Kag

JD

a

ARR3A

KABCKPW

1

tiaumurmtcbccbsnab

1d

Biochemical Engineering Journal 49 (2010) 422–428

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journa l homepage: www.e lsev ier .com/ locate /be j

inetic analysis on the production of polyhydroxyalkanoates from volatile fattycids by Cupriavidus necator with a consideration of substrate inhibition, cellrowth, maintenance, and product formation

in Wang, Zheng-Bo Yue, Guo-Ping Sheng, Han-Qing Yu ∗

epartment of Chemistry, University of Science & Technology of China, Hefei 230026, China

r t i c l e i n f o

rticle history:eceived 11 October 2009eceived in revised form1 December 2009ccepted 9 February 2010

eywords:ctivated sludge

a b s t r a c t

Volatile fatty acids (VFAs) could be utilized by Cupriavidus necator (previously named as Ralstoniaeutropha) as its sole carbon and energy source for growth and polyhydroxyalkanoates (PHAs) synthe-sis. In such a biosynthesis, VFAs at a high concentration had an inhibition on cell growth and deterioratemetabolite production. In this work the kinetics of PHAs production and consumption of VFAs by C. necatorwith simultaneous considerations of substrate inhibition, cell growth, maintenance and product forma-tion were explored. The growth of C. necator and production of PHAs were significantly affected by theinitial VFAs concentration. The cell growth was inhibited under initial substrate-sufficient conditions,and the cell activities could be resumed with the consumption of VFAs in batch cultures. Experimen-tal results verify that the model established in this work was able to appropriately describe the PHA

iopolymers

upriavidus necatorinetics

production from VFAs by C. necator.© 2010 Elsevier B.V. All rights reserved.

olyhydroxyalkanoatesaste treatment

. Introduction

The Gram-negative, facultative bacterium Cupriavidus neca-or has been intensively investigated for almost 50 years. Todayt is one of the best studied producer of poly(3-hydroxybutyriccid) [1]. It prefers accumulating polyhydroxyalkanoates (PHAs)nder nitrogen-limited or unbalanced conditions [2]. The poly-ers are accumulated in large amounts in the cell and can be

sed for various applications ranging from replacement of fossilesource-based bulk plastics to high-value special purpose poly-ers, thus providing tremendous market potential [1,3,4]. But,

he high production costs, which are mainly associated with theosts of carbon source, prevent their wide utilization. One possi-le approach to reduce the costs is the utilization of inexpensivearbon sources for the biosynthesis of PHAs [5]. Organic wastesontaining a high carbon content, such as palm oil and starch, haveeen used for the production of PHAs [5,6]. Since the molecular

tructures of most of the organic wastes are complicated, they can-ot be transformed to PHAs directly by microorganisms. However,fter acidogenic reactions under anaerobic conditions, they cane metabolized into volatile fatty acids (VFAs) so as to produce

∗ Corresponding author. Fax: +86 551 3601592.E-mail address: hqyu@ustc.edu.cn (H.-Q. Yu).

369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2010.02.005

PHAs [7]. Therefore, one feasible solution to the costs problem isto convert organic wastes into VFAs such as acetic, propionic, andbutyric acids, and subsequently to produce the PHAs from theseVFAs.

C. necator can utilize the volatile fatty acids (acetic, propionic,and butyric acids) as its sole carbon and energy source for growthand PHA synthesis [7,8]. On the other hand, acetate and other short-chain fatty acids inhibit cell growth and deteriorate metaboliteproduction as they uncouple the trans-membrane protein potentialand thus interfere with efficient energy metabolism [9]. However,as VFAs are gradually consumed, cell activity may resume. Sub-strate limitation can also result in slow acid utilization rates at lowinitial VFAs levels. When VFAs are present at appropriate levels,the undissociated fatty acids molecules yet can enter the cyto-plasm, are activated, and metabolized into CO2, cell biomass, orPHA [8,10]. Mathematical models can be used to optimize this bio-process and describe the metabolic activities of microorganismsinvolved. Therefore, the main contribution of this work was toestablish a model for the production of PHAs from a mixture ofVFAs by C. necator. In this model, the biomass formation, main-tenance and product formation were taken into account together,

which is important for performing a kinetic analysis on substratemetabolism in a PHA-producing system. Furthermore, by combina-tion of the model and experiments, the inhibition of a high initialVFAs concentration on the growth of C. necator and production ofPHAs was evaluated.

