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Production of biodegradable polymer by A. eutrophus using volatile fatty acids from acidified wastewater
Process Biochemistry 39 (2003) 295 /299 www.elsevier.com/locate/procbio
Production of biodegradable polymer by A. eutrophus using volatile fatty acids from acidified wastewater Wenquan Ruan *, Jian Chen, Shiyi Lun School of Biotechnology, Wuxi University of Light Industry, Wuxi 214036, China Received 29 July 2002; received in revised form 9 January 2003; accepted 20 February 2003
Abstract A new process of production of biodegradable polymer poly(hydroxyalkanoates) (PHAs) was studied with Alcaligenes eutrophus using volatile fatty acids (VFA) from acidified wastewater. The process was divided into two distinct stages, cell growth with fructose and PHAs polymerization with VFA. Cell growth was much better with fructose than with VFA. After using fructose for growth, A. eutrophus utilized VFA as the substrate to polymerization. The concentration of PHAs reached 16.7 g/l by fed-batch cultivation. Using VFA from acidified wastewater, A. eutrophus had a good performance for polymerization, and the concentration of cell and PHAs in broth reached 15.9 and 9.6 g/l, respectively. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Poly (hydroxyalkanoates ); Acidification; Fermentation; VFA
1. Introduction Poly(hydroxyalkanoates) (PHAs), a kind of biodegradable polymer, have very attractive properties which are ideal substitute products of some synthetic plastics. But PHAs still can not enter into our routine life in the present time and a major drawback to the industrial realization of bioplastic PHAs production is its higher production cost compared with conventional petrochemical polymers [1,2]. One of the important factors in determining the economics of PHAs production on an industrial scale is the high raw materials price. Among the substrates required, the carbon source is of prime significance in the case of PHAs production, since PHAs are composed only of C, H and O atoms. The main materials used for the production of PHAs by Alcaligenes eutrophus are fructose and volatile fatty acids * Corresponding author. Tel./fax: /86-510-588-8301. E-mail address: [email protected] (W. Ruan).
(VFA) which are both expensive. Significant research has been focused on using VFA from acidified wastewater from anaerobic systems [3] by which cheaper raw materials for the production of PHAs could be obtained. A. eutrophus first attracted scientific investigation because of its ability to grow on completely inorganic nutrients. Doi and colleagues [4] investigated the characteristics of several microorganisms on the production of PHAs using organic acids as sole carbon source during fermentation. Lee and Yu [5] verified that A. eutrophus could use VFA as carbon source to synthesize PHAs. The VFA used in their experiment were obtained from an anaerobic system in which biodegradable components in wastes were digested under anaerobic conditions by acidogenic bacteria, VFA such as acetic acid, propionic acid, butyric acid and other soluble organic compounds could be harvested from the effluent. Jin and Chen [6] set up a composite anaerobic acidification /fermentation system to produce PHAs. They found butyric acid was the most suitable acid for A. eutrophus growth and PHAs accumulation. VFA
0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00074-8
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were the carbon source for both growth of microorganism and polymerization of PHAs in their research. In this paper, a new process of production of biodegradable polymer (PHAs) was developed with A. eutrophus using VFA from acidified wastewater.
