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Bioconversion of chicken wastes to value-added products
Bioresource Technology 36 (1991) 229-234
Bioconversion of Chicken Wastes to Value-Added Products S. Barik, T. Forgacs & J. Isbister ARCTECH, Inc., 5390 Cherokee Avenue, Alexandria, Virginia 22312, USA
Abstract Increasing quantities of chicken waste concerns the poultry industry because of escalating disposal costs and the potential for environmental pollution. Biological conversion of these wastes to valuable products such as methane and/or chemical feedstocks appears to be feasible. Biomethanation of chicken waste by a sewage sludge microbial consortium produced as much as 69 mol% methane in the gas phase. Acetic and propionic acids were the major acids produced during the bioconversion. Addition of chelating agents and other micronutrients enhanced methane production and shifted the ratios of intermediates accumulated. Preliminary data indicate that more than 60% of the chicken waste carbon was converted and that the nitrogen-rich residue may have potential as a soil additive. Key words: Chicken waste, bioconversion, biomethanation, methane.
INTRODUCTION The poultry industry is made up of large-scale operations, primarily in the southern regions of the United States because of the comparative advantages of climate, low-priced land, and the economics of aggregation and scale (USDA, 1983). Although the increased size of poultry operations has resulted in economical poultry products for the consumer, it has also resulted in the generation of substantial volumes of chicken waste at each location (USDA, 1978). These large volumes of chicken waste pose significant environmental problems associated with the need for proper disposal (Washington Post, 1988). Stringent disposal requirements are now being imposed by environmental agencies resulting in
increased costs for disposal and increased poultry prices for the consumer. Chicken waste was commonly used as a fertilizer in the past when the volume of waste to be disposed was relatively small and widely dispersed. These wastes are undesirable as fertilizers if offensive odors are allowed to develop and because the high ammonia content can harm crops. Thus, the poultry operations are faced with disposal of increasing volumes of wastes which are an economic and environmental liability. Biological production of methane and other byproducts from poultry waste (Jones & Ogden, 1984; Shih, 1984) and from a mixture of cattle manure and chicken waste (L. Gornall, 1989 pers. comm.) has been demonstrated. Feasibility studies on in-situ utilization of fermentation products from chicken waste have been conducted (Jiang et al., 1987; Yang & Chandrasekharan, 1988); however, no technology has been developed thus far to implement this bioconversion process for use by the poultry industry. The development of such a process converting chicken wastes into valueadded products could turn a problem into an economic opportunity for the poultry industry. This paper presents research data to support the technical feasibility for bioconversion of chicken wastes to methane and organic acids.
MATERIALS AND M E T H O D S Chicken waste
Fresh, non-fermented chicken waste samples were collected from Seaboard Farm, Roanoke, Virginia, and Purdue Chicken Farm, Salisbury, Virginia. Ultimate analyses of the Purdue chicken waste were conducted by Commercial Testing and Engineering Co., Norfolk, Virginia. The feed and other foreign materials were separated and the chicken wastes were air-dried before they were
229 Bioresource Technology 0960-8524/91/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
230
S. Barik, T. Forgacs, J. Isbister
ground to - 100 mesh and stored in air-tight containers at room temperature until used. Microbial culture Fresh anaerobic sewage sludge was collected from the primary digestor of the city of Alexandria, Virginia, and was transported to the laboratory under anaerobic conditions. This culture was used as a 20% (v/v) inoculum in all experiments. A methanogenic enrichment culture containing acetate-utilizing Methanothrix sp. and acetateand H2-CO2-utilizing Methanosarcina sp. developed at A R C T E C H was also used in later experiments. These cultures were grown in a specific methanogenic medium as described earlier (Corder et al., 1983). Bacterial identification was based on morphologies using phase contrast and epifluorescence microscopy.
