Biotechnology Letters 23: 16131617, 2001. 2001 Kluwer Academic Publishers. Printed in the Netherlands.

1613

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha
G. Du, Y. Si & J. Yu
Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, P.R. China Author for correspondence (Fax: 852-2358-0054; E-mail: kejianyu@ust.hk)
Received 15 May 2001; Revisions requested 31 May 2001; Revisions received 25 July 2001; Accepted 25 July 2001

Key words: biodegradable polymers, medium-chain-length fatty acid, polyhydroxyalkanoates, Ralstonia eutropha, volatile fatty acid

Abstract Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.

Introduction Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy reserve materials accumulated by a number of bacteria under the conditions of restricted growth. They have been considered as promising candidates for the production of biodegradable thermoplastics. The major obstacle to their wide acceptance is the high production cost, particularly the raw material cost (Lee 1996). Novel processes have been investigated to produce PHAs from organic wastes in wastewater (Chua et al. 1997), industrial wastes (Rusendi & Sheppard 1995), and municipal wastes (Takabatake et al. 2000). Production of PHAs from organic wastes can provide multiple benets to the environment and promote sustainable development. Organic wastes, however, are usually in complex forms that cannot be directly utilized by PHA-producing microbes such as Ralstonia eutropha (Lee & Yu 1997). The rst step to overcome this problem is hydrolysis and acidogenesis of the wastes producing short-chain volatile fatty acids such as acetic, propionic and butyric acids that can be used by R. eutropha for synthe-

sis of poly(hydroxybutyrate-co-hydroxyvalerate) (Yu 2001). Some medium-chain-length fatty acids, such as nonanoic acid (9:0) and octanoic acid (8:0), may also be formed from the digestion of oily and fatty matters via -oxidation. Little is known about their toxicity to R. eutropha in cell growth and PHA accumulation, compared with the well-known inhibitive effect of volatile fatty acids on the bacterium (Chung et al. 1997, Yu & Wang 2001). In contrast to a recent report on the positive effect of oleic acid on PHA synthesis by R. eutropha (Marangoni et al. 2000), inhibitive effects of nonanoic acid and octanoic acid on PHA synthesis by R. eutropha are reported in this paper.

Materials and methods Microorganism and growth conditions Ralstonia eutropha ATCC 17699 was grown in a nutrient-rich medium for 24 h in a rotary shaker at 200 rpm and 30 C. The cells were harvested and transferred to a mineral solution for PHA accumula-