2. Materials and methods 2.1. Strain and media A.eutrophus WSH8 (a mutant strain of A. eutrophus DSM545) was used in this study. The bacteria were stored on nutrient broth slants with yeast extract 10 g/l, peptone 10 g/l, meat extract 5 g/l and ammonium sulfate 5 g/l, agar 20 g/l. The strain was activated before using, and enlarged in flasks containing the medium of fermentation. In a 2 l fermentor, cells were cultivated in synthetic medium of the same compositions as in the literature [5,6] except that fructose, acetic acid, propionic acid, butyric acid were used as a carbon source in this study. 2.2. Assays The determination of cell dry weight (DCW) was made via a dry ice vacuum process. The amount of PHAs was measured by GC [7]: about 40 mg of dry bacterial mass was weighed in a tightly sealable vial (volume 10 ml). A 2-ml volume of dichlorine ethane (DCE), 2 ml propanol containing hydrochloric acid (1 volume concentrated hydrochloric acid/4 volume propanol) and 200 ul of internal standard solution (2.0 g benzoic acid in 50 ml propanol) were added and the whole kept for 3.5 /4 h in an incubator at 80 8C. The mixture was shaken at the beginning and also during the incubation from time to time. After cooling to room temperature, 4 ml of water is added, and the mixture shaken for 20/30 s. The heavier phase (DCE /propanol) was injected directly into the gas chromatograph. Quantitative evaluation was effected by means of the quotient of the peak areas of hydroxybutyric acid and benzoic acid. VFA were measured by GC (Hewlett /Packard 5890 series II) equipped with a Nukol fused silica capillary column (30 m /0.25 mm, Supelco, Ballefonte, USA) and flame ionization detector (FID). Injector and detector temperatures were at 220 and 250 8C, respectively. The volatile organic compounds were eluted by helium at 10 ml/min with a temperature program of 10 8C/min from 150 to 190 8C. The water samples were first filtered through a 0.45 um cellulose nitrate membrane and acidified to pH 3 with concentrated phosphoric acid prior to GC analysis. COD concentration was determined using a standard method. Ammonium sulfate concentration was mea-
sured as the ammonium ion concentration by the Nesslers reaction [8].
3. Results and discussion 3.1. Characteristic of cell growth of A. eutrophus using fructose and volatile fatty acid Jin and Chen [6] found that butyric acid was more suitable for the polymerization of A. eutrophus than other VFA, such as acetic acid and propionic acid. For comparison of the cell growth using both VFA and fructose, butyric acid was used in the cultivation of A. eutrophus . Fig. 1 shows the result of cell growth using fructose and butyric acid. Because of its ill-effects on cell growth of high concentration of VFA, the concentration of butyric acid in the start medium was controlled at 10 g/l, and fructose was 20 g/l. Fructose was consumed after 22 h fermentation and the cell concentration in the broth reached 12.3 mg/l. With the same cultivation condition and using butyric acid as the carbon source, the cell concentration reached only 2.4 mg/l in the broth after 30 h. Butyric acid also produced a long lag stage of the cell growth. The number of cells in broth is an important factor for the production of PHAs. In order to produce a high concentration of cells in broth, it is necessary to use fructose instead of VFA as the carbon source of cultivation of A. eutrophus. VFA was, therefore, used for synthesis of PHAs by A. eutrophus . 3.2. PHAs production of A. eutrophus using fructose and butyric acid In the fermentation of A. eutrophus , an efficient system was developed using nitrogen as the limitation
Fig. 1. A. eutrophus growth on fructose and butyric acid. /j /, Fructose; /m /, buytric acid; /I /, DCW from fructose; /k /, DCW from butyric acid.
W. Ruan et al. / Process Biochemistry 39 (2003) 295 /299
factor of PHAs production [6,7]. By controlling the concentration of ammonia in the fermentation broth, two different stages of A. eutrophus fermentation are evident, cell growth and PHAs polymerization. With high concentration of ammonia in broth (balanced growth conditions), cells mainly use nutrient substrates to grow and under unbalanced growth conditions (limitation of ammonia in broth), microorganisms use most of the nutrient to polymerize PHAs. A fermentation strategy of A. ertrophus was obtained in this research by controlling the concentration of ammonia. In the cell growth stage under conditions of balanced culture, fructose was used to produce a high concentration of cells in broth, then the fermentation was switched to the stage of polymerization by controlling the ammonia concentration at 70 /90 mg/l (unbalanced culture). At the beginning of the second stage, VFA were added into broth as the only carbon source for polymerization. Fig. 2 illustrates the time course of variation of DCW, butyric acid, PHAs and fructose during the fermentation. The processing was divided into two stages by controlling the ammonia concentration in broth [6,9,10]. Fructose was digested by A. eutrophus for cell growth and in the first 20 h, the concentration of fructose in broth decreased from 20 to 1.8 g/l, and cell concentration increased to 11.3 g/l. Fructose could not be detected after 22 h. Butyric acid was added to the broth at 20 h while the ammonia concentration decreased to below 90 mg/l. The fermentation was switched into the stage of polymerization. The ammonia ion concentration in broth was maintained at a level of 60/80 mg/l during the polymerization stage. DCW in broth had a small increment, from 11.3 g/l at 20 h to 17 g/l at 50 h of fermentation. The concentration of PHAs in broth increased from 3 to 11.3 g/l. The increasing rate of PHAs was 0.27 g/l h, much higher than that of cell growth (0.16 g/l h) during polymerization.