Media and culture conditions Media and culture conditions were essentially those previously described (Bryant, 1972; Balch & Wolfe, 1976; McInerney et al., 1979). The basal salts medium contained low-sulfate minerals, B-vitamins, trace metals and 0.1% yeast extract. The medium was buffered with 40 m u sodium bicarbonate and had a gas phase of 4:1 N2-CO 2. The pH was 7.4 and sodium sulfide (final concentration, 1 mmol liter- ~) was used to reduce the medium before use. Liquid medium was dispensed in 8 ml amounts in butyl rubber stoppered serum tubes (18 x 150 mm) or in 32 ml amounts in 100 ml serum bottles. The tubes or bottles containing medium were crimp sealed and autoclaved for 20 min at 121°C. Chicken waste, in appropriate amounts, was weighed into pre-sterilized serum stoppered tubes or bottles. Autoclaved medium was then added to the tubes or bottles containing chicken waste and was inoculated with freshly collected sewage digestor sludge (20% v/v). Additions of chemicals and methane inhibitors, BESA (2-bromoethanesulfonic acid) or monensin, at specified concentrations were made as required. These procedures were performed in an anaerobic chamber. All samples were inoculated in triplicate and were incubated at 37°C under static conditions. Appropriate controls were included with each set of experiments. Analytical methods Total gas production for each sample was determined using a syringe displacement method. Methane concentration was determined using a
Varian 3700 gas chromatograph fitted with a thermal conductivity detector. Gas samples (125 pl) were injected into a stainless steel column (3 m x 3.2 mm OD) packed with 100/200 mesh Carbosieve S-II (Supelco, Inc., Bellefonte, Pennsylvania). The operating conditions were: oven temperature, 210°C; detector and column temperatures, 220°C; and helium as carrier gas (6 ml min- 1). The peak areas were integrated using a HP 3396A computing integrator. Aliquots of the liquid phase were taken periodically to determine the accumulation of short chain fatty acids and alcohols. These samples were centrifuged, diluted with equal amounts of 0"05 M phosphoric acid, and placed in sample vials. Analysis employed a Hewlett-Packard 5880A gas chromatograph fitted with a flame ionization detector (300°C), split/splitless injector (205°C), a 3-m SP-1000 borosilicate glass precolumn (Supelco) and a 15-m Nukol fused silica capillary column with a 0.5-/xm film thickness (Supelco). The oven temperature was programmed from 48°C for 0.7 min to 200°C using three different rates (15°C min-1 to 120°C, 10°C min -~ to 155°C and 5°C min -~ to 200°C). Helium was used as the carrier gas. Injections (2 pl) were made in the split mode (25: 1) using a HP 7672A automatic sampler. Peak areas were integrated using the HP 5880A data terminal. RESULTS AND DISCUSSION Analysis of the Purdue chicken waste to determine the carbon content of the proposed substrate was the first priority after acquisition of the waste. Results of the analyses (Table 1) show that chicken waste contains more than 37% carbon, sufficient carbon substrate(s) for bioconversion to value-added products. A relatively high nitrogen content (greater than 4%) in the chicken waste could be of importance as a component of the fermentation residue which may have potential as a soil additive. The studies investigating bioconversion of chicken waste were designed using two conversion approaches (Fig. 1). In one approach, the chicken waste was fermented with selected microbial cultures under anaerobic conditions for production of methane. In a second approach, the fermentation of the chicken waste occurred anaerobically in the presence of methane inhibitors to force the accumulation of organic acids and/or alcohols by preventing the production of
231
Bioconversion of chicken wastes to value-added products
Table 1. Ultimate analysis* of Purdue chicken waste Dry weight basis, %
Carbon Hydrogen Nitrogen Sulfur Ash Oxygen (by difference)
37.3 5"3 4.6 0"9 19.2 32.8
*Analysis by Commercial Testing and Engineering Co,
Chickenwaste ]
• Organic waste
Organic"
"~* [ ()rganicchemicals ]
I Soil additives I
production observed at 10% and 20% solids loadings.
Experiment 2 Bioconversion of the Purdue chicken wastes (2.5% w/v) and Seaboard Farm chicken waste (2"5% w/v) was essentially the same over a 60-day incubation period, although bioconversion of the P u r d u e chicken waste showed a shorter lag period (Fig. 2). This short lag period followed by rapid methane production may be attributed to the native microflora or to the bioavailability of the carbon in P u r d u e chicken waste, or both. As m u c h as 109 cm 3 methane g - t of chicken waste was generated within 30 days from the P u r d u e chicken waste with a final production of 140 cm 3 methane g-~ chicken waste within 60 days. Bioconversion of the Seaboard Farms chicken waste reached 127 cm 3 methane g - i chicken waste during a 60-day fermentation.
