Process Biochemistry 37 (2002) 731 738 www.elsevier.

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Kinetics modeling of inhibition and utilization of mixed volatile fatty acids in the formation of polyhydroxyalkanoates by Ralstonia eutropha
Jian Yu a,*, Yingtao Si b, Wan Keung R. Wong c
b

Department of Ocean and Resources Engineering, Uni6ersity of Hawaii at Manoa, Honolulu, H196822, USA Department of Chemical Engineering, Hong Kong Uni6ersity of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Peoples Republic of China c Department of Biochemistry, Hong Kong Uni6ersity of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Peoples Republic of China Received 18 May 2001; accepted 21 July 2001

Abstract Acetic, propionic and butyric acids are the major fermentation acids produced on acidogenesis of organic wastes such as food scraps. They can be utilized by Ralstonia eutropha as sole carbon sources for cell growth and polyhydroxyalkanoate (PHA) synthesis. The acids, however, are inhibitory and toxic to the bacterium depending on the medium pH and the total acid concentration. The inhibition and utilization kinetics of the total acids and individual acids in mixed acid media were investigated in ask batch cultures, and simulated by modied Michaelis Menten models based on variable cell activity. Cell activity, a function of the total acid concentration, is dened as the active fraction of the measurable residual biomass (RBM) under specic culture conditions, which might be as low as 20% at high acid concentrations. The overall production rate of PHA is contributed from the utilization rates of individual acids as well as the interaction of two acids such as acetate and propionate, depending on the predominant acids in the medium. R. eutropha preferred propionic acid for cell mass synthesis and butyric acid for polymer synthesis, the latter giving the highest polymer yield (0.39 g/g) among the three acids. In mixtures of predominant acetic and butyric acids, a high PHA formation rate (60 mg/g RBM per h) was achieved, compared with the relatively low PHA formation rate (35 mg/g RBM per h) in the mixtures of predominant acetic and propionic acids. Acetic acid in acid mixtures reduced the metabolism of propionic acid, which resulted in a high rate ratio (up to 0.8) of hydroxyvalerate (HV) formation to the overall PHA formation. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Polyhydroxyalkanotes; PHAs; Biodegradable polymers; Volatile fatty acids; Acid inhibition modeling; R. eutropha

Nomenclature A k kA K Ki r S t cell activity () rate coefcient of Michaelis Menten model (mmol/g RBM per h) rate coefcient of cell activity recovery (l2/g RBM per mmol per h) half rate concentration of Michaelis Menten model (mmol/l) inhibition coefcient of Michaelis Menten model (l/mmol) Specic rate of acid utilization and polymer production based on RBM (mmol/g RBM per h or mg/g RBM per h) acid concentration (mmol/l) time (h)

* Corresponding author. Tel.: + 808-956-8110; fax: + 808-956-3498. E-mail address: jianyu@oc.soest.hawaii.edu (J. Yu). 0032-9592/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 6 4 - 3

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X Y Subscripts a b 0 p t

residual biomass concentration (g RBM per l) PHA yield (g PHA per g acid)

acetic acid butyric acid initial condition of batch culture propionic acid total organic acid

1. Introduction Polyhydroxyalkanoates (PHAs), a family of bacterial polyesters, are formed and accumulated by various bacterial species under unbalanced growth conditions. PHAs have thermomechanical properties similar to synthetic polymers such as polypropylene, but are truly biodegradable in the environment [1]. The major barrier to their wide acceptance is the high cost, particularly the costs of carbonaceous raw materials (\ 40%) and polymer recovery (\ 26%) [2]. Innovative processes have been investigated to produce PHAs from the organic matters in wastewater [3,4], in industrial waste [5,6] and in municipal waste [7,8]. Producing PHA from organic wastes may provide multiple benets to the environment and sustainable development. Ralstonia eutropha (formerly Alcaligenes eutrophus) is the most extensively studied bacterium in both basic and applied research on the formation of PHAs. This species can accumulate PHAs up to 80% (wt.) of dry cell mass using various carbon sources including carbohydrates, alcohols and organic acids [9]. Organic wastes such as food scraps usually have a complicated composition and form that cannot be directly utilized by PHA-producers such as R. eutropha. Hydrolysis and acidogenesis of the raw wastes are often the rst step to produce organic acids including acetic, propionic and butyric acids that can be utilized by PHA-producers for PHA synthesis [10,11]. Depending on the organic loading rate, the acidogenic microbial population may produce an acid solution of predominant acetic and propionic acids or a solution of predominant acetic and butyric acids [12]. The fermentation acids can be utilized by R. eutrophus for growth and PHA synthesis, but are also toxic or inhibitory to the bacterium depending on pH and acid concentration. The toxicity of the volatile fatty acids is attributed to their undissociated lipophilic molecules that penetrate freely the cell membrane, dissociate and acidify the cytoplasm [13]. As a result, the gradient of protons through the membrane cannot be maintained, and the production

