) Active biomass; ( ) PHB; (�) Ammonia; (þ) Cumulative carbon dioxide evolution; (�)

Cumulative oxygen uptake.


From the individual net biomass yields as identified inSBR 1 and SBR 2-I, the value for fP. acidivoranswas estimatedto be 0.47 in SBR 3.

Figure 7 shows that the model provides a gooddescription of the measurements for both SBR and fed-batch experiments in this study. The developed model canaccurately describe the measurement data in all cases. Themodel derived yields, biomass specific rates and otherkinetic parameters are listed in Table III.

The overall process in the different reactors was highlycomparable. During the feast phase of each SBR cycle, mostof the substrate was used for PHB synthesis (Y feast;Obs

PHB;S wasaround 0.64 Cmol/Cmol). Growth contributed only a smallfraction of the total substrate consumption during thisphase, since the Y

feast;ObsX;S was only about 0.05 Cmol/Cmol.

The remaining part of the consumed substrate was respiredto CO2 (Y feast;Obs

CO2;Swas about 0.32 Cmol/Cmol). All enrich-

ments showed a very high biomass specific substrate uptakerate ðqmax

S Þ, with a lowest value of 1.8 Cmol/Cmol/h forthe Thauera sp. dominating SBR 2-I and SBR 3. On the samesubstrate, the novel gammaproteobacterium showed amore than two times higher specific lactate uptake rate.The performance of the different enrichments during thefamine phase was also remarkably similar; k-value whichindicates the PHB degradation rate during the famine phasewas about �0.15 ((Cmol/Cmol)1/3/h)in all enrichments.

Biomass synthesis was inhibited in the accumulationexperiment due to the limitation of ammonia. Theparameters obtained from the feast phase of the cycleexperiments were highly comparable to those obtained inthe accumulation experiments. PHB synthesis accounted forthe major part of the substrate consumption; Yacc;Obs

PHB;S wasaround 0.64 Cmol/Cmol. The only exceptional parameterwas the mAPT estimated from the SBR 2-II accumulationexperiment. In most experiments this parameter had anegligible value, but in the novel gammaproteobacteriumdominated culture, maintenance appeared to play animportant role. It cannot be excluded that this is due tofutile cycles or other imbalances in the energy metabolism.

In both the cycle measurement and the fed-batchexperiment in SBR 3, the model based estimation of theinitial relative abundance of P. acidivoranswas found slightlyless than 50%. This was consistent with the FISHobservation (Supplementary Appendix C).

Discussion

Impact of the Selective Pressure on PHB Production

A strong selective pressure was applied to enrich the PHBproducing mixed microbial cultures in this study. Allenrichments showed superior PHB producing capacity, eventhough the microbial community structure was stronglydifferent. The mixed culture enriched in SBR 2-II on lactate

showed the highest reported PHB producing capacity interms of both maximum content and production rate ascompared to other studies (Dias et al., 2006; Johnson et al.,2009a; Slater et al., 1988). These observations confirm thevalidity of the ecological selection principle proposed forestablishment of a microbial community with a superiorPHB producing capacity, based on a low number of cyclesper SRT in a SBR.

The operational conditions used in this study weredirectly derived from the work of Johnson et al. (2009a),who achieved the first significant breakthrough in final PHBcontent that can be established in a mixed culture process.The maximum PHB content of their enrichment wasincreased from 65 to 89wt% PHB of cell dry weight byoperating the SBR at long cycle length (12 h), short SRT (1day), carbon limiting conditions and a relatively hightemperature (308C). Jiang et al. (2010a) continued this workand proposed that the key selective pressure was based onthe low number of operational cycles per SRT (i.e., a highsubstrate to biomass ratio after feeding and a short SRT). Itwas estimated that at an SRT: cycle length ratio of 2, bacteriahave to accumulate 50wt% PHB in order to stay in thereactor. Independent on the substrate used in this study, thePHB content at the end of the feast phase was indeed alwaysaround 50wt%, substantiating the proposed relation.