J. Wang et al. / Biochemical Engineeri

Nomenclature

X biomass concentration (Cmmol l−1)XR residual biomass concentration (Cmmol l−1)S acid concentration (Cmmol l−1)S0 the initial acid concentration (Cmmol l−1)P PHAs concentration (Cmmol l−1)t time (h)qs the specific rate of acid utilizationYXR

/S the yield coefficient about active cell growth on sub-strate (Cmmol Cmmol−1)

YPi/S the yield coefficient about product on substrate

(Cmmol Cmmol−1)YX/S the yield coefficient about total biomass on sub-

strate (Cmmol Cmmol−1)A cell activityA0 the initial cell activityKS the saturation constant for the substrate (Cmmol

Acids l−1)KA the rate coefficient of the second-order activity

recovery(l2 Cmmol Acids−1 Cmmol RBM−1 h−1)K1 the constants related to active cell growth rateK2 the constants related to production accumulation

ratem maintenance energya the constant related to the cell growth rateb the constant related to the cell mass

Greek letters˛ the yield of product from biomass (Cmmol

PHAs Cmmol RBM−1)ˇ the specific rate of product formation (Cmmol

PHAs Cmmol RBM−1 h−1)�R the specific growth rate (Cmmol RBM Cmmol

acid−1 h−1) of XR

2

fiepc

q

wptsq

−

aisp

On the other hand, the relationship between the substrate con-

�m the maximum specific growth rate (CmmolRBM Cmmol acid−1 h−1)

. Model development

The growth of microorganisms requires carbon sources, growthactors and other nutrients. The substrate utilized by microorgan-sms can be transformed to various intracellular metabolites andnergy, which are used for biomass formation, maintenance androduct formation. Thus, the rate of substrates consumption (qs)an be described as:

s = K1�R + m + K2dP/dt

XR(1)

here K1 and K2 are the constants related to cell growth rate androduct accumulation rate, respectively; m is the energy neededo maintain cell activity; P is the concentration of PHAs; �R is thepecific growth rate of XR; qs and �R can be respectively defined ass = −(dS/dt)/XR and �R = (dXR/dt)/XR.

Rearrangement of Eq. (1) leads to:

dS

dt= K1

dXR

dt+ mXR + K2

dP

dt(2)

The biomass (X) has two components [11]: the catalytically

ctive component, XR, and the inert component in the cell, P, whichs the concentration of PHAs. The active component comprises theynthetic part of the cell, where metabolism occurs. Therefore, itlays a major role in cell growth.

ng Journal 49 (2010) 422–428 423

Since the accumulation of PHAs partially couples with cellgrowth, the accumulation rate of PHAs could be calculated as fol-lows [12]:

dP

dt= ˛

dXR

dt+ ˇXR (3)

Substitution of dP/dt into Eq. (2) with Eq. (3) results in:

−dS

dt= (K1 + K2˛)�RXR + K2ˇXR + mXR (4)

Defining a = K1 + K2˛ and b = K2ˇ + m, Eq. (4) can be rewritten asfollows:

−dS

dt= a�RXR + bXR (5)

where a is a constant related to the cell growth rate, and b is aconstant related to the cell mass.

The growth rate of XR can be modeled with the Monod equation:

�R = �mS

KS + S(6)

Substitution of Eq. (6) into Eq. (5) yields:

−dS

dt= a

�mS

KS + SXR + bXR (7)

Taking into account the inhibitory effect of VFAs, cell activity(A) is introduced here. This item was defined as the fraction of theresidual biomass (XR) with usual cell activity at a specific VFAs con-centration [10]. By integrating item A into Eq. (7), the followingequations, which describe the utilization rate and specific utiliza-tion rate of VFAs based on the measurable residual biomass (XR),respectively, can be obtained:

−dS

dt= a

�mS

KS + SXRA + bXRA (8a)

qs = a�mS

KS + SA + bA (8b)