of energy and the transport system dependent on this gradient are decoupled [14]. The dissociation also induces an anion accumulation, resulting in increased internal osmotic pressure of cells [15]. In response to the accumulation of fatty acids, microorganisms release free energy via ATPase and expel protons out of cells in order to maintain the proton gradient. This results in an overall decline of microbial activity including acid utilization rate, growth rate and yield [16]. Studies have been conducted on utilization of individual acids for PHA production such as acetic, propionic or butyric acid [17 19], but little is known of the utilization of mixed acids such as the kinetics at inhibitory high acid concentrations, the utilization of individual acid in a mixed acid medium, the interactions among the acids and the preference of R. eutropha for different acids for synthesis of polymer and residual biomass. In this study, kinetic models are established and used to analyze the complicated phenomena of inhibition, utilization and interaction of three major volatile fatty acids (acetic, propionic and butyric acids) and formation of PHAs by R. eutropha. The models not only help understand the interrelated biochemical phenomena, but also design a novel bioprocess for PHA production from organic waste digestion.

2. Materials and methods

2.1. Media and cell culti6ation

R. eutropha (A. eutrophus ATCC 17699) was maintained on agar slants containing per l: 10 g fructose, 10 g yeast extract, 10 g peptone, 10 g beef extract and 15 g agar. The bacterium was cultivated in a nutrient-rich medium containing (per l) 5 g yeast extract, 5 g peptone, 2.5 g (NH4)2SO4 and 2.5 g beef extract at 180 rpm, 30 C for 24 h. The cell mass was harvested by centrifugation at 10 000 g for 10 min, washed with 0.8% NaCl solution and re-suspended at predetermined turbidity in a mineral solution containing (per l) 3.8 g Na2HPO4, 2.65 g

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KH2PO4, 0.4 g MgSO4 7H2O and 1 ml trace element solution. The ingredients of the trace solution included (in mg/l), 200 Fe(NH4)SO4, 5 ZnSO4 7H2O, 5 MnCl2 4H2O, 2 CuSO4 5H2O, 2 NaB4O2 10H2O and 2 NaMoO4 2H2O. The medium with a C/N mass ratio around 80 contained the same amount of acetic acid ( 100 mmol/l), but different propionic acid (060 mmol/l) and/or butyric acid (040 mmol/ l). The initial acid concentrations were determined after sterilization at 120 C for 15 min. For one mixed acid composition, 13 500-ml conical asks (100 ml medium in each) were set up and shaken at 200 rpm and 30 C. Flasks were taken at 4-h intervals to measure the concentrations of organic acids, dry cell mass (DCM), RBM, PHA and the content of hydroxyvalerate (HV) in the copolymers. The medium pH was around 7.5 at the beginning and gradually increased to 8.5 during cultivation, which minimized the toxic effect of low pH.

3. Results and discussion

3.1. Time courses and rate calculation

Fig. 1 shows the time courses of the organic acids, DCM and PHA in a typical batch culture of acid mixture. In the presence of propionic acid, PHA was a co-polymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and the concentration of HV was also monitored as shown in Fig. 1. In a binary acid mixture of acetic and butyric acid, the homopolmer polyhydroxybutyrate (PHB) was produced. The experimental data points were rst tted with the polynomial curves (0.96B R 2 B 0.98) as shown in Fig. 1. The curve derivates were further used to calculate the specic rates based on RBM. The rates in the rst 4 h and near the zero acid concentrations are not included in the following modeling to control the curve tting error at the boundary points. This data-processing is also based on the fact that little acid were consumed at the beginning and end of the batch cultures.