This operational procedure has been validated underdifferent conditions. Jiang et al. (2010a) enriched a mixedculture dominated by Zoogloea sp. by operating the SBR at208C, 12 h cycle length and 1 day SRT on acetate. Themaximum PHB content of that enrichment was 75wt% ofcell dry weight. Moralejo-Garate et al. (submitted) usedglycerol as substrate to enrich a PHB producing mixedculture and operated the SBR at 308C, 24 h cycle length and2 days SRT. Although 10wt% glycogen was present in thebiomass, the enrichment was still capable of producing morethan 80wt% PHB.

With the aim of improving the reaction rates, a short SRT(1 day) and a relatively high temperature (308C) wereadopted. Applying a shorter SRT results in a higher biomassproduction rate and less inert biomass in the reactor. Aslightly higher temperature can generally increase allreaction rates (Johnson et al., 2010). As a result of theimproved reaction rates, the length of the feast phase can beshortened and the famine phase is prolonged, increasing theselective pressure for bacteria with a high storage capacity.Albuquerque et al. (2010) suggested that a small feast-to-famine ratio (F/F ratio) is considered as an indication of aprocess with high PHB producing capacity. The F/F ratio ofall enrichments in this study was very small (less than 0.13),which was consistent with the high PHB producing capacity.

In most studies, a long SRT (10 days) has been used forenriching PHA producing mixed cultures (Albuquerqueet al., 2010; Bengtsson, 2009; Bengtsson et al., 2010; Lemoset al., 2006; Serafim et al., 2004). This development ofthe PHA production process is derived from activatedsludge processes, where a long SRT is used for minimizingsludge production. Dias et al. (2006) state that ‘‘It can be



anticipated that the shorter the SRT the higher the cellgrowth rate and the less the substrate is used for storage,’’which would can be interpreted as a preference for long SRTfor PHA production systems. Beun et al. (2002) also suggestthat the formation of PHA is higher at longer SRT. Theysuggest that a long SRT is associated with a low growth ratein the feast phase and consequently a low biomass yield onsubstrate according to Equation (1) (neglecting main-tenance).

Y feast;ObsX;S ¼ mfeast

qfeastS

(1)

At a lower value for Y feast;ObsX;S , a larger fraction of substrate

is available for PHA production, stimulating the PHAproducing capacity of the biomass. However, the equationshows that the value for Y feast;Obs

X;S is not only a function of thegrowth rate, but also of the biomass specific substrate uptakerate. In an SBR system the selection of bacteria is primarilybased on the substrate uptake rate and extremely high qmax

S

values were found in our systems (Table III). The qmaxS of the

Thauera sp. was about 1.8 Cmol/Cmol/h and was the lowestvalue in all three enriched bacterial communities, but stillsignificantly higher than the values reported in literature(Dias et al., 2006; Dionisi et al., 2004; Lemos et al., 2006;Serafim et al., 2004). This observation indicates that theYfeast;ObsX;S value we found is not higher than those observed in

other studies, despite the potentially slightly higher growthrate. In all our experiments the fraction of substrate taken upduring the feast phase and used for growth is very small(0–10%). Moreover, the prolonged SRT results in a decreasein the overall biomass yield which is considered anadvantage during waste water treatment, but is a cleardisadvantage in the system described here that aims at highrate production of biomass with a superior PHA producingcapacity.

Impact of Substrate on the Microbial Diversity

In this work, the microbial community structure wasstrongly dependent on the substrate composition. Only onepredominant bacterial species was enriched when a singlesubstrate was used. Taken into account that the overallbiomass yields on the same substrate are highly similar fordifferent microorganisms, the microbial competition will bedetermined by differences in the maximum specificsubstrate uptake rate ðqmax

S Þ. The results from short-termsubstrate shift experiments demonstrated that P. acidivoransis specialized in acetate consumption, whereas the Thauerasp. and the novel gammaproteobacterium are lactateconsumers. Therefore, P. acidivorans dominated the acetatefed SBR (SBR 1), while Thauera sp. and the novelgammaproteobacterium dominated the lactate fed SBR(SBR 2). The microbial composition shift from Thauera sp.to the novel gammaproteobacterium in SBR 2 confirmed thecrucial role of qmax

S . The novel gammaproteobacteriumoutcompeted Thauera sp. due to its more than two times

higher lactate uptake rate (Table III). The fact that onepredominant bacterial species was enriched on a singlesubstrate has been reported before. Jiang et al. (2010a)enriched in a highly comparable system Zoogloea sp. as theonly dominant bacterial species on acetate at 208C.However, the coexistence of more than one bacterial speciesin a SBR fed with only one substrate has also been reported(Lemos et al., 2008). In their study, both the substrate andthe nitrogen source were limited. Moreover, the pH was notcontrolled and long SRT (10 days) was used. As a result, theselective pressure is no longer only based on substrate uptakerate, which might explain the more diverse microbialpopulation.