Fig. 2. The fermentation processing of A. eutrophus with fructose and butyric acid. /j /, Fructose; /2 /, butyric acid; /" /, ammonium; /m /, DCW from fructose; /' /, content of PHA.
297
From this experiment, it was clear that this fermentation process was operative for the production of PHAs. With a large concentration of cells in broth, a high concentration of PHAs could be obtained by A. eutrophus using butyric acid. 3.3. Volatile acids as the production carbon source The VFA used in this research were acetic acid, propionic acid, butyric acid. These acids are the main products of an acidification system of wastewater [3,6]. The polymerizing characteristic of A. eutrophus using these three acids was compared in this research. The experiment was conducted in three individual flasks after producing sufficient cell concentration from fermentation with fructose. Fig. 3 illustrates the comparison of polymerization of A. eutrophus using three VFA with an initial concentration 10.5 g/l in broth. Butyric acid was better for utilization than other two acids for the polymerization, and propionic acid, the odd carbon acid, better than acetic acid. 3.4. The fermentation process of A. eutrophus by fedbatch cultivation The characteristic of growth and polymerization of A. eutrophus was studied by fed-batch fermentation. Butyric acid was used as the carbon source of polymerization by A. eutrophus in this research. Fig. 4 shows the fermentation processing of A. eutrophus by the fedbatch cultivation. The acid was added into reactor from 20 to 80 h at 2 ml/h at a concentration of 350 g/l while ammonium was maintained at a level of 80 mg/l. DCW increased continuously after 20 h at a rate of 0.222 g/l h composed with a rate of 0.525 g/l h during the first 20 h. The cell concentration reached 23 g/l at the end of fermentation. The increment of DCW was mainly attributed to the synthesis of PHAs in cells. The content of PHAs in cell was about 70% of DCW. The
Fig. 3. Comparsion of polymerization of A. eutrophus using VFA. /j /, Butyric acid; /" /, proponic acid; /' /, acetic acid.
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Fig. 4. The production of PHAs with butyric acid by fed-batch cultivation. /" /, DCW; /m /, fructose; /k /, residual butyric acid; /' /, PHA; /^ /, ammonium.
concentration of PHAs in broth changed from 2.7% at 20 h to 16.65% at the end, with a 5.1-times increase. 3.5. Volatile fatty acids in acidified effluent
VFA based on COD was around 0.28 g VFA/g COD over the COD loading. Glucose was almost totally acidified at an HRT of 3 h. The major products in acidified effluent were acetic acid, lactic acid, butyric acid and propionic acid. The concentration of each acid varied greatly with change in HRT. Since the best acid for A. eutrophus cells growth and PHAs accumulation was butyric acid, the condition of acidification was controlled for the transfer from glucose to butyric acid. When the HRT was 3 h, the concentration of lactic acid was the highest in the effluent, reaching 1.43 g/l, however, butyric acid was only 0.8 mg/l. As the HRT changed from 3 to 12 h, lactic acid decreased dramatically, was almost zero at 12 h of HRT, and the concentration of butyric acid reached 8.6 g/l, higher than others. The acidified effluent was centrifuged and then was concentrated to 150 g/l for use of polymerization. 3.6. Polymerization of A. euthophus with VFA from acidified effluent
Formation of VFA from synthetic glucose wastewater was studied in a thermophilic (55 8C) upflow anaerobic sludge blanket (UASB) reactor. The distribution of organic acids (especially butyric and propionic) in the effluent was dependent on chemical oxygen demand (COD) loading rate, pH and hydraulic retention time (HRT) of wastewater in the reactor. The thermophilic UASB reactor showed a stable performance on hydrolysis and acidogenesis of glucose as well as suspended solid removal at short HRT during operation. The production of VFA was proportional to COD loading rate. Fig. 5 shows the performance of acidification dependant on HRT in a thermophilic UASB with artificial wastewater containing 3% glucose. The yield of