2.2. Measurements
Solution turbidity was measured with a spectrophotometer at 620 nm. The composition of the organic acids was determined by gas chromatography (Hewlett Packard 5890II) equipped with a Nukol fused silica capillary column (f0.25 mm 30 m, Supelco). The aqueous samples were acidied to pH 2 with a phosphoric acid solution and ltered through a 0.45-mm cellulose nitrate membrane. The acids were eluted with helium at 10 ml/min while the temperature arose from initial 120 to 190 C at 10 C/min. DCM of R. eutropha was determined after freeze-drying and its PHA content was determined after chloroform extraction. About 0.1 g DCM was extracted in 8 ml chloroform in a tightly sealed glass tube at 60 C for 24 h. The residual DCM was dened as RBM and removed with glass ber lter. The polymer mass was determined after the solvent was completely vaporized at room temperature. The extraction and recovery efciency for polymer measurement was greater than 96%. The content of HV of PHA copolymers was determined with a modied GC method [20]. About 100 mg lyophilized biomass was mixed with 2 ml chloroform and 2 ml acidied methyl alcohol, and allowed for 4 h at 80 C. Two milliliters water was added to the cooled solution and mixed completely. The samples were analyzed with a GC equipped with a HP5 column under a temperature program: initially maintained at 60 C for 3 min., then increased to 200 C at 10 C/min, and kept at 200 C for 3 min. The relative amount of the methyl esters of hydroxybutyric and hydroxyvaleric acids was determined with the method developed by Comeau and coworkers [21]. Standard PHB was purchased from Aldrich (Milwaukee, USA).

Fig. 1. The experimental data points and polynomial tting curves of the time courses in a typical batch culture of triple acid medium. (Top) The concentrations of acetic, propionic and butyric acids. (Bottom) The concentrations of DCM, RBM, PHA and HV. The polynomial tting curves are used to calculate the specic rates based on RBM.

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or

dSt k tS t (X A) = dt Kt + St dSt kS = t t A Xdt Kt + St

(1a)

rt =

(1b)

where St refers to the total acid concentration (mmol/l); kt (mmol/g RBM per h) and Kt (mmol/l) are the two model parameters for total acid utilization. X is the measurable RBM by deducting PHA mass from DCM (g RBM per l) and XA is the active biomass under acid inhibition. The cell activity (A) was recovered with time in batch cultures due to acid consumption by the active biomass, and its recovery can be described with a second-order rate model (Eq. (2)) between the total acids and the active cell mass. dA =kASt(XA) dt
Fig. 2. The effect of total acid mole concentration on the utilization rate of total acid including acetic acid (Ac), propionic acid (Pr) and butyric acid (Bu) with 4 mol compositions.

(2)

3.2. Total acid utilization and inhibition modeling

The three volatile fatty acids (acetic, propionic and butyric acids) have almost the same pKa (4.75 4.87) and dissociate to the same extent in the cytoplasm after the undissociated acid molecules penetrate into the cells, which caused the same inhibitory or toxic effect on the cell activity. Fig. 2 shows the inhibitory effect of total acids on the overall utilization rate at high acid molar concentrations. Depending on the initial amount of seed cell mass in each batch culture, which might affect the tolerance of cells to the organic acids as found previously [17], the trends were very similar even though the compositions of propionic and butyric acids in the acid mixtures were quite different. This fact implies that the overall inhibition of total acid utilization was caused by the dissociation of free acids in cytoplasm regardless of the molecule size of the volatile fatty acids. In the batch cultures, the initial high acid concentrations depressed cell activity and hence reduced the acid utilization rate. The cell activity, however, recovered gradually with decline in acid concentration due to acid consumption. At the low acid concentrations, the utilization rate declined again because of the limitation of acid substrates. The cell activity (A) at a specic total acid concentration is dened as the fraction of the residual biomass, which has the normal cell activity. By assuming a Michaelis Menten type model with a variable cell activity (A), Eqs. (1a) and (1b) give the decline rate of the total acid concentration and the specic utilization rate of total acid based on the measurable residual biomass (X).

where kA (l2/g RBM per mmol per h) is the rate coefcient of the second-order activity recovery. The relationship between the cell activity and total acid concentration is established by dividing Eq. (1a) by Eq. (2). or A=A0 + dA kA = (K + St) dSt kt t kA 1 Kt (St0 St)+ (St0 S 2) t 2 kt (3)

(4)

where A0 is the cell activity at the initial total acid concentration and may have different values in each batch culture. St0 and St are the total acid concentrations at the beginning and a time of a batch culture (mmol/l). Combining Eqs. (1b) and (4) gives the specic rate (Eq. (5)) of total acid utilization based on the measurable residual biomass (X) rt = ktSt k 1 A0 + A Kt(St0 St)+ (S 2 S 2) t Kt + St kt 2 t0

n"

(5)

This modied MichaelisMenten rate model describes the two effects of acid concentration on acid utilization rate, the substrate effect and the toxicity effect. The latter increased with acid consumption in a batch culture from the minimum value A0. If the initial acid concentration was so high that A0 became zero, the complete inhibition of cell activity by volatile fatty acids could be predicted. This phenomenon has been observed and reported in our previous report [17]. A non-linear MATLAB optimization program was used to t the data with Eq. (5) and estimate the values of Kt, kt and kA. The parameters were further used in the following sections for individual acid utilization in various mixed acid solutions. In addition to the experimen-

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tal data points, Fig. 2 also shows the trend curves given by Eq. (5) with the parameter values: Kt =26.4 mmol/l; kt = 7.25 (mmol/g RBM per h); and kA =1.83 10 4 (l/mol per h). A0 was about 0.21, implying that the activity of RBM was reduced by 80% at the initial total acid concentration around 150 mmol/l.