1614 tion (Yu & Wang 2001). As the sole carbon source, the fatty acids included three volatile fatty acids (acetic, propionic and butyric acids) and two medium-chainlength fatty acids, nonanoic acid (9:0) and octanoic acid (8:0). About 0.15 g cells (in dry weight) were resuspended in 100 ml mineral solution and cultivated in a rotary shaker at 200 rpm and 30 C for 30 h. Individual fatty acids were added at the pre-determined concentrations to examine their toxic effect on cell growth and PHA synthesis. Batch fermentations were further carried out in a 2 l bioreactor for the time courses. About 2.3 g cell mass harvested from the nutrient-rich culture was re-suspended in 1.5 l mineral solution that contained nonanoic acid, a mixture of acetic and propionic acids, or a mixture of nonanoic, acetic and propionic acids. In each case, the initial concentrations of individual acids were controlled at 2.0 to 2.5 g l1 . Dissolved O2 concentration was maintained at higher than 20% of air saturation by aeration and agitation. The mediums pH and temperature were controlled at 7.5 and 30 C, respectively. Analytical procedures The active biomass and PHA content of Ralstonia eutropha were determined as follows. A culture broth (50 ml) was centrifuged at 10 000 g for 10 min and lyophilized to constant weight. PHA was extracted from the dry cell mass in an excess of chloroform (0.2 g cell mass in 10 ml solvent) at 60 C for 24 h. The residual solid, dened as the active biomass, was recovered by ltration through a glass lter. After the chloroform was evaporated at room temperature, the PHA mass was weighed. The PHA content was calculated as the percentage of the PHA mass in the dry cell mass. The volatile fatty acids were measured by GC with direct injection of acidied aqueous samples (pH 2 to 3) on to a capillary column (Supelco, USA). The nonanoic and octanoic acids as well as the PHA composition were determined by GC-MS after methanol esterication of the fatty acids and polymers (Lageveen et al. 1988). chain-length fatty acids. On the volatile fatty acids, the cell growth or the active biomass was increased with the initial acid concentration and it approached a plateau in a concentration range from 5 to 11 g l1 . Propionic acid was the best carbon source for cell growth. The PHA content also increased with the initial acid concentration to the maximal levels of individual acids. Butyric acid was the best carbon source for PHA accumulation (up to 60% of cell mass, w/w). On the medium-chain-length fatty acids, both the active biomass and PHA synthesis were maximal at low initial acid concentrations (22.5 g l1 ) and complete inhibition occurred at high acid concentrations (data not shown). The maximal PHA content (2025% wt) was much lower than the content (4560% wt) obtained from volatile fatty acids. Like acetic and butyric acids, octanoic acid (8:0) produced a homopolymer, polyhydroxybutyrate (PHB). Like the propionic acid, the nonanoic acid (9:0) produced a copolymer, P(3HVco-3HB) (PHBV) with 60.5 mol% HB unit and 39.5 mol% HV unit. Utilization of nonanoic acid and mixed acids Figure 2 shows the time course of nonanoic acid utilizaton as the sole carbon source by R. eutropha. Acetic and propionic acids were released into the medium at high nonanoic acid concentrations and taken up again at the low acid concentrations. No other intermediate acids with more than three carbon atoms were detected, indicating that nonanoic acid was quickly degraded via -oxidation by sequential removal of two C2 units to propionic acid. Because three mol acetate and one mol propionate could be produced from one mol nonanoic acid (9:0), the stoichiometric composition of P(HB-co-HV) should be 50 mol% HB unit and 50 mol% HV unit by assuming there were two acetates for one HB unit and one acetate plus one propionate for one HV unit (Lee 1996). The PHA accumulated in the cells, however, contained 60 mol% HB unit and 40 mol% HV unit. This implies that more intermediate propionate than acetate was used for synthesis of the biomass rather than the P(HBco-HV), as observed in the utilization of individual acids. Figure 3 shows the time courses of growth when acetic and propionic acids were added as the cosubstrates to nonanoic acid. In contrast to the case of nonanoic acid as the sole carbon source (Figure 2), the utilization of nonanoic acid in the mixed acids was delayed with its constant concentration in the rst 8 h

Results and discussion Utilization of individual fatty acids Ralstonia eutropha was grown on the individual fatty acids at different initial concentrations (see Figure 1). Two different patterns of cell growth and PHA accumulation were observed on volatile and medium-

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Fig. 1. Utilization of individual acids for cell growth (a) and PHA synthesis (b) by R. eutropha at different initial acid concentrations: acetic acid ( ); propionic acid ( ); butyric acid ( ); octanoic acid ( ); and nonanoic acid ( ).

Fig. 2. Time course of nonanoic acid ( ) as the sole carbon source in a batch culture. Acetic ( ) and propionic ( ) acids were the metabolic intermediates.

Fig. 3. Time courses of externally added acids in a batch culture: nonanoic acid ( ), acetic acid ( ) and propionic acid ( ).

and complete consumption in 35 h. By comparing the time courses of nonanoic acid in Figures 2 and 3, it is clear that R. eutropha preferred the volatile fatty acids, which may be attributed to the inhibition of oxidation of medium-chain-length fatty acids by the external metabolic intermediate acids. Comparison of acid utilization Figure 4 shows the specic consumption rate of nonanoic acid, the active biomass and the PHA content in the presence or absence of the external acetic and propionic acids. Without the external volatile fatty acids, the specic consumption rate of nonanoic acid reached its maximal value, 0.12 g h1 , in 11 h. With the external volatile acids, however, the rate was almost zero in the rst 8 h, and increased to its maximal value, 0.048 g h1 , in 25 h (Figure 4a). This implies