Table 2. Bioconversion of chicken waste to methane at different solids ioadings*
Fig. 1. Proposed pathway for bioconversion of chicken waste to value-added products.
crn~Methane g chicken waste (CW)
% Solids (w/v)
Autoclaved
methane. In both approaches, value-added products and fermentation residue would be generated. The production of methane, organic acids and alcohols was monitored during the feasibility studies. Experiment 1 Methane p r o d u c t i o n from autoclaved and nonautoclaved P u r d u e chicken waste by the sewage sludge inocula was studied at solids loadings ranging from 0 to 20% (w/v). No significant methane was generated from m e d i u m c o m p o n e n t s when chicken waste was not a d d e d to the reaction mixture (Table 2). M e t h a n e p r o d u c t i o n from the non-autoclaved chicken waste was slightly higher than from the autoclaved chicken waste samples at 2 . 5 - 1 0 % solids loading, indicating that the native microflora could be important for biodegradation of the organic matter present in the chicken waste or that autoclaving alters the bioavailability of the carbon in the chicken waste. T h e conversion of carbon in chicken waste to methane appears to be most efficient when the chicken waste is provided at a solids loading of 5.0%, with significantly decreased m e t h a n e
Non-autoclaved
0(Control,noCW) 2.5 2"5 2-5 142'8 162"0 5.0 126.6 172.5 10.0 85.7 107.9 20.0 43.9 17.2 *Sewage sludge culture, Purdue chicken waste, 45 days incubation, static conditions, 37°C.
0
125. 100. T 75.
~
50.
~ / ~
Q.
o -5
/
25. 0¢
CW
O--O
•
•--
•
PCW
~.~o 10
2'0
3'o
4'o
go
Incubotion(doya) Fig. 2. Biological production of methane from chicken wastes. CW, Seaboard Farm chicken waste; PCW, Purdue Farm chicken waste. Sewage sludge culture; static conditions, 37°C; cm3methane g- =dry wt.
232
S. Barik, T. Forgacs, J. Isbister
Experiment 3 Bioconversion of chicken waste (2.5% solids, w/v), at pH 5 and pH 6, by the sewage sludge microorganisms to acids and alcohols in the presence of methane inhibitors (BESA or monensin) was investigated. Acidic pH has been shown to result in accumulation of short chain organic acids and alcohols (Barik et al., 1988; US DOE-PETC, 1989). Under both pH regimes, acetic acid was the major product formed, although small concentrations of propionic acid were also detected (Table 3) with minimal production of methane. The accumulation of propionic acid in the control (no inhibitor) at pH 5 was probably due to inhibition in methane production at this pH. However, propionic acid was perhaps converted to methane at pH 6. Short chain organic alcohols were not accumulated in the medium during these experiments.
Experiment 4 Methanogens require trace metals and inorganic nutrients for growth and methanogenesis (Patel et al., 1978; Schoneit et al., 1979; Scherer & Sahm, 1981 a, b; Speece, 1983). The presence of optimal concentrations of micronutrients in chicken wastes and their bioavailability during the fermentation are unknown. The addition of chelating agents (to solubilize trace metals) and the addition of selected inorganic nutrients to the fermentation broth were evaluated for enhancing bioconversion of chicken waste to methane. The addition of ferrous sulfate (0.1 mmol liter-1), nickel chloride (10 mmol liter-~) and EDTA (120 mmol liter-1)
resulted in only slightly increased methane production (Table 4). The addition of nickel chloride has been shown to increase biogas formation in a laboratory scale poultry digestor (Williams et al., 1986). The addition of known methanogens to the sewage sludge consortium (no chelator or inorganic nutrient addition) showed a similar small increase in methane production when compared to the control (no chemical or microbial addition) samples. The fermentation broth from the experiments designed to evaluate methane production in the presence of chelators and/or inorganic nutrients was analyzed for the accumulation of short chain acids and alcohols. Although the addition of 0.1 mM ferrous sulfate resulted in a small increase in methane production (Fig. 3), the addition of this trace metal at concentrations of 0-1 and 0.5 mmol liter-~ resulted in decreased organic acid accumulation in the medium. Methane production appeared to increase slightly as the concentration of nickel chloride increased (Fig. 4). Accumulation of acetic acid was greatly enhanced by the presence of 10 mM nickel chloride, while the addition of nickel chloride resulted in a decrease in Table 4. Effect of additives on production of methane from Purdue chicken waste*
Additives, concentration
Methane (mol%)
No additions (control) Fe sulfate -- 0.1 mM Fe chloride - - 5.0 mM Ni chloride -- 10"0 mM Na selenite 0.1 mM Nitrilotriacetate - - 5-0 mM Na citrate - - 5'0 mM E D T A - - 10"0 mM Methanogens
69.0 74.0 69'5 76.9 62-9 71.4 70' 1 73"7 74.8
-
Table 3. Effect of B E S A and monensin on bioconversion of chicken waste"
Sample
Methane (mol%)
Acids, mg g of chicken waste Acetate
*Sewage sludge inoculum, 45 days incubation.