In addition, the cell activity (A) can be resumed in batch cul-tures, attributed to the acid consumption by the active biomass. Thechanges of cell activity herewith can be described with a second-order model between the total VFAs and active cell mass [10]:

dA

dt= KAS(XRA) (9)

The relationship between the cell activity and total VFA concen-tration can be established by dividing Eq. (8a) by Eq. (9):

−dA

dS= KAS

(a�mS/(KS + S)) + b(10)

Thus,

A = A0 + KAKS

a�m + b(S − S0) − abKAK2

S �m

(a�m + b)3ln

bKS + (a�m + b)SbKS + (a�m + b)S0

+ KA

2(a�m + b)2((a�m + b)(S + S0) − 2bKS)(S − S0) (11)

The model established above, as shown in Eqs. (8a) and (11),indicates that the specific consumption rate of VFAs is a functionof VFAs concentration (S), in addition to the residual biomass (XR)and cell growth rate (�m).

sumption rate and cell growth is described as following:

−dS

dt= �RXR

YXR/S(12)

4 ineering Journal 49 (2010) 422–428

e

aaKb

3

3

mbw(tt0c

1tMTcwr

wsrwVh

3

ttTaR

mC4RahwCP

4

4

tc

24 J. Wang et al. / Biochemical Eng

Substitution of Eq. (12) into Eq. (5) leads to a following linearquation:

1�R

= 1b

1YXR/S

− a

b(13)

The kinetic constants a and b can be estimated from a regressionnalysis between the reciprocal of cell growth rate constant (�R)nd reciprocal of cell yields coefficient (YXR

/S). The values of A0, �m,A and KS can also be estimated by a non-linear regression modelased on Eqs. (8a), (8b) and (11).

. Materials and methods

.1. Strain cultivation

C. necator ATCC 17699 was maintained in agar slants of aedium containing 10 g l−1 yeast extract, 10 g l−1 peptone, 5 g l−1

eef extract, and 5 g l−1 (NH4)2SO4. The same medium without agaras used for seed culture, which was cultivated in 500-ml flasks

100 ml medium in each) on a rotary shaker with 180 rpm. Theemperature was kept at 30 ◦C for 24 h. The cells were harvestedhrough centrifugation at 10,000 × rpm for 10 min, washed with.8% NaCl solution and then re-suspended in a mineral solutionontaining VFAs with a predetermined concentration.

The composition of the mineral solution is: 1.2 g l−1 MgSO4,.7 g l−1 citrate, 13.3 g l−1 KH2PO4, and 10 ml trace element solu-ion [(g l−1): FeSO4·7H2O 10, ZnSO4·7H2O 2.25, CuSO4·5H2O 2.25,

nSO4·5H2O 0.5, CaCl2·2H2O 2, H3BO4 0.3, and (NH4)6Mo7O24 0.1].he medium with a C/N molar ratio of 60 contained various VFAsoncentrations. A mixture of acetic, butyric, and propionic acidsas used as the carbon source in this work. Their concentration

anged from 1 to 9 g l−1 for each individual acid.All the experiments were conducted in a biofermentor with a

orking capacity of 3 l at 30 ◦C with an initial pH of 7.0. The dis-olved oxygen (DO) concentration was controlled using an aerationate of 4.5 l min−1 and a stirring speed rate of 400 rpm. Samplesere taken at each 4-h intervals to measure the concentrations ofFAs, dry cell weight (DCW), residual biomass (RBM), PHAs andydroxyvalerate (HV) in the copolymers.

.2. Analyses and calculation

After centrifugation of the samples at 3000 × rpm for 10 min,he supernatant was taken for the determination of VFAs, andhe sediment was used for the measurement of DCW and PHAs.he concentration of VFAs, PHAs, and ammonium was measuredccording to Wang and Yu [13] as well as the values of DCW andBM.