The situation turned more complex when a mixture ofsubstrates was used. Two predominant bacteria,P. acidivorans and Thauera sp., were found in the SBRfed with the acetate and lactate mixture (SBR 3). Both ofthese two bacterial species were also individually enrichedon sole acetate (SBR 1) and lactate (SBR 2-I). It isunexpected that the novel gammaproteobacterium was notenriched in SBR 3, although it has a much higher lactateuptake rate than the Thauera sp. The reason for thisphenomenon remains unclear. Several possibilities couldexplain the absence of the novel gammaproteobacterium inSBR 3:

� SBR 3 has not been operated long enough to enrich thenovel gammaproteobacterium. It was observed that thenovel gammaproteobacterium was present in SBR 3 invery low numbers by FISH (Supplementary Appendix D).The novel gammaproteobacterium may need longerperiod to outcompete Thauera species.

� Thauera sp. can utilize acetate more efficiently than thenovel gammaproteobacterium. It has been reported thatThauera sp. can consume acetate as substrate (Macy et al.,1993). The competitive disadvantage of Thauera sp. to thenovel gammaproteobacterium on lactate can be com-pensated by consuming a part of acetate.

� The novel gammaproteobacterium might be sensitive toacetate. The presence of acetate could inhibit the lactateuptake rate of the novel gammaproteobacterium. How-ever, this inhibition was not observed in this study(Supplementary Appendix E).

To obtain a conclusive answer, the novel gammaproteo-bacterium is currently being isolated for furthercharacterization.

The impact of a mixture of substrates on the microbialcomposition has been studied in continuous stirred tankreactors (CSTR). It was reported that mixotrophs, which arecapable of consuming multiple substrates, have in general acompetitive advantage over specialists, which only degradeone type of substrate (Kovarova-Kovar and Egli, 1998;Temudo et al., 2008). Since the substrates are continuouslypresent at a low concentration in a CSTR, the microbialselection is based on a combination of qmax

S and substrate


affinity (KS). Mixotrophs can maintain a higher growth rateat lower substrate concentrations than specialists andthereby outcompete the specialists. The situation is verydifferent in a pulse-fed SBR, where the substrates are presentin excess. Here qmax

S plays an important role in thecompetition if the overall yield of biomass on substrate isconstant. The observations in this study suggested thatspecialists are enriched in pulse-fed reactor with amixture ofsubstrates. Different results were obtained by Villano et al.(2010) who enriched amixed culture dominated by only onebacterial species, Lampropedia hyalina, in a SBR fed with amixture of acetate and propionate (pH 7.5). Jiang et al.(2011) found that P. acidivorans dominated SBRs fed with amixture of acetate and propionate, independent on the ratioof these two types of substrate. Additionally, P. acidivoranswas also found to dominate enrichments on mixtures ofacetate and butyrate mixed in any ratio (data not shown). P.acidivorans can utilize C2–C10 volatile fatty acids (VFAs) ascarbon and energy source (Jiang et al., 2010b).

In summary, it is suggested that the microbial communitystructure is directly related to the type of substrate utilized.In comparable operational conditions enrichments estab-lished on lactate (this study), VFAs (Jiang et al., 2011;Villano et al., 2010) or carbohydrates (i.e., glycerol)(Moralejo Garate et al., submitted) provide a highly distinctmicrobial community structure.

Impact of Substrate on the Kinetics

The metabolic model developed in this study can accuratelydescribe the biochemical conversions involved in both theSBR and the fed-batch reactor. However, the model wasimplemented based on several assumptions. Whether theseassumptions were correct still needs to be deliberated.