Propionate
100-
pH 5"0 Control ~' + BESA + Monensin
80.
65"2 1'4 3'2
0'4 244'7 213'6
2"5 77"6 77'8
pH 6.0 Control h + BESA + Monensin
_~ s0 ~"
74.6 0"9 2"3
0-2 136.4 136"9
0.04 51'9 62"8
aSewage sludge culture, Purdue chicken waste, static conditions, 37°C, 45 days incubation. BESA: 2-bromoethanesulfonic acid. Note: no alcohols were detected in these samples. hNo inhibitors.
-
40.
20. 0
-2500
Control 0.1 mM FeSO4 5 . 0 mM FeSO4
- 2000
111 Methane
Acetate
I
1000
X
5O0
Propionate
Fig. 3. Effect of ferrous sulfate on bioconversion of chicken waste. Sewage sludge culture; Purdue Farm chicken waste; static conditions; 37°C; 45 days incubation.
Bioconversion of chicken wastes to value-added products -2500 100-
Control m~l 0.1 mM NiCI2 1 5.0 rnM NiCI 2 10 mM NiCt 2
-2000
80-6
0 Methone
lml lID Acetote
1500
I
1000
500
Prooionote
Fig. 4. Effect of nickel chloride on bioconversion of chicken waste. Sewage sludge culture; Purdue Farm chicken waste; static conditions; 37°C; 45 days incubation.
propionic acid accumulation. The addition of sodium citrate into the fermentation medium did not enhance methane production (data not shown) nor the accumulation of propionic acid in the medium. However, accumulation of acetic acid in the medium was significantly greater when sodium citrate was present at 5 and 10 mmol liter-~. These data suggest that manipulation of the medium components as well as medium pH could provide selective pressures for accumulation of specific products during bioconversion of chicken wastes.
Experiment 5 To evaluate the efficacy of adding methanogens to the microbial consortium converting the chicken wastes to methane, the sewage sludge consortium was supplemented with an enrichment culture containing acetate-utilizing Methanothrix sp. and acetate- and H2-CO2-utilizing Methanosarcina sp. developed at ARCTECH. The results indicate that the addition of these methanogens to the sewage sludge consortium resulted in a 10% increase in methane production (Table 4) from Purdue chicken waste.
CONCLUSIONS This research confirms that a bioprocess for conversion of the carbon in chicken waste to methane is technically feasible. Chicken waste and the sewage sludge cultures used in these studies were able to convert the carbon in both the Purdue and Seaboard farm chicken waste to methane and organic acids; however, no alcohols were produced. Based on theoretical calculations, bioconversion of more than 60% of the carbon in the
233
Purdue chicken waste to methane and organic acids was demonstrated. The addition of chelators to the fermentation medium did not result in enhanced methane production, although a slightly higher concentration of methane was observed when 10 mM EDTA was added. Methane production increased with increasing nickel chloride concentration, confirming results reported by Williams et al. (1986). In the presence of 10 mM nickel chloride, propionate concentrations were decreased, while the acetic acid concentration increased by more than twofold. This organic acid accumulation was accompanied by only a slight increase in methane production, suggesting that acetate-cleaving microorganisms were inactive or present in insufficient numbers to efficiently produce methane from acetate. The addition of 0-1 mM ferrous sulfate resulted in a slight increase in methane production coupled with decreases in both acetate and propionate concentrations. The data obtained in these preliminary studies indicate that manipulation of medium components and the addition of methanogens could result in a consortium for use in a commercial process converting chicken waste to value-added products. Successful development of a biotechnology for economical conversion of chicken wastes to methane and other value-added products could significantly enhance the economics of poultry operations. Waste disposal costs could be dramatically decreased and environmental pollution reduced.