The biomass formula used in this study is based on a for-ula of C. necator ATCC 17697 reported previously [14]:

4H6.9O1.64N0.98, hence the carbon content in biomass is× 12/(4 × 12 + 1 × 6.9 + 16 × 1.64 + 14 × 0.98) = 0.506 g-carbon/g-BM. As for PHAs, PHB has a formula of C4H6O2 (MW = 86 g/mol),nd its C content = 48/86 = mol/g = 0.558 g-carbon/g-PHB; PHBVas a formula of C4H6O2–C5H8O2 (MW = (1 − ˛)86 + ˛ × 100),here ˛ is the weight content of PHV in the copolymer. The PHBVcontent = [48 × (1 − ˛) + 60 × ˛]/[(1 − ˛)86 + ˛ × 100] g-carbon/g-

HBV.

. Results

.1. Overall performance in a typical batch test

When C. necator was cultured in a mineral VFAs-containing solu-ion, a homopolymer of PHB was produced from the acids of evenarbon numbers, whereas a copolymer of 3-hydroxybutyrate (HB)

Fig. 1. Time courses of substrates and products in a typical batch culture [acetic acid(Ac):propionic acid (Pr):butyric acid (Bu) = 11 Cmmol:9 Cmmol:4 Cmmol].

and HV units was produced from acids of odd carbon numbers [15].In the presence of propionic acid, PHAs were a copolymer of HBand HV units. Fig. 1 shows the time courses of VFAs, NH4-N, DCW,RBM, and PHAs in a typical batch test using a mixture of VFAs as asubstrate. The butyric acid was rapidly utilized in the initial 12-hcultivation, resulting in a slight increase in PHB concentration. Sub-sequently, the co-consumption of propionic acids and acetic acidsled to a significant increase in PHV concentration, which peaked athour 32. From hour 28, since nitrogen was exhausted and only lit-tle propionic acids remained in the culture, the residual acetic acidwas mainly used for PHB production. Therefore, the PHB concentra-tion significantly increased in the subsequent cultivation. However,the PHAs concentration dropped in the last 4-h cultivation becauseof the exhaustion of substrate. This indicates that the cells couldutilize the intracellular accumulated PHAs as an energy source tomaintain their bioactivity. As shown in Fig. 1, the PHAs accumula-tion seemed to partially couple with cell growth. The increase inDCW was mainly associated with the increase in PHAs concentra-tion, while the residual cell concentration increased slowly underthe nitrogen-limited conditions. This result was in good agreementwith previous report [7], in which a mixture of fatty acids from theanaerobic digestion of food wastes was used as a carbon and energysource.

4.2. Kinetics of VFAs consumption and PHAs production

Fig. 2 illustrates the specific consumption rates of VFAs and spe-cific formation rates of PHAs. As shown in Fig. 2(a), the maximumutilization rate of butyric acid of 0.042 h−1 was achieved at hour8, while the corresponding value was 0.053 h−1 for propionic acidat hour 20, and 0.042 h−1 for acetic acid at hour 24, respectively.This indicates that butyric acid had a higher initial consumptionrate than acetic and propionic acids. As shown in Fig. 2(b), there

were two peaks for the specific formation rate of PHB, while onlyone was observed for the specific formation rate of PHV in the cul-tivation. The first significant increase in PHB was caused by theconsumption of butyric acid, and the rapid increase in PHV wasresulted from the co-consumption of propionic and acetic acids.

J. Wang et al. / Biochemical Engineering Journal 49 (2010) 422–428 425

FP

AdgcraiiapabaPi

4

(lf

TP

Fig. 3. Pathways of PHBV synthesis in C. necator from acetic acid, propionic acid,and butyric acids [4,5].

ig. 2. (a) Specific consumption rates of VFAs and (b) specific production rates ofHAs in a typical batch culture (Ac:Pr:Bu = 11 Cmmol:9 Cmmol:4 Cmmol).

fter peaking, the specific PHV formation rate decreased with aecrease in the specific consumption rate of propionic acid, sug-esting that the biosynthesis of PHV was largely related to theonsumption of propionic acid. Later, the specific PHB productionate re-increased because of the rapid consumption of the residualcetic acid in the medium. Thus, more PHB units were incorporatednto PHAs, which was responsible for the decrease in PHV contentn PHAs from 54.2% to 44.1%. The low content of PHV might bettributed to the substrate specificity of PHB synthase, or the com-lexity of metabolism pathway of fatty acids. This might also bessociated with the utilization of acetic and propionic acids for theiomass growth, or the regeneration of reducing equivalents suchs NADH and NADPH (Fig. 3). The reasons for the low content ofHV units in PHAs at the beginning of cultivation warrant a furthernvestigation.