VFA from the thermophilic UASB was concentrated to 150 g/l which, as feeding substrate, was fed to a 2 l reactor from 20 to 70 h at the rate of 0.3 /0.5 g/l h. The residual acids in broth were controlled at the level of 2/ 6 g/l. The fermentation processing with concentrated acids is shown in Fig. 6. DCW increased from 9.8 g/l at 20 h to 15.9 g/l at the end of fermentation. Accordingly, the concentration of PHAs in broth changed from 1.9 to 9.8 g/l. From the concentration variation of each acid, it was obvious that butyric acid was the easiest acid for the polymerization of PHAs by A. eutrophus . The biosynthesis of PHAs with odd carbon acid or even carbon acid is different. With even carbon acids (acetic and butyric acid), A. eutrophus will mainly synthesis the PHB, and with odd acid (propionic acid)
Fig. 5. The distribution of VFA in anaerobic reactor as the change of HRT. /2 /, Glucose; /' /, butyric acid; /j /, acetic acid; /m /, lactic acid; /" /, proponic acid.
Fig. 6. The processing of PHAs production by feeding VFA. /" /, DCW; /m /, ammonium; /^ /, proponic acid; /' /, PHA; /k /, butyric acid; /j /, fructose; /I /, acetic acid.
W. Ruan et al. / Process Biochemistry 39 (2003) 295 /299
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yield of PHAs increased greatly during the fed-batch fermentation with concentrated VFA from acidified wastewater by A. eutrophus , the concentrations of DCW and PHAs were 15.9 and 9.6 g/l, respectively.
Acknowledgements The authors thank National Natural Science Foundation of China for providing financial support.
Fig. 7. The composition of PHAs by the fermentation with acidified effluent. /" /, PHA; /j /, HB; /' /, HV.
the cell will produce both HB and HV sections together that make biopolymer more flexible and more competitive. Fig. 7 describes the situation of PHAs, HB and HV which were synthesized by A. eutrophus with concentrated acidified effluent contained the odd carbon acid /propionic acid. The content of HV section increased in polymer during the fermentation. The ratio of HV:HB was about 1:10.1 at the end of the processing. It will be quite significant to obtain more propionic acid in acidified effluent for the better properties of PHAs products.
4. Conclusion A. eutrophus had the ability to use fructose to grow and VFA to polymerize. A new process of cell growth with fructose and PHAs polymerization with VFA was efficient. The most suitable organic acid utilized for PHAs accumulation was butyric acid, the concentration of PHAs reached 11.2 g/l, higher than propionic acid and acetic acid. VFA could be obtained from an acidified thermophilic UASB reactor, which was controlled for the formation of butyric acid in effluent. The
References [1] Byrom D. Polymer synthesis by microorganisms: technology and economics. Trends Biotechnol 1987;5:246 /50. [2] Yamane T. Yield of poly-D-( /)-3-hydroxybutyrate from various carbon sources: a theoretical study. Biotechnol Bioeng 1993;41:165 /70. [3] Wiegant WM, Lettinga G. Thermophilic anaerobic digestion of sugars in upflow anaerobic sludge blanket reactor. Biotechnol Bioeng 1985;27:1603 /7. [4] Doi Y, Kunioka M, Nakamura Y, Soga K. Biosynthesis of copolyesters in Alcaligenes eutrophus H16 from 13C-labelled acetate and proprionate. Macromolecules 1987;20:2988 /91. [5] Lee S, Yu J. Production of biodegradable thermoplastics from municipal sludge by a two-stage bioprocess. Resour Conserv Recycl 1997;19:151 /64. [6] Jin DY, Chen J, Lun SY. Production of poly(hyoxyalkanoate) by a composite anaerobic acidification /fermentation system. Process Biochem 1999;34:829 /33. [7] Riis V, Mai W. Gas chromatographic determination of poly-betahydroxybutyric acid in microbial biomass after hydrochloric acid propanolysis. J Chromatogr 1988;445:285 /9. [8] Johnson MJ. Isolation and properties of a pure yeast polypeptidase. J Biol Chem 1941;137:575 /86. [9] Shi HD, Shiraishi M, Shimizu K. Metabolic flux analysis for biosynthesis of poly-beta-hydroxybutyric acid in A. ertrophus from various carbon sources. J Ferment Bioeng 1997;84(6):579 / 87. [10] Ramsay BA, Lomaliza K, Chavaric C, Dube B, Bataille P, Ramsay JA. Production of poly-(beta-hydroxybutyric-co-betahydroxyvaleric) acids. Appl Environ Microbiol 1990;56:2093 /8.