3.3. Utilization of indi6idual acids

Partially inhibited by the total acid in the medium, the cells could utilize the individual acids at retarded rates in different metabolic pathways. Acetic acid is the major fermentation acid from acidogenesis of organic matters by mixed microbial populations and has a quite consistent concentration. Propionic acid or butyric acid, however, may be the second complementary acid depending on the organic loading rate, microbial population, and acidogenic reactor design and operation [12]. Fig. 3 shows the specic rate of acetate utilization in mixed acid media with the similar initial acetate concentration but different composition of propionic and butyric acids. By using a modied Michaelis Menten type model with variable activity of acetate utilization, Eqs. (6a) and (6b) give the rate of acetate utilization in a mixed acid solution, ra = or ra = kaSa A Ka + Sa a kaSa k 1 A + A K (S St) + (S 2 S 2) t 2 t0 Ka + Sa a0 kt t t0 (6a)

n"
(6b)

Fig. 4. The effects of propionic acid concentration (top) and butyric acid concentration (bottom) on their specic utilization rate in mixed acids, respectively, (mole composition, A, acetic acid; P, propionic acid; and B, butyric acid).

where ka and Ka are the two model parameters for acetate utilization. Aa refers to the cell activity for acetic acid utilization. The overall effect of total acid concentration on cell activity (A) has been determined

Fig. 3. The effect of acetic acid concentration on acetate utilization rate in mixed acid media (mole composition, A, acetic acid; P, propionic acid; and B, butyric acid).

as above, except the initial activity for acetate utilization (Aa0), which depends on individual acids and the initial culture conditions such as the total acid and cell mass. In addition to the experimental data points, Fig. 3 also shows the trend curves predicted by Eq. (6b) with the following parameters, ka = 6.2 (mmol Hac per g RBM per h), and Ka = 7.2 (mmol Hac per l). The values of initial acetate utilization activity Aa0 ranged from 0.03 to 0.07 as estimated by the nonlinear optimization MATLAB program. The predominance and utilization of propionic or butyric acid in the mixed acid solution are important to PHA synthesis from organic wastes via acidogenesis. Propionic acid contributes HV content of PHAs, which determines to a great extent the thermo-mechanical properties of PHAs such as melting point, impact strength and elongation at break [1]. Butyrate, on the other hand, does not change polymer composition by producing the homopolymer, PHB, in parallel with acetic acid. It, however, contains the greatest amount of energy among the three acids, which may improve the low yields of polymer production and cell growth as the cells are grown on sole acetic acid. Fig. 4 shows the utilization rates of propionic and butyric acids versus

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their concentrations, respectively. To adopt a MichaelisMenten type model with a variable activity for propionate utilization, it was found that the inhibition of acetic acid should also be included in addition to the overall cell activity effect (Eq. (7)). rp = kpSp kA 1 Ap0 + Kt(St0 St) + (S 2 S 2) t 2 t0 1 + KiaSa kt

cal macromolecules. On the other hand, this propionate consumption for biomass could lead to less propionate available for formation of HV in PHA copolymers.

n

3.5. PHA formation rate 6ersus acid utilization rates

In a mixed acid solution, the accumulation of PHA was at the expense of individual acids and/or their combinations with different yields. Eq. (9) gives the general contributions from the individual acids and their binary interaction. No contribution was assumed from the interactions among the three acids [9]. rPHA = Yara + Yprp + Ybrb + Yaprarp + Yabrarb + Ybprbrp (9) The signicance of each contribution and the corresponding yield depend on the composition of mixed acid medium, and are determined statistically by using non-linear correlation of the experimental results with Eq. (9). Two equations were found for two types of acid mixtures: Eq. (10) for the solutions of predominant acetate and propionate with minor butyrate (B 10% mol), and Eq. (11) for the solutions of predominant acetate and butyrate with minor propionate (B 10% mol).