.3. Model verification

A set of the data in the present work (Case 1) and data from [10]Case 2) were employed for the verification of the model estab-ished in this study. The experimental conditions and parametersor the two cases calculated with Eqs. (8a), (8b) and (11) are sum-

able 1arameter comparison in the two studies.

Parameters Case 1 Case 2

S0 (Cmmol l−1) 230 400X0 (Cmmol l−1) 93 73Ac:Pr:Bu (Cmmol:Cmmol:Cmmol) 11:9:4 20:15:4�m (Cmmol Acids Cmmol RBM−1 h−1) 0.24 0.40KA (l2 Cmmol Acids−1 Cmmol RBM−1 h−1) 2.29E−6 7.93E−6KS (Cmmol l−1) 84 210A0 0.11 0.09m (Cmmol Acids Cmmol RBM−1 h−1) 0.0036 0.0040

Fig. 4. Effect of total acid carbon concentration on the utilization rate of total VFAs(Ac:Pr:Bu = 11 Cmmol:9 Cmmol:4 Cmmol).

marized in Table 1. Figs. 4 and 5 show the measured and simulatedresults of Case 1 and Case 2, respectively.

The higher value of KS in Case 2 was likely to be attributed tothe higher VFAs concentration, compared with Case 1. Furthermore,the �m in Case 2 was slightly higher than that in Case 1. This mightbe associated with the higher S0/XR0 value as shown in Table 1. Thedifferent initial cell activities should be related with the differentVFAs compositions.

5. Discussion

As shown in Figs. 1 and 2, a copolymer of HB and HV unitscould be produced using acetic, propionic, and butyric acids. More-

over, monomer HB and HV content varied with the consumptionof different acids. Rapid consumption of propionic acid led to anincrease in HV content, while HB gradually increased because of theconsumption of acetic acid. These results indicate that initial VFAcomposition and dosage affected the PHA composition greatly.

426 J. Wang et al. / Biochemical Engineeri

F(

wtePbgopaWtaofvtftepIbmmpttaho

stibtupnit[iw

ig. 5. Effect of total acid carbon concentration on the utilization rate of total VFAsAc:Pr:Bu = 25 Cmmol:15 Cmmol:4 Cmmol) (data from [10]).

To further enhance productivity and to allow tailor-made PHAsith different monomer compositions by metabolic engineering,

he knowledge of metabolic pathways and of the biochemical prop-rties of the enzymes involved is essential. In native producers,HA bioplastics are produced intracellularly as storage granulesy a number of enzyme pathways, encoded by distinct sets ofenes, which convert cellular intermediates such as acetyl-CoAr intermediates of fatty acid pathways found in all cell types toolymer [3]. The experimental results were interpreted using thenabolism pathways of the three acids as shown in Fig. 3 [4,5].hen the precursor substrate for PHBV biosynthesis is an acid mix-

ure, e.g., acetic, propionic and butyric acids used in this work, thenabolism for PHB unit is relatively complicated. Acetoacetyl-CoA isbtained from the acids through their respective pathways (Fig. 3)or the synthesis of 3HB unit. As for acetic acid, it is initially con-erted to acetyl-CoA. Two acetyl-CoA moieties are then condensedo acetoacetyl-CoA. 3HB units can also be directly synthesizedrom butyric acid via acetoacetyl-CoA without prior degradationo acetyl-CoA [16]. When propionic acid is present in the medium,limination of the carbonyl carbon of propionyl-CoA always takeslace and results in the synthesis of both 3HB and 3HV units.

n addition, propionic acid is converted to 3HV, in addition toeing catabolized to pyruvic acid or succinyl-CoA, which are inter-ediates of the central metabolism [17]. Butyric acid needs lessetabolic steps for the synthesis of the macromolecules than pro-

ionic or acetic acids (Fig. 3). This might be due to the fact thathe carbon skeleton of butyric acid is similar to 3-hydroxybutyrate,he precursor of PHB. Butyric acid is a good carbon source for PHBccumulation, and a butyric acid-containing culture would lead to aigher polymer yield by C. necator. Therefore, the consumption ratef VFAs was also affected by the characteristics of carbon sources.