ACKNOWLEDGEMENTS We thank Dr Daman S. Walia for supporting this work through ARCTECH in-house Research and Development funding. We also thank Mr Fred Principe for his interest and encouragement.
REFERENCES Balch, W. E. & Wolfe, R. S. (1976). New approach to the cultivation of methanogenic bacteria: 2-mercaptoethane sulfonic acid (HS-COM) dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl. Environ. Microbiol., 32, 781-91. Barik, S., Prieto, S., Harrison, S. B., Clausen, E. C. & Gaddy, J. L. (1988). Biological production of alcohols from coal through indirect liquefaction. Appl. Biochem. Biotechnol., 18, 363-8. Bryant, M. P. (1972). Commentary on the Hungate techniques for culture of anaerobic bacteria. Am. J. Clin. Nutr., 25, 1324-8.
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Corder, R. E., Hook, L. A., Larkin, J. M. & Frea, J. 1. (1983). Isolation and characterization of two methane producing cocci: Methanogenium olentangyi, sp. nov., and Methanococcus deltae, sp. nov. Archiv. Microbiol., 134, 28-32. Jiang, Z. H., Steinsberger, S. C. & Shih, J. C. H. (1987). In situ utilization of biogas on a poultry farm: heating, drying and animal brooding. Biomass, 14, 269-81. Jones, H. B. & Ogden, E. A. (1984). Biomass energy potential from livestock and poultry wastes in the Southern United States. Biomass, 6, 25-35. McInerney, M. J., Bryant, M. P. & Pfennig, N. (1979). Anaerobic bacterium that degrades fatty acids in syntrophic association with methanogens. Archiv. Microbiol., 122,129-35. Patel, G. B., Khan, A. W. & Roth, L. A. (1978). Optimum levels of sulfate and iron for the cultivation of pure cultures of methanogens in synthetic media. J. Appl. Microbiol., 5, 347-56. Scherer, P. & Sahm, M. (1981a). Influence of sulfur containing compounds on the growth of Methanosarcina barkeri in a defined medium. Eur. J. Appl. Microbiol. Biotechnol., 12,28-35. Scherer, P. & Sabra, H. ( 1981 b). Effect of trace elements and vitamins on the growth of Methanosarcina barkeri. Acta Biotechnol., 1, 57-65. Schoneit, P., Moll, J. & Thauer, R. K. (1979). Nickel, cobalt and molybdenum requirement for growth of Methano-
bacterium thermoautotrophicum. Archiv. MicrobioL, 123, 105-7. Shih, J. C. H. (1984). Economical production and utilization of biogas and by-products from the NCSU poultry waste digester system. In Bioenergy 84, Proceedings of a Conf., Vol. 111Biomass Conversion, pp. 310-16. Speece, R. E. ( 1983). Anaerobic biotechnology for industrial wastewater treatment. Environ. Sci. Technol., 17, 416A-17A. USDA (1978). Estimating US Livestock, Poultry Manure and Nutrient Production, ESCS- 1,2. USDA (1983). The US Poultry Industry: Changing Economics and Structure, Report No. 502. US DOE-PETC (1989). Development of a novel approach for coal bioconversion to alcohol fuels. Topical Report, Contract No. DE-AC22-88PC88815, October. Washington Post (1988). Virginia's largest egg producer says it will cut its work force by half, 21 March. Williams, C. M., Shih, J. C. H. & Spears, J. W. (1986). Effect of nickel on biological methane generation from a laboratory poultry waste digester. Biotechnol. Bioeng., 28, 1608-10. Yang, P. Y. & Chandrasekharan, M. (1988). On-site hybrid anaerobic treatment of particulated poultry wastes. In Alternate Waste Treatment Systems, ed. R. J. Bhamidimarri, P. Y. Yang & M. Chandrasekharan. Elsevier Applied Science, London, pp. 154-65.
Bioconversion of Chicken Wastes to Value-Added Products S. Barik, T. Forgacs & J. Isbister ARCTECH, Inc., 5390 Cherokee Avenue, Alexandria, Virginia 22312, USA
Abstract Increasing quantities of chicken waste concerns the poultry industry because of escalating disposal costs and the potential for environmental pollution. Biological conversion of these wastes to valuable products such as methane and/or chemical feedstocks appears to be feasible. Biomethanation of chicken waste by a sewage sludge microbial consortium produced as much as 69 mol% methane in the gas phase. Acetic and propionic acids were the major acids produced during the bioconversion. Addition of chelating agents and other micronutrients enhanced methane production and shifted the ratios of intermediates accumulated. Preliminary data indicate that more than 60% of the chicken waste carbon was converted and that the nitrogen-rich residue may have potential as a soil additive. Key words: Chicken waste, bioconversion, biomethanation, methane.