PHB was observed as the only polymer formed fromlactate, which suggested that all lactate was converted toacetyl-CoA. One CO2 molecule is released during thisconversion (R2 in Table II). According to carbon balance, itcan readily be predicted that the theoretical maximal yield ofPHB on lactate ðY feast;max

PHB;Lac Þ cannot be higher than 0.67 Cmol/Cmol. However, the yield calculated according to theequation in Supplementary Appendix A was 0.74 Cmol/Cmol by assuming a P/O ratio of 2.0. This observationsuggests that the P/O ratio is overestimated, or that the ATPconsumption in the lactate storage pathway is higher thanexpected from common biochemistry (Johnson et al.,2009b).

In the current model, the P/O ratio was assumed to be 2.0in all experiments. When acetate was the only substrate, theTCA cycle is always needed for NADH2 generation, which issubsequently used for ATP production. In contrast, thepathway involved in PHB production from lactate directlygenerates NADH2 (Table II). If the P/O ratio is sufficientlyhigh, NADH2 respiration may supply adequate amounts ofATP for the storage of PHA and/or growth, excluding theneed for the TCA cycle. Overestimation of the P/O ratio

therefore in the proposed model results in a reversed TCAcycle, causing CO2 conversion to acetyl-CoA. It can beestimated that the TCA cycle is not needed at a P/O ratio of1.3. However, the measured biomass production during thefamine phase suggests that the stoichiometric yield ofbiomass on PHB (Y famine;max

X;PHB , Supplementary Appendix A) isunderestimated at such low values for the P/O ratio. Thisobservation suggests that the value for the P/O ratio can bedifferent during the feast phase and the famine phase.Another explanation could be an increased need for ATP inthe lactate uptake. Increasing the ATP requirements forlactate uptake (e.g., from 0.67 to 1 ATP/Cmol) results in anestimated P/O ratio of 2.0 in absence of the TCA cycle(Table II). The knowledge on the ATP consumption duringthe lactate uptake is still very limited.

Several authors have reported that the estimation of P/Oratio is associated with the estimation of mATP in themetabolic model (Johnson et al., 2009b; Lopez-Vazquezet al., 2009). An overestimated P/O ratio results in anoverestimated mATP and vice versa. The exceptional mATP

observed from SBR 2-II could be the result of the large P/Oratio adopted in this study. The value of mATP can beimproved to a more reasonable range by lowering the P/Oratio. Although P/O ratio has a significant influence onmaximal stoichiometric yields in Supplementary AppendixA and mATP, its impact on other parameters is negligible(Supplementary Appendix F). The parameters listed inTable III are therefore still reliable, except mATP.

Conclusions

The feast–famine strategy is specifically effective for selectingPHA producing bacterial from natural environment. Thecomposition of substrates has a direct impact on microbialcomposition, but has no significant effect on the function-ality of PHB production process. After P. acidivorans wasdiscovered as a new genus of bacteria with superior PHBproducing capacity, a novel gammaproteobacterium whichcan accumulate over 80wt% PHB within 3 h and with amaximum PHB storage capacity of more than 90wt% wasenriched on lactate in this study. This is currently the bestresult of PHB production by mixed culture in terms of bothcapacity and rate. The selective pressure for enriching PHAproducing mixed culture has been confirmed by the resultof this study, ensuring a stable functionality of PHAproduction by mixed culture. The bottleneck of PHAproduction by mixed culture biotechnology has beeneliminated. Considering the benefits from cheap substrate,non-sterile operation, no genetic modification investmentand no interfere with food production, the PHA productionby mixed culture biotechnology shows a high potential toreplace the PHA production by pure culture biotechnology.We are expecting for more remarkable findings in the case ofPHA production by mixed culture at large scale in thefuture.