Microbial growth inhibition can be categorized into two types:ubstrate-limited and substrate-sufficient. The former is limited byhe insufficient supply of the carbon source, whereas the latters restrained by the lack of other nutrients or by product inhi-ition [18]. Furthermore, the substrate consumption rate underhe substrate-sufficient conditions is significantly higher than thatnder the substrate-deficient conditions [19]. However, in theresent work the substrate consumption rate by C. necator wasot high under both conditions. Since the C/N molar ratio in this

nitial culture was high and C. necator preferred the accumula-

ion of PHAs under the nitrogen-limited or unbalanced conditions2,7,18], the culture used in this work seemed to be categorizednto the substrate-sufficient case at the beginning of cultivation,

hereas the culture was shifted to the substrate-deficient type

ng Journal 49 (2010) 422–428

at the end of cultivation, attributed to the low VFAs concentra-tion (Figs. 4 and 5). The low specific VFAs consumption rate underthe substrate-sufficient conditions indicated the inhibitory effectof VFAs under the acid-sufficient conditions.

The consumption rate of energy and carbon sources is governedby the rates at which the extracellular substrate enters cell andis subsequently metabolized [20]. C. necator can metabolize sev-eral VFAs as additional carbon sources under nitrogen deficiency,which could be toxic depending on their concentration and pH. AtpH levels below the pKa’s for VFAs, the undissociated form pre-dominates and could readily cross the cell membrane. Inside cellsthey rapidly become dissociated and acidify the cytoplasm. A highlevel of VFA could reduce the proton gradient across the membrane,increase osmotic pressure, and reduce acid utilization rate, growthrate, and microbial yield. Moreover, VFAs would be in the dissoci-ated form in the medium for C. necator at pH levels closer to theoptimum. While the anions would not be transported readily, onceinside the cell they would not cause an adverse effect of the undis-sociated form [8]. VFAs concentration is also an important factorat more neutral pH levels, as reported by others [8,21,22]. Thus,VFAs could be effective carbon sources only when pH and VFA con-centration were carefully regulated [8]. For the two cases above(Figs. 4 and 5), the substrate utilization rates were initially low,suggesting the inhibitory effect of VFAs on the total acid utiliza-tion. This might be caused by the accumulated undissociated freeacids in cytoplasm. With a decrease in acid concentration, both cellactivity (A) and specific rate (qs) recovered gradually.

The cell activity could be estimated by substituting the calcu-lated parameter into Eq. (11). As shown in Fig. 6(a), the cell activityresumed gradually with the consumption of VFAs, regardless of thelow qs near the end of cultivation. This can be confirmed by theresults in Fig. 6(b) and (c), which clearly show that both yields ofproducts and total biomass increased gradually with the increas-ing cell activity. Most physiological maintenance requirements, i.e.,energy associated with osmoregulation, cell motility, proofreadingand internal turnover of macromolecular compounds, and defensemechanisms, are related to cell activity [23]. In this work, the main-tenance energy coefficient in Case 1 was 0.0036, slightly lowerthan 0.0040 in Case 2. On the other hand, the initial cell activityin Case 1 was 0.11 slightly higher than 0.09 in Case 2. This indi-cates that the initial VFAs concentration had an effect on the cellactivity and maintenance energy required (Table 1). Furthermore,mixed microorganisms that contained PHA-producers had similarcoefficients of 0.0027 and 0.0035 by using propionate and acetateas substrates, respectively [24]. The different maintenance coef-ficients were attributed to their maintenance energies when thebacteria grew on different VFAs [25].

Since the consumed VFAs are mainly used for cell growth, thePHAs production and maintenance, the correlation between prod-uct i and substrate could thus be expressed as follows based on theconservation of mass [26]:

dPi

dt= −YPi

dS

dt(14)

dPi = −YPidS

∫ Pi

0

dPi = −YPi

∫ S

S0

dS

Pi = YPi(S0 − S) (15)

where YPiis the yield of product i. Solution to Eq. (15) is plotted in

Fig. 6(b).