Production of biodegradable polymer by A. eutrophus using volatile fatty acids from acidified wastewater Wenquan Ruan *, Jian Chen, Shiyi Lun School of Biotechnology, Wuxi University of Light Industry, Wuxi 214036, China Received 29 July 2002; received in revised form 9 January 2003; accepted 20 February 2003
Abstract A new process of production of biodegradable polymer poly(hydroxyalkanoates) (PHAs) was studied with Alcaligenes eutrophus using volatile fatty acids (VFA) from acidified wastewater. The process was divided into two distinct stages, cell growth with fructose and PHAs polymerization with VFA. Cell growth was much better with fructose than with VFA. After using fructose for growth, A. eutrophus utilized VFA as the substrate to polymerization. The concentration of PHAs reached 16.7 g/l by fed-batch cultivation. Using VFA from acidified wastewater, A. eutrophus had a good performance for polymerization, and the concentration of cell and PHAs in broth reached 15.9 and 9.6 g/l, respectively. # 2003 Elsevier Science Ltd. All rights reserved. Keywords: Poly (hydroxyalkanoates ); Acidification; Fermentation; VFA
1. Introduction Poly(hydroxyalkanoates) (PHAs), a kind of biodegradable polymer, have very attractive properties which are ideal substitute products of some synthetic plastics. But PHAs still can not enter into our routine life in the present time and a major drawback to the industrial realization of bioplastic PHAs production is its higher production cost compared with conventional petrochemical polymers [1,2]. One of the important factors in determining the economics of PHAs production on an industrial scale is the high raw materials price. Among the substrates required, the carbon source is of prime significance in the case of PHAs production, since PHAs are composed only of C, H and O atoms. The main materials used for the production of PHAs by Alcaligenes eutrophus are fructose and volatile fatty acids * Corresponding author. Tel./fax: /86-510-588-8301. E-mail address: [email protected] (W. Ruan).
(VFA) which are both expensive. Significant research has been focused on using VFA from acidified wastewater from anaerobic systems [3] by which cheaper raw materials for the production of PHAs could be obtained. A. eutrophus first attracted scientific investigation because of its ability to grow on completely inorganic nutrients. Doi and colleagues [4] investigated the characteristics of several microorganisms on the production of PHAs using organic acids as sole carbon source during fermentation. Lee and Yu [5] verified that A. eutrophus could use VFA as carbon source to synthesize PHAs. The VFA used in their experiment were obtained from an anaerobic system in which biodegradable components in wastes were digested under anaerobic conditions by acidogenic bacteria, VFA such as acetic acid, propionic acid, butyric acid and other soluble organic compounds could be harvested from the effluent. Jin and Chen [6] set up a composite anaerobic acidification /fermentation system to produce PHAs. They found butyric acid was the most suitable acid for A. eutrophus growth and PHAs accumulation. VFA
0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00074-8
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were the carbon source for both growth of microorganism and polymerization of PHAs in their research. In this paper, a new process of production of biodegradable polymer (PHAs) was developed with A. eutrophus using VFA from acidified wastewater.