Nomenclature

fP. acidivorans the fraction of P. acidivorans in the total microbial

community (Cmol/Cmol)

fmaxPHB the maximum fraction of PHB on active biomass (Cmol/

Cmol)

k rate constant of PHB degradation ((Cmol/Cmol)1/3/h)

KS half-saturation constant for substrate (Cmol/L)

mATP biomass specific ATP requirement for maintenance (mol/

Cmol/h)

p exponent of PHB inhibition term

qfeastS biomass specific substrate uptake rate (Cmol/Cmol/h)

qmaxS maximum biomass specific substrate uptake rate (Cmol/

Cmol/h)

qmaxPHB maximum biomass specific PHB production rate (mol/

Cmol/h)

Yacc;Obsi;j observed yield of compound i on compound j in the

accumulation experiment (Cmol/Cmol)

Yfamine;maxi;j stoichiometric yield of compound i on compound j in the

famine phase ((C)mol/Cmol)

Yfamine;Obsi;j observed yield of compound i on compound j in the famine

phase (Cmol/Cmol)

Yfeast;maxi;j stoichiometric yield of compound i on compound j in the

feast phase ((C)mol/Cmol)

Yfeast;Obsi;j observed yield of compound i on compound j in the feast

phase (Cmol/Cmol)

mfeast biomass specific growth rate in the feast phase (1/h)

mmax maximum biomass specific growth rate in the feast phase

(1/h)

We thank Ben Abbas for his help in phylogenetic analysis and Gert van

der Steen for the PHA analysis. These investigations were supported

by The Netherlands Organisation for Scientific Research (NWO) in

the NWO-ACTS research programme B-Basic and by the Foundation

for Technical Sciences (STW).

References

Albuquerque MGE, Eiroa M, Torres C, Nunes BR, Reis MAM. 2007.

Strategies for the development of a side stream process for polyhy-

droxyalkanoate (PHA) production from sugar cane molasses. J Bio-

technol 130(4):411–421.

Albuquerque MGE, Torres CAV, Reis MAM. 2010. Polyhydroxyalkanoate

(PHA) production by a mixed microbial culture using sugar molasses:

Effect of the influent substrate concentration on culture selection.

Water Res 44(11):3419–3433.

Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA. 1990.

Combination of 16s ribosomal-RNA-targeted oligonucleotide probes

with flow-cytometry for analyzing mixed microbial-populations. Appl

Environ Microbiol 56(6):1919–1925.

Anderson AJ, Dawes EA. 1990. Occurrence, metabolism, metabolic role,

and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev

54(4):450–472.

Beccari M, Dionisi D, Giuliani A, Majone M, Ramadori R. 2002. Effect of

different carbon sources on aerobic storage by activated sludge. Wat Sci

Technol 45(6):157–168.

Bengtsson S. 2009. The utilization of glycogen accumulating organisms for

mixed culture production of polyhydroxyalkanoates. Biotechnol

Bioeng 104(4):698–708.

Bengtsson S, Hallquist J, Werker A, Welander T. 2008. Acidogenic fermen-

tation of industrial wastewaters: Effects of chemostat retention time

and pH on volatile fatty acids production. Biochem Eng J 40(3):492–

499.

Bengtsson S, Pisco AR, Reis MAM, Lemos PC. 2010. Production of

polyhydroxyalkanoates from fermented sugar canemolasses by a mixed

culture enriched in glycogen accumulating organisms. J Biotechnol

145(3):253–263.

Beun JJ, Dircks K, Van Loosdrecht MCM, Heijnen JJ. 2002. Poly-beta-

hydroxybutyrate metabolism in dynamically fed mixed microbial

cultures. Water Res 36(5):1167–1180.

Chen GQ. 2010. Plastics from bacteria, nature functions and application.

Berlin-Heidelberg: Springer.

Choi JI, Lee SY. 1997. Process analysis and economic evaluation for poly(3-

hydroxybutyrate) production by fermentation. Bioprocess Eng

17(6):335–342.

Dabrock B, Bahl H, Gottschalk G. 1992. Parameters affecting solvent

production by Clostridium-Pasteurianum. Appl Environ Microbiol

58(4):1233–1239.

Daims H, Bruhl A, Amann R, Schleifer KH, Wagner M. 1999. The domain-

specific probe EUB338 is insufficient for the detection of all bacteria:

Development and evaluation of a more comprehensive probe set. Syst

Appl Microbiol 22(3):434–444.

Dias JML, Lemos PC, Serafim LS, Oliveira C, Eiroa M, Albuquerque MGE,

Ramos AM, Oliveira R, Reis MAM. 2006. Recent advances in poly-

hydroxyalkanoate production by mixed aerobic cultures: From the

substrate to the final product. Macromol Biosci 6(11):885–906.