(7) The inhibition effect of acetate on propionate utilization might come from the fact that both acids are activated by the same enzyme, acetate-CoA ligase (EC 6.2.1.1), in their rst step of metabolic pathways. The trend curves of propionate utilization in three acid compositions are given by Eq. (7) and shown in Fig. 4 with the parameter values: Kia =0.45 (l/mmol HAc), kp = 6.5 (l/g RBM per h) and Ap0 ranging from 0.05 to 0.1. In contrast to propionic acid, butyric acid is activated by a different ligase, butyrate CoA ligase, in its rst step of metabolism. Correspondingly, the Michaelis Menten type model did not contain the acetate inhibition except the toxic effect of total acids. Eq. (8) gives the rate model of butyrate utilization with variable cell activity. rb = kbSb Ab0 +

kA 1 K (S St) + (S 2 S 2) t 2 t0 kt t t0

n"

(8)

The trend curves given by Eq. (8) are shown in Fig. 4, giving the specic rate kb =0.32 (mmol per Hbu per g RBM per h) and Ab0 of 0.07.

3.4. Cell growth and PHA accumulation

After the undissociated acid molecules penetrate into the cells, they are activated, metabolized and ended up in three nal products, CO2, PHAs and residual biomass. Although the chemically dened medium had limited nitrogen nutrient (C/N = 80 mass), the cells utilized the limited nitrogen and the organic acid as the carbon source for cell growth at the beginning of batch cultures (Fig. 5). Although PHA was also formed at the same time, it seems that the acids were mainly used for residual biomass synthesis. After the nitrogen was exhausted, the PHA synthesis took over and became the major product in the rest of batch time. Therefore, in the following correlation of PHA formation rate with the acid utilization rates, the data obtained with negligible increase in the residual biomass was used. Fig. 5 also shows an interesting trajectory of the two utilization rates in a batch culture of acetic and propionic acids. In the clockwise direction, the propionate utilization rate increased faster than the acetate utilization rate, which corresponded to the biomass growth stage as shown in the 3-D plot (Fig. 5). It might be attributed to consumption of propionate for synthesis of biologi-

Fig. 5. (Top) The clockwise trajectory of variation in the utilization rates of acetic (ra) and propionic (rp) acids in a batch culture of binary acid solution (HAc:HPr =100:60 mol). (Bottom) The corresponding variation in the synthesis rates of RBM () and PHA () in the batch culture.

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Fig. 7. Effect of the relative utilization rates of propionate to acetate on the formation of HV monomer in poly(3-hydroxybutyrate-co-3hydroxyvalerate).

Fig. 6. The specic formation rates of PHA vs. the utilization rates of volatile fatty acids in two types of triple acid solutions; predominant acetate and propionate with minor butyrate (top) and predominant acetate and butyrate with minor propionate (bottom). The curves are given by Eqs. (10) and (11), respectively.

rPHA =0.28ra + 0.051rp +0.0019rprb rPHA =0.15ra + 0.39rb +0.010rarp

(10) (11)

Fig. 6 shows 3-D plots for correlation of PHA formation rate with the acid utilization rates in the two types of acid mixtures. Judged by the yield, butyrate is the most favorable acid for PHA synthesis compared with acetic and propionic acids, both of which need extra metabolic step(s) for two-molecule condensation to form C4 or C5 monomer unit before they can be added onto the growing PHA chains [9]. Although the maximum uptake rates of butyric and propionic acids were not different (Fig. 4) the maximum formation rate of PHA from acetate plus butyrate ( 60 mg/g RBM per h) was much faster than that from acetate plus propionate (35 mg/g RBM per h). The minor acid in the triple acid solutions was taken up by cells and utilized in a way that more or less relies on the active enzyme(s) of major acid metabolism. For example, the small amount of propionic acid in a triple acid solution was used mainly for HV synthesis via condensation with acetic acid (Eq. (11)).

Formation of HV in the copolymers depends not only on the availability of propionate, but also on the availability of acetic acid, because the C5 monomer comes from condensation of acetic and propionic acids. When pure propionate was used as the sole carbon source, a signicant amount of propionate lost one carbon, which resulted in a PHBV copolymer of about 40% wt. HV [22]. In a binary acid solution with acetic and propionic acids as the major acids as shown in Fig. 7, little propionate was decomposed and hence the relative HV formation rate over the overall PHA formation rate could reach 0.8 corresponding to the high relative consumption rate of propionate to acetate. This fact implies that the HV formation rate could reach about 80% of the overall PHA formation rate, or that the PHA copolymer from mixed acids can contain maximum 80% wt. of HV by controlling the relative utilization rates of propionic and acetic acids. Furthermore, in a triple acid solution with minor butyrate as shown in Fig. 7, the amount of butyrate was utilized quickly at the beginning of the batch cultures, leading to a fast formation of PHB, and hence lower relative HV formation rate. Acknowledgements The work described in this paper was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. HKUST61 15/97P). References
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