J. Wang et al. / Biochemical Engineeri

Foc

r

Y

Y

t

Y

tttblV

[

[

[

[

[

ig. 6. (a) Effects of total acid carbon concentration on the cell activity A; (b) effectsf substrate consumption on the production of PHAs; and (c) time courses of yieldoefficient (X = XR + PHAs) in the cultivation.

Rearrangements of Eqs. (12) and (14) lead to Eqs. (16) and (17),espectively:

XR/S = −�R

qS= dXR/dt

dS/dt(16)

Pi= −dPi/dt

dS/dt(17)

Defining YX/S = YXR/S + YP (PHAs), the yield coefficient for theotal biomass can be given by:

X/S = −dX/dt

dS/dt(18)

Fig. 6(c) shows the changes of yield coefficient (YX/S) duringhe cultivation. As illustrated in Fig. 6, with the VFAs consump-

ion, the inhibitory effect of a high initial VFAs concentration onhe cell activity was relieved and the yield of products and totaliomass increased gradually. Hence, the cell growth and intracel-

ular PHAs accumulation might be triggered by the relatively highFAs concentrations.

[

[

ng Journal 49 (2010) 422–428 427

6. Conclusions

In this work the kinetics of PHAs production and consumptionof VFAs by C. necator are explored, and the following results aredrawn:

• A model was established to describe the production kineticsof PHAs from VFAs by taking into account substrate inhibition,cell growth, maintenance, and product formation simultane-ously. Furthermore, experimental results in this work andliterature both verify that the model established was able toappropriately describe the PHAs production from VFAs by C. neca-tor.

• The growth of C. necator and production of PHAs were sig-nificantly affected by the VFAs concentration. The cell growthwas inhibited under initial VFAs-sufficient conditions. InitialVFAs concentrations had an effect on the cell activity andmaintenance energy required. VFAs could be effective carbonsources only when VFAs concentration was carefully regu-lated.

Acknowledgements

The authors wish to thank the Natural Science Foundation ofChina (50708106 and 50738006), and the Tai-Lake Water SpecificProgram of Jiangsu Province (BS2007098) for the support of thisstudy.

References

[1] F. Reinecke, A. Steinbuchel, Ralstonia eutropha strain H16 as model organism forPHA metabolism and for biotechnological production of technically interestingbiopolymers, J. Mol. Microbiol. Biotechnol. 16 (2009) 91–108.

[2] J. Wang, F. Fang, H.Q. Yu, Substrate consumption and biomass growth of Ral-stonia eutropha at various S0/X0 levels in batch cultures, Bioresour. Technol. 98(2007) 2599–2604.

[3] D.S. Kristi, P.P. Oliver, PHA bioplastic: a value-added coproduct for biomassbiorefineries, Biofuels Bioprod. Bioref. 3 (2009) 456–467.

[4] A. Steinbüchel, T. Lütke-Eversloh, Metabolic engineering and pathway con-struction for biotechnological production of relevant polyhydroxyalkanoatesin microorganisms, Biochem. Eng. J. 16 (2003) 81–96.

[5] A.S. Aldor, J.D. Keasling, Process design for microbial plastic factories: metabolicengineering of polyhydroxyalkanoates, Curr. Opin. Biotechnol. 14 (2003)475–483.

[6] J.M.B.T. Cavalheiro, M.C.M.D. de Almeida, C. Grandfils, M.M.R. da Fonsecab,Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glyc-erol, Process Biochem. 44 (2009) 509–515.

[7] G.C. Du, J. Yu, Green technology for conversion of food scraps to biodegrad-able thermoplastic polyhydroxyalkanoates, Environ. Sci. Technol. 36 (2002)5511–5516.

[8] P. Chakraborty, W. Gibbons, K. Muthukumarappan, Conversion of volatile fattyacids into polyhydroxyalkanoate by Ralstonia eutropha, J. Appl. Microbiol. 106(2009) 1996–2005.

[9] K. Shimizu, Toward systematic metabolic engineering based on the analysisof metabolic regulation by the integration of different levels of information,Biochem. Eng. J. 46 (2009) 235–251.