2. Materials and methods 2.1. Strain and media A.eutrophus WSH8 (a mutant strain of A. eutrophus DSM545) was used in this study. The bacteria were stored on nutrient broth slants with yeast extract 10 g/l, peptone 10 g/l, meat extract 5 g/l and ammonium sulfate 5 g/l, agar 20 g/l. The strain was activated before using, and enlarged in flasks containing the medium of fermentation. In a 2 l fermentor, cells were cultivated in synthetic medium of the same compositions as in the literature [5,6] except that fructose, acetic acid, propionic acid, butyric acid were used as a carbon source in this study. 2.2. Assays The determination of cell dry weight (DCW) was made via a dry ice vacuum process. The amount of PHAs was measured by GC [7]: about 40 mg of dry bacterial mass was weighed in a tightly sealable vial (volume 10 ml). A 2-ml volume of dichlorine ethane (DCE), 2 ml propanol containing hydrochloric acid (1 volume concentrated hydrochloric acid/4 volume propanol) and 200 ul of internal standard solution (2.0 g benzoic acid in 50 ml propanol) were added and the whole kept for 3.5 /4 h in an incubator at 80 8C. The mixture was shaken at the beginning and also during the incubation from time to time. After cooling to room temperature, 4 ml of water is added, and the mixture shaken for 20/30 s. The heavier phase (DCE /propanol) was injected directly into the gas chromatograph. Quantitative evaluation was effected by means of the quotient of the peak areas of hydroxybutyric acid and benzoic acid. VFA were measured by GC (Hewlett /Packard 5890 series II) equipped with a Nukol fused silica capillary column (30 m /0.25 mm, Supelco, Ballefonte, USA) and flame ionization detector (FID). Injector and detector temperatures were at 220 and 250 8C, respectively. The volatile organic compounds were eluted by helium at 10 ml/min with a temperature program of 10 8C/min from 150 to 190 8C. The water samples were first filtered through a 0.45 um cellulose nitrate membrane and acidified to pH 3 with concentrated phosphoric acid prior to GC analysis. COD concentration was determined using a standard method. Ammonium sulfate concentration was mea-
sured as the ammonium ion concentration by the Nesslers reaction [8].
3. Results and discussion 3.1. Characteristic of cell growth of A. eutrophus using fructose and volatile fatty acid Jin and Chen [6] found that butyric acid was more suitable for the polymerization of A. eutrophus than other VFA, such as acetic acid and propionic acid. For comparison of the cell growth using both VFA and fructose, butyric acid was used in the cultivation of A. eutrophus . Fig. 1 shows the result of cell growth using fructose and butyric acid. Because of its ill-effects on cell growth of high concentration of VFA, the concentration of butyric acid in the start medium was controlled at 10 g/l, and fructose was 20 g/l. Fructose was consumed after 22 h fermentation and the cell concentration in the broth reached 12.3 mg/l. With the same cultivation condition and using butyric acid as the carbon source, the cell concentration reached only 2.4 mg/l in the broth after 30 h. Butyric acid also produced a long lag stage of the cell growth. The number of cells in broth is an important factor for the production of PHAs. In order to produce a high concentration of cells in broth, it is necessary to use fructose instead of VFA as the carbon source of cultivation of A. eutrophus. VFA was, therefore, used for synthesis of PHAs by A. eutrophus . 3.2. PHAs production of A. eutrophus using fructose and butyric acid In the fermentation of A. eutrophus , an efficient system was developed using nitrogen as the limitation
Fig. 1. A. eutrophus growth on fructose and butyric acid. /j /, Fructose; /m /, buytric acid; /I /, DCW from fructose; /k /, DCW from butyric acid.