Dionisi D,MajoneM, Tandoi V, Beccari M. 2001. Sequencing batch reactor:

Influence of periodic operation on performance of activated sludges in

biological wastewater treatment. Ind Eng Chem Res 40(23):5110–5119.

Dionisi D, Majone M, Papa V, Beccari M. 2004. Biodegradable polymers

from organic acids by using activated sludge enriched by aerobic

periodic feeding. Biotechnol Bioeng 85(6):569–579.

Dionisi D, Beccari M, Di Gregorio S, MajoneM, PapiniMP, Vallini G. 2005.

Storage of biodegradable polymers by an enriched microbial commu-

nity in a sequencing batch reactor operated at high organic load rate. J

Chem Technol Biotechnol 80(11):1306–1318.

Dionisi D, Majone M, Vallini G, Di Gregorio S, Beccari M. 2006. Effect of

the applied organic load rate on biodegradable polymer production by

mixed microbial cultures in a sequencing batch reactor. Biotechnol

Bioeng 93(1):76–88.

Dionisi D, Majone M, Vallini G, Di Gregorio S, Beccari M. 2007. Effect of

the length of the cycle on biodegradable polymer production and

microbial community selection in a sequencing batch reactor. Biotech-

nol Progr 23:1064–1073.

Gao MT, Hirata M, Toorisaka E, Hano T. 2009. Development of a

fermentation process for production of calcium-L-lactate. Chem Eng

Process 48(1):464–469.

Hofvendahl K, Hahn-Hagerdal B. 2000. Factors affecting the fermentative

lactic acid production from renewable resources. Enzyme Microb

Technol 26(2–4):87–107.

Jiang Y, Hebly M, Kleerebezem R, Muyzer G, van Loosdrecht MCM. 2011.

Metabolic modeling of mixed substrate uptake for polyhydroxyalk-

anoate (PHA) production. Water Res 45(3):1309–1321.

Jiang Y, Marang L, Kleerebezem R,Muyzer G, van LoosdrechtMCM. 2010a.

Effect of temperature and cycle length on microbial competition in

PHB-producing sequencing batch reactor. ISME J DOI: 10.1038/

ismej.2010.174.

Jiang Y, Sorokin D, Kleerebezem R, Muyzer G, van Loosdrecht MCM.

2010b. Plasticicumulans acidivorans gen. nov., sp. nov., a polyhydrox-

yalkanoate-accumulating gammaproteobacterium from a sequencing-

batch bioreactor. Int J Syst Evol Microbiol DOI: ijs.0.021410-0.

Johnson K, Jiang Y, Kleerebezem R, Muyzer G, van Loosdrecht

MCM. 2009a. Enrichment of a mixed bacterial culture with a high

polyhydroxyalkanoate storage capacity. Biomacromolecules 10(4):670–

676.

Johnson K, Kleerebezem R, van Loosdrecht MCM. 2009b. Model-based

data evaluation of polyhydroxybutyrate producing mixed microbial

cultures in aerobic sequencing batch and fed-batch reactors. Biotechnol

Bioeng 104(1):50–67.


Johnson K, van Geest J, Kleerebezem R, van Loosdrecht MCM. 2010. Short-

and long-term temperature effects on aerobic polyhydroxybutyrate

producing mixed cultures. Water Res 44(6):1689–1700.

Kleerebezem R, van Loosdrecht MCM. 2007. Mixed culture biotechnology

for bioenergy production. Curr Opin Biotechnol 18(3):207–212.

Kovarova-Kovar K, Egli T. 1998. Growth kinetics of suspended microbial

cells: From single-substrate-controlled growth to mixed-substrate

kinetics. Microbiol Mol Biol Rev 62(3):646–666.

Lee SY. 1996. Plastic bacteria? Progress and prospects for polyhydroxyalk-

anoate production in bacteria. Trends Biotechnol 14(11):431–438.

Lemos PC, Serafim LS, Reis MAM. 2006. Synthesis of polyhydroxyalk-

anoates from different short-chain fatty acids by mixed cultures sub-

mitted to aerobic dynamic feeding. J Biotechnol 122(2):226–238.