INTRODUCTION The poultry industry is made up of large-scale operations, primarily in the southern regions of the United States because of the comparative advantages of climate, low-priced land, and the economics of aggregation and scale (USDA, 1983). Although the increased size of poultry operations has resulted in economical poultry products for the consumer, it has also resulted in the generation of substantial volumes of chicken waste at each location (USDA, 1978). These large volumes of chicken waste pose significant environmental problems associated with the need for proper disposal (Washington Post, 1988). Stringent disposal requirements are now being imposed by environmental agencies resulting in
increased costs for disposal and increased poultry prices for the consumer. Chicken waste was commonly used as a fertilizer in the past when the volume of waste to be disposed was relatively small and widely dispersed. These wastes are undesirable as fertilizers if offensive odors are allowed to develop and because the high ammonia content can harm crops. Thus, the poultry operations are faced with disposal of increasing volumes of wastes which are an economic and environmental liability. Biological production of methane and other byproducts from poultry waste (Jones & Ogden, 1984; Shih, 1984) and from a mixture of cattle manure and chicken waste (L. Gornall, 1989 pers. comm.) has been demonstrated. Feasibility studies on in-situ utilization of fermentation products from chicken waste have been conducted (Jiang et al., 1987; Yang & Chandrasekharan, 1988); however, no technology has been developed thus far to implement this bioconversion process for use by the poultry industry. The development of such a process converting chicken wastes into valueadded products could turn a problem into an economic opportunity for the poultry industry. This paper presents research data to support the technical feasibility for bioconversion of chicken wastes to methane and organic acids.
MATERIALS AND M E T H O D S Chicken waste
Fresh, non-fermented chicken waste samples were collected from Seaboard Farm, Roanoke, Virginia, and Purdue Chicken Farm, Salisbury, Virginia. Ultimate analyses of the Purdue chicken waste were conducted by Commercial Testing and Engineering Co., Norfolk, Virginia. The feed and other foreign materials were separated and the chicken wastes were air-dried before they were
229 Bioresource Technology 0960-8524/91/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
230
S. Barik, T. Forgacs, J. Isbister
ground to - 100 mesh and stored in air-tight containers at room temperature until used. Microbial culture Fresh anaerobic sewage sludge was collected from the primary digestor of the city of Alexandria, Virginia, and was transported to the laboratory under anaerobic conditions. This culture was used as a 20% (v/v) inoculum in all experiments. A methanogenic enrichment culture containing acetate-utilizing Methanothrix sp. and acetateand H2-CO2-utilizing Methanosarcina sp. developed at A R C T E C H was also used in later experiments. These cultures were grown in a specific methanogenic medium as described earlier (Corder et al., 1983). Bacterial identification was based on morphologies using phase contrast and epifluorescence microscopy.