10] J. Yu, Y.T. Si, W.K.R. Wong, Kinetics modeling of inhibition and utilization ofmixed volatile fatty acids in the formation of polyhydroxyalkanoates by Ral-stonia eutropha, Process Biochem. 37 (2002) 731–738.

11] P. Patwardhan, A.K. Srivastava, Fed-batch cultivation of Wautersia eutropha,Bioresour. Technol. 99 (2008) 1787–1792.

12] S. Khanna, A.K. Srivastava, A simple structured mathematical model forbiopolymer (PHB) production, Biotechnol. Prog. 21 (2005) 830–838.

13] J. Wang, H.Q. Yu, Biosynthesis of polyhydroxybutyrate (PHB) and extracellularpolymeric substances (EPS) by Ralstonia eutropha ATCC 17699 in batch cultures,Appl. Microbiol. Biotechnol. 75 (2007) 871–878.

14] T. Katoh, D. Yuguchi, H. Yoshii, H.D. Shi, K. Shimizu, Dynamics and modelingon fermentative production of poly(ˇ-hydroxybutyric acid) from sugars vialactate by a mixed culture of Lactobacillus delbrueckii and Alcaligenes eutrophus,J. Biotechnol. 67 (1999) 113–134.

15] D. Dennis, M. McCoy, A. Stangl, H.E. Valentin, Z. Wu, Formation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by PHA synthase from Ralstoniaeutropha, J. Biotechnol. 64 (1998) 177–186.

16] Y. Doi, A. Tamaki, M. Kunioka, Production of copolyesters of 3-hydroxybutyrateand 3-hydroxyvalerate by Alcaligenes eutrophus from butyric and pentanoicacids, Appl. Microbiol. Biotechnol. 28 (1988) 330–334.

4 ineeri

[

[

[

[

[

[

[

[

2778.

28 J. Wang et al. / Biochemical Eng

17] X.W. Liu, H.H. Wang, J.Y. Chen, X.T. Li, G.Q. Chen, Biosynthesis ofpoly(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichiacoli harboring propionyl-CoA synthase gene (prpE) or propionate permeasegene (prpP), Biochem. Eng. J. 43 (2009) 72–77.

18] S. Muller, T. Bley, W. Babel, Adaptive responses of Ralstonia eutropha to feastand famine conditions analysed by flow cytometry, J. Biotechnol. 75 (1999)81–97.

19] Y. Liu, G.H. Chen, J.L. Rols, A kinetic model incorporating energy spilling for sub-strate removal in substrate-sufficient batch culture of activated sludge, Appl.

Microbiol. Biotechnol. 52 (1999) 647–651.

20] G. Gottschalk, Bacterial Metabolism, 2nd ed., Springer Press, New York, NY,1986.

21] Y.J. Chung, H.J. Cha, J.S. Yeo, Y.J. Yoo, Production of poly(3-hydroxybutyric-co-3-hydroxyvaleric) acid using propionic acid by pH regulation, J. Ferment. Bioeng.83 (1997) 492–495.

[

[

ng Journal 49 (2010) 422–428

22] J.H. Kim, B.G. Kim, C.Y. Choi, Effect of propionic acid on poly(ˇ-hydroxybutyric-co-hydroxyvaleric acid) production by Alcaligenes eutrophus, Biotechnol. Lett.14 (2005) 903–906.

23] P. Van Bodegom, Microbial maintenance: a critical review on its quantification,Microb. Ecol. 53 (2007) 513–523.

24] A. Oehmen, R.J. Zeng, A.M. Saunders, L.L. Blackall, J. Keller, Z.G. Yuan, Anaer-obic and aerobic metabolism of glycogen accumulating organisms selectedwith propionate as the sole carbon source, Microbiology 152 (2006) 2767–

25] H.D. Shi, M. Shiraishi, K. Shimizu, Metabolic flux analysis for biosynthesisof poly(ˇ-hydroxybutyric acid) in Alcaligenes eutrophus from various carbonsources, J. Ferment. Bioeng. 84 (1997) 579–587.

26] Y. Mu, G. Wang, H.Q. Yu, Kinetic modeling of batch hydrogen production processby mixed anaerobic cultures, Bioresour. Technol. 97 (2006) 1302–1307.