that the volatile fatty acids inhibited the metabolism of nonanoic acid. Corresponding to the inhibition by volatile fatty acids, R. eutropha showed differences in cell growth and PHA synthesis (Figure 4b). Because of the extra carbon source of volatile fatty acids, more biomass (2.55 g l1 ) was formed from the mixed acids than that (2.12 g l1 ) from nonanoic acid as the sole carbon source. It was surprising, however, that a similar PHA content (2123% wt) was accumulated even though the extra acetic and propionic acids were 2.54 and 2.61 g l1 , respectively. The small increase in PHA accumulation from the extra volatile fatty acids on the same amount of nonanoic acid (1.96 g l1 ) implies that the toxic effect of nonanoic acid might result in wastage of acetic and propionic acids. Figure 5 compares the specic consumption rates of acetic and propionic acids, the active biomass and PHA content in the presence or absence of nonanoic

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Fig. 4. Effects of acetic and propionic acids on (a) the specic consumption rate of nonanoic acid ( ), and (b) the cell active biomass ( ) and PHA synthesis ( ). The batch cultures had initially nonanoic acid 1.95 g l1 as the sole carbon source (unlled symbols) or with acetic and propionic acids added as co-substrates at 2.6 g l1 and 2.5 g l1 , respectively (lled symbols).

Fig. 5. Effects of nonanoic acid on (a) the specic consumption rates of acetic acid ( ) and propionic acid ( ) and (b) the cell active biomass ( ) and PHA synthesis ( ). The batch cultures had initially acetic acid 2.6 g l1 and propionic acid 2.5 g l1 without nonanoic acid (unlled symbols) or with nonanoic acid 2 g l1 (lled symbols).

acid. Without nonanoic acid, R. eutropha utilized the two volatile fatty acids in a short period of time (<20 h), at a high maximal specic rate of 0.17 0.18 g h1 . With nonanoic acid, the maximal rates were almost halved to 0.09 g h1 , and it took a long time (up to 35 h) for the cells to consume the two acids completely (see Figure 5a). Nonanoic acid had a positive effect on the biomass increase, with a maximal concentration of 1.8 g l1 from the two volatile acids versus 2.6 g l1 from the same amount of volatile acids plus nonanoic acid (Figure 5b). The most signicant inhibitive effect of nonanoic acid on R. eutropha was in PHA synthesis. Without nonanoic acid, the PHA content from the two volatile fatty acids was 52% (w/w) of dry cell mass. In the presence of nonanoic acid, however, this polymer content was decreased to 23% on the same amount of volatile fatty acids (see Figure 5b). It seems that the metabolism of acetic and propionic acids for

the biomass was not inuenced by nonanoic acid but the pathway of PHA synthesis was inhibited by the medium-chain-length fatty acid. In R. eutropha, PHA is synthesized from acetic and propionic acids by a sequence of three reactions catalyzed by 3-ketothiolase, reductase and PHA synthase (Lee 1996). Nonanoic acid might inhibit one or all of them which then blocks the carbon ux to PHA synthesis but further analysis is required to understand the inhibition mechanism. R. eutropha was also cultivated on octanoic acid (8:0), and the specic consumption rate reached a maximal 0.13 g h1 in 9 h (data not shown), which was similar to the utilization of nonanoic acid. The oxidation of octanoic acid released acetic and butyric acids as the metabolic intermediates that were detected in the culture broth and consumed completely at the end of batch fermentation. In a mixture of acetic, butyric and octanoic acids, R. eutropha consumed the volatile fatty acids rst and then octanoic acid in a

1617 very similar way to the case of nonanoic acid with its metabolic intermediates. It was also observed that the utilization of acetic and butyric acids was inhibited by octanoic acid and their specic consumption rates reached the maximals of 0.079 g h1 in 13 h and 0.062 g h1 in 16 h, respectively. But in the absence of octanoic acid, the maximal rates were 0.20 g h1 and 0.18 g h1 , respectively. Furthermore, the maximal PHB content was only 25% (w/w) of the dry cell mass from a mixture of three fatty acids, compared with 65% (w/w) with a mixture of acetic and butyric acids. It can be concluded that the inhibition by medium-chain-length fatty acids on PHA synthesis is not determined by the number of carbon atoms of the substrates nor by the -oxidation intermediates of these acids. References
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Acknowledgement The work described in this paper was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administration Region, China (Project No. HKUST 6115/97P).