Media and culture conditions Media and culture conditions were essentially those previously described (Bryant, 1972; Balch & Wolfe, 1976; McInerney et al., 1979). The basal salts medium contained low-sulfate minerals, B-vitamins, trace metals and 0.1% yeast extract. The medium was buffered with 40 m u sodium bicarbonate and had a gas phase of 4:1 N2-CO 2. The pH was 7.4 and sodium sulfide (final concentration, 1 mmol liter- ~) was used to reduce the medium before use. Liquid medium was dispensed in 8 ml amounts in butyl rubber stoppered serum tubes (18 x 150 mm) or in 32 ml amounts in 100 ml serum bottles. The tubes or bottles containing medium were crimp sealed and autoclaved for 20 min at 121°C. Chicken waste, in appropriate amounts, was weighed into pre-sterilized serum stoppered tubes or bottles. Autoclaved medium was then added to the tubes or bottles containing chicken waste and was inoculated with freshly collected sewage digestor sludge (20% v/v). Additions of chemicals and methane inhibitors, BESA (2-bromoethanesulfonic acid) or monensin, at specified concentrations were made as required. These procedures were performed in an anaerobic chamber. All samples were inoculated in triplicate and were incubated at 37°C under static conditions. Appropriate controls were included with each set of experiments. Analytical methods Total gas production for each sample was determined using a syringe displacement method. Methane concentration was determined using a
Varian 3700 gas chromatograph fitted with a thermal conductivity detector. Gas samples (125 pl) were injected into a stainless steel column (3 m x 3.2 mm OD) packed with 100/200 mesh Carbosieve S-II (Supelco, Inc., Bellefonte, Pennsylvania). The operating conditions were: oven temperature, 210°C; detector and column temperatures, 220°C; and helium as carrier gas (6 ml min- 1). The peak areas were integrated using a HP 3396A computing integrator. Aliquots of the liquid phase were taken periodically to determine the accumulation of short chain fatty acids and alcohols. These samples were centrifuged, diluted with equal amounts of 0"05 M phosphoric acid, and placed in sample vials. Analysis employed a Hewlett-Packard 5880A gas chromatograph fitted with a flame ionization detector (300°C), split/splitless injector (205°C), a 3-m SP-1000 borosilicate glass precolumn (Supelco) and a 15-m Nukol fused silica capillary column with a 0.5-/xm film thickness (Supelco). The oven temperature was programmed from 48°C for 0.7 min to 200°C using three different rates (15°C min-1 to 120°C, 10°C min -~ to 155°C and 5°C min -~ to 200°C). Helium was used as the carrier gas. Injections (2 pl) were made in the split mode (25: 1) using a HP 7672A automatic sampler. Peak areas were integrated using the HP 5880A data terminal. RESULTS AND DISCUSSION Analysis of the Purdue chicken waste to determine the carbon content of the proposed substrate was the first priority after acquisition of the waste. Results of the analyses (Table 1) show that chicken waste contains more than 37% carbon, sufficient carbon substrate(s) for bioconversion to value-added products. A relatively high nitrogen content (greater than 4%) in the chicken waste could be of importance as a component of the fermentation residue which may have potential as a soil additive. The studies investigating bioconversion of chicken waste were designed using two conversion approaches (Fig. 1). In one approach, the chicken waste was fermented with selected microbial cultures under anaerobic conditions for production of methane. In a second approach, the fermentation of the chicken waste occurred anaerobically in the presence of methane inhibitors to force the accumulation of organic acids and/or alcohols by preventing the production of
231
Bioconversion of chicken wastes to value-added products
Table 1. Ultimate analysis* of Purdue chicken waste Dry weight basis, %
Carbon Hydrogen Nitrogen Sulfur Ash Oxygen (by difference)
37.3 5"3 4.6 0"9 19.2 32.8
*Analysis by Commercial Testing and Engineering Co,
Chickenwaste ]
• Organic waste
Organic"
"~* [ ()rganicchemicals ]
I Soil additives I
production observed at 10% and 20% solids loadings.
Experiment 2 Bioconversion of the Purdue chicken wastes (2.5% w/v) and Seaboard Farm chicken waste (2"5% w/v) was essentially the same over a 60-day incubation period, although bioconversion of the P u r d u e chicken waste showed a shorter lag period (Fig. 2). This short lag period followed by rapid methane production may be attributed to the native microflora or to the bioavailability of the carbon in P u r d u e chicken waste, or both. As m u c h as 109 cm 3 methane g - t of chicken waste was generated within 30 days from the P u r d u e chicken waste with a final production of 140 cm 3 methane g-~ chicken waste within 60 days. Bioconversion of the Seaboard Farms chicken waste reached 127 cm 3 methane g - i chicken waste during a 60-day fermentation.
Table 2. Bioconversion of chicken waste to methane at different solids ioadings*
Fig. 1. Proposed pathway for bioconversion of chicken waste to value-added products.