Lemos PC, Levantesi C, Serafim LS, Rossetti S, Reis MAM, Tandoi V.

2008. Microbial characterisation of polyhydroxyalkanoates storing

populations selected under different operating conditions using

a cell-sorting RT-PCR approach. Appl Microbiol Biotechnol

78(2):351–360.

Lopez-Vazquez CM, Oehmen A, Hooijmans CM, Brdjanovic D, Gijzen HJ,

Yuan ZG, van Loosdrecht MCM. 2009. Modeling the PAO-GAO

competition: Effects of carbon source, pH and temperature. Water

Res 43(2):450–462.

Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar,

Buchner A, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S,

Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T,

Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A,

Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH.

2004. ARB: A software environment for sequence data. Nucleic Acids

Res 32:1363–1371.

Macy JM, Rech S, Auling G, Dorsch M, Stackebrandt E, Sly LI. 1993.

Thauera selenatis gen. nov, sp. nov, a member of the beta-subclass of

proteobacteria with a novel type of anaerobic respiration. Int J Syst

Bacteriol 43(1):135–142.

Manz W, Amann R, Ludwig W, Wagner M, Schleifer KH. 1992.

Phylogenetic oligodeoxynucleotide probes for the major subclasses

of proteobacteria—Problems and solutions. Syst Appl Microbiol

15(4):593–600.

Moralejo-Garate H, Mar’atusalihat E, Kleerebezem R, van Loosdrecht

MCM. (submitted). Microbial community engineering for biopolymer

production from glycerol. Appl Microbiol Biotechnol.

Reis MAM, Serafim LS, Lemos PC, Ramos AM, Aguiar FR, Van Loosdrecht

MCM. 2003. Production of polyhydroxyalkanoates by mixed microbial

cultures. Bioprocess Biosyst Eng 25(6):377–385.

Schafer H, Muyzer G. 2001. Denaturing gradient gel electrophoresis in

marine microbial ecology. Method Microbiol 30(30):425–468.

Serafim LS, Lemos PC, Oliveira R, Reis MAM. 2004. Optimization of

polyhydroxybutyrate production by mixed cultures submitted to aero-

bic dynamic feeding conditions. Biotechnol Bioeng 87(2):145–160.

Shi HD, Shiraishi M, Shimizu K. 1997. Metabolic flux analysis for biosynth-

esis of poly(beta-hydroxybutyric acid) in Alcaligenes eutrophus from

various carbon sources. J Ferment Bioeng 84(6):579–587.

Silva CC, Jesus EC, Torres APR, Sousa MP, Santiago VMJ, Oliveira VM.

2010. Investigation of bacterial diversity in membrane bioreactor and

conventional activated sludge processes from petroleum refineries

using phylogenetic and statistical approaches. J Microbiol Biotechnol

20(3):447–459.

Slater SC, Voige WH, Dennis DE. 1988. Cloning and expression in

Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybu-

tyrate biosynthetic-pathway. J Bacteriol 170(10):4431–4436.

Steinbuchel A. 1991. Polyhydroxyalkanoic acids. In: Byrom D, editor.

Biomaterials: Novel materials from biological sources. New York:

Stockton, pp. 124–213.

Temudo MF, Kleerebezem R, van Loosdrecht M. 2007. Influence of the pH

on (open) mixed culture fermentation of glucose: A chemostat study.

Biotechnol Bioeng 98(1):69–79.

Temudo MF, Muyzer G, Kleerebezem R, van Loosdrecht MCM. 2008.

Diversity of microbial communities in open mixed culture fermenta-

tions: Impact of the pH and carbon source. Appl Microbiol Biotechnol

80(6):1121–1130.

Villano M, Beccari M, Dionisi D, Lampis S, Miccheli A, Vallini G, Majone

M. 2010. Effect of pH on the production of bacterial polyhydroxyalk-

anoates by mixed cultures enriched under periodic feeding. Process

Biochem 45(5):714–723.

Vishniac W, Santer M. 1957. Thiobacilli. Bacteriol Rev 21(3):195–213.



Documents

Polyhydroxybutyrate production from lactate using a mixed microbial culture