W. Ruan et al. / Process Biochemistry 39 (2003) 295 /299
factor of PHAs production [6,7]. By controlling the concentration of ammonia in the fermentation broth, two different stages of A. eutrophus fermentation are evident, cell growth and PHAs polymerization. With high concentration of ammonia in broth (balanced growth conditions), cells mainly use nutrient substrates to grow and under unbalanced growth conditions (limitation of ammonia in broth), microorganisms use most of the nutrient to polymerize PHAs. A fermentation strategy of A. ertrophus was obtained in this research by controlling the concentration of ammonia. In the cell growth stage under conditions of balanced culture, fructose was used to produce a high concentration of cells in broth, then the fermentation was switched to the stage of polymerization by controlling the ammonia concentration at 70 /90 mg/l (unbalanced culture). At the beginning of the second stage, VFA were added into broth as the only carbon source for polymerization. Fig. 2 illustrates the time course of variation of DCW, butyric acid, PHAs and fructose during the fermentation. The processing was divided into two stages by controlling the ammonia concentration in broth [6,9,10]. Fructose was digested by A. eutrophus for cell growth and in the first 20 h, the concentration of fructose in broth decreased from 20 to 1.8 g/l, and cell concentration increased to 11.3 g/l. Fructose could not be detected after 22 h. Butyric acid was added to the broth at 20 h while the ammonia concentration decreased to below 90 mg/l. The fermentation was switched into the stage of polymerization. The ammonia ion concentration in broth was maintained at a level of 60/80 mg/l during the polymerization stage. DCW in broth had a small increment, from 11.3 g/l at 20 h to 17 g/l at 50 h of fermentation. The concentration of PHAs in broth increased from 3 to 11.3 g/l. The increasing rate of PHAs was 0.27 g/l h, much higher than that of cell growth (0.16 g/l h) during polymerization.
Fig. 2. The fermentation processing of A. eutrophus with fructose and butyric acid. /j /, Fructose; /2 /, butyric acid; /" /, ammonium; /m /, DCW from fructose; /' /, content of PHA.
297
From this experiment, it was clear that this fermentation process was operative for the production of PHAs. With a large concentration of cells in broth, a high concentration of PHAs could be obtained by A. eutrophus using butyric acid. 3.3. Volatile acids as the production carbon source The VFA used in this research were acetic acid, propionic acid, butyric acid. These acids are the main products of an acidification system of wastewater [3,6]. The polymerizing characteristic of A. eutrophus using these three acids was compared in this research. The experiment was conducted in three individual flasks after producing sufficient cell concentration from fermentation with fructose. Fig. 3 illustrates the comparison of polymerization of A. eutrophus using three VFA with an initial concentration 10.5 g/l in broth. Butyric acid was better for utilization than other two acids for the polymerization, and propionic acid, the odd carbon acid, better than acetic acid. 3.4. The fermentation process of A. eutrophus by fedbatch cultivation The characteristic of growth and polymerization of A. eutrophus was studied by fed-batch fermentation. Butyric acid was used as the carbon source of polymerization by A. eutrophus in this research. Fig. 4 shows the fermentation processing of A. eutrophus by the fedbatch cultivation. The acid was added into reactor from 20 to 80 h at 2 ml/h at a concentration of 350 g/l while ammonium was maintained at a level of 80 mg/l. DCW increased continuously after 20 h at a rate of 0.222 g/l h composed with a rate of 0.525 g/l h during the first 20 h. The cell concentration reached 23 g/l at the end of fermentation. The increment of DCW was mainly attributed to the synthesis of PHAs in cells. The content of PHAs in cell was about 70% of DCW. The
Fig. 3. Comparsion of polymerization of A. eutrophus using VFA. /j /, Butyric acid; /" /, proponic acid; /' /, acetic acid.
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Fig. 4. The production of PHAs with butyric acid by fed-batch cultivation. /" /, DCW; /m /, fructose; /k /, residual butyric acid; /' /, PHA; /^ /, ammonium.
concentration of PHAs in broth changed from 2.7% at 20 h to 16.65% at the end, with a 5.1-times increase. 3.5. Volatile fatty acids in acidified effluent
VFA based on COD was around 0.28 g VFA/g COD over the COD loading. Glucose was almost totally acidified at an HRT of 3 h. The major products in acidified effluent were acetic acid, lactic acid, butyric acid and propionic acid. The concentration of each acid varied greatly with change in HRT. Since the best acid for A. eutrophus cells growth and PHAs accumulation was butyric acid, the condition of acidification was controlled for the transfer from glucose to butyric acid. When the HRT was 3 h, the concentration of lactic acid was the highest in the effluent, reaching 1.43 g/l, however, butyric acid was only 0.8 mg/l. As the HRT changed from 3 to 12 h, lactic acid decreased dramatically, was almost zero at 12 h of HRT, and the concentration of butyric acid reached 8.6 g/l, higher than others. The acidified effluent was centrifuged and then was concentrated to 150 g/l for use of polymerization. 3.6. Polymerization of A. euthophus with VFA from acidified effluent
Formation of VFA from synthetic glucose wastewater was studied in a thermophilic (55 8C) upflow anaerobic sludge blanket (UASB) reactor. The distribution of organic acids (especially butyric and propionic) in the effluent was dependent on chemical oxygen demand (COD) loading rate, pH and hydraulic retention time (HRT) of wastewater in the reactor. The thermophilic UASB reactor showed a stable performance on hydrolysis and acidogenesis of glucose as well as suspended solid removal at short HRT during operation. The production of VFA was proportional to COD loading rate. Fig. 5 shows the performance of acidification dependant on HRT in a thermophilic UASB with artificial wastewater containing 3% glucose. The yield of
VFA from the thermophilic UASB was concentrated to 150 g/l which, as feeding substrate, was fed to a 2 l reactor from 20 to 70 h at the rate of 0.3 /0.5 g/l h. The residual acids in broth were controlled at the level of 2/ 6 g/l. The fermentation processing with concentrated acids is shown in Fig. 6. DCW increased from 9.8 g/l at 20 h to 15.9 g/l at the end of fermentation. Accordingly, the concentration of PHAs in broth changed from 1.9 to 9.8 g/l. From the concentration variation of each acid, it was obvious that butyric acid was the easiest acid for the polymerization of PHAs by A. eutrophus . The biosynthesis of PHAs with odd carbon acid or even carbon acid is different. With even carbon acids (acetic and butyric acid), A. eutrophus will mainly synthesis the PHB, and with odd acid (propionic acid)
Fig. 5. The distribution of VFA in anaerobic reactor as the change of HRT. /2 /, Glucose; /' /, butyric acid; /j /, acetic acid; /m /, lactic acid; /" /, proponic acid.
Fig. 6. The processing of PHAs production by feeding VFA. /" /, DCW; /m /, ammonium; /^ /, proponic acid; /' /, PHA; /k /, butyric acid; /j /, fructose; /I /, acetic acid.
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yield of PHAs increased greatly during the fed-batch fermentation with concentrated VFA from acidified wastewater by A. eutrophus , the concentrations of DCW and PHAs were 15.9 and 9.6 g/l, respectively.
Acknowledgements The authors thank National Natural Science Foundation of China for providing financial support.
Fig. 7. The composition of PHAs by the fermentation with acidified effluent. /" /, PHA; /j /, HB; /' /, HV.
the cell will produce both HB and HV sections together that make biopolymer more flexible and more competitive. Fig. 7 describes the situation of PHAs, HB and HV which were synthesized by A. eutrophus with concentrated acidified effluent contained the odd carbon acid /propionic acid. The content of HV section increased in polymer during the fermentation. The ratio of HV:HB was about 1:10.1 at the end of the processing. It will be quite significant to obtain more propionic acid in acidified effluent for the better properties of PHAs products.
4. Conclusion A. eutrophus had the ability to use fructose to grow and VFA to polymerize. A new process of cell growth with fructose and PHAs polymerization with VFA was efficient. The most suitable organic acid utilized for PHAs accumulation was butyric acid, the concentration of PHAs reached 11.2 g/l, higher than propionic acid and acetic acid. VFA could be obtained from an acidified thermophilic UASB reactor, which was controlled for the formation of butyric acid in effluent. The
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