crn~Methane g chicken waste (CW)
% Solids (w/v)
Autoclaved
methane. In both approaches, value-added products and fermentation residue would be generated. The production of methane, organic acids and alcohols was monitored during the feasibility studies. Experiment 1 Methane p r o d u c t i o n from autoclaved and nonautoclaved P u r d u e chicken waste by the sewage sludge inocula was studied at solids loadings ranging from 0 to 20% (w/v). No significant methane was generated from m e d i u m c o m p o n e n t s when chicken waste was not a d d e d to the reaction mixture (Table 2). M e t h a n e p r o d u c t i o n from the non-autoclaved chicken waste was slightly higher than from the autoclaved chicken waste samples at 2 . 5 - 1 0 % solids loading, indicating that the native microflora could be important for biodegradation of the organic matter present in the chicken waste or that autoclaving alters the bioavailability of the carbon in the chicken waste. T h e conversion of carbon in chicken waste to methane appears to be most efficient when the chicken waste is provided at a solids loading of 5.0%, with significantly decreased m e t h a n e
Non-autoclaved
0(Control,noCW) 2.5 2"5 2-5 142'8 162"0 5.0 126.6 172.5 10.0 85.7 107.9 20.0 43.9 17.2 *Sewage sludge culture, Purdue chicken waste, 45 days incubation, static conditions, 37°C.
0
125. 100. T 75.
~
50.
~ / ~
Q.
o -5
/
25. 0¢
CW
O--O
•
•--
•
PCW
~.~o 10
2'0
3'o
4'o
go
Incubotion(doya) Fig. 2. Biological production of methane from chicken wastes. CW, Seaboard Farm chicken waste; PCW, Purdue Farm chicken waste. Sewage sludge culture; static conditions, 37°C; cm3methane g- =dry wt.
232
S. Barik, T. Forgacs, J. Isbister
Experiment 3 Bioconversion of chicken waste (2.5% solids, w/v), at pH 5 and pH 6, by the sewage sludge microorganisms to acids and alcohols in the presence of methane inhibitors (BESA or monensin) was investigated. Acidic pH has been shown to result in accumulation of short chain organic acids and alcohols (Barik et al., 1988; US DOE-PETC, 1989). Under both pH regimes, acetic acid was the major product formed, although small concentrations of propionic acid were also detected (Table 3) with minimal production of methane. The accumulation of propionic acid in the control (no inhibitor) at pH 5 was probably due to inhibition in methane production at this pH. However, propionic acid was perhaps converted to methane at pH 6. Short chain organic alcohols were not accumulated in the medium during these experiments.
Experiment 4 Methanogens require trace metals and inorganic nutrients for growth and methanogenesis (Patel et al., 1978; Schoneit et al., 1979; Scherer & Sahm, 1981 a, b; Speece, 1983). The presence of optimal concentrations of micronutrients in chicken wastes and their bioavailability during the fermentation are unknown. The addition of chelating agents (to solubilize trace metals) and the addition of selected inorganic nutrients to the fermentation broth were evaluated for enhancing bioconversion of chicken waste to methane. The addition of ferrous sulfate (0.1 mmol liter-1), nickel chloride (10 mmol liter-~) and EDTA (120 mmol liter-1)
resulted in only slightly increased methane production (Table 4). The addition of nickel chloride has been shown to increase biogas formation in a laboratory scale poultry digestor (Williams et al., 1986). The addition of known methanogens to the sewage sludge consortium (no chelator or inorganic nutrient addition) showed a similar small increase in methane production when compared to the control (no chemical or microbial addition) samples. The fermentation broth from the experiments designed to evaluate methane production in the presence of chelators and/or inorganic nutrients was analyzed for the accumulation of short chain acids and alcohols. Although the addition of 0.1 mM ferrous sulfate resulted in a small increase in methane production (Fig. 3), the addition of this trace metal at concentrations of 0-1 and 0.5 mmol liter-~ resulted in decreased organic acid accumulation in the medium. Methane production appeared to increase slightly as the concentration of nickel chloride increased (Fig. 4). Accumulation of acetic acid was greatly enhanced by the presence of 10 mM nickel chloride, while the addition of nickel chloride resulted in a decrease in Table 4. Effect of additives on production of methane from Purdue chicken waste*
Additives, concentration
Methane (mol%)
No additions (control) Fe sulfate -- 0.1 mM Fe chloride - - 5.0 mM Ni chloride -- 10"0 mM Na selenite 0.1 mM Nitrilotriacetate - - 5-0 mM Na citrate - - 5'0 mM E D T A - - 10"0 mM Methanogens
69.0 74.0 69'5 76.9 62-9 71.4 70' 1 73"7 74.8
-
Table 3. Effect of B E S A and monensin on bioconversion of chicken waste"
Sample
Methane (mol%)
Acids, mg g of chicken waste Acetate
*Sewage sludge inoculum, 45 days incubation.
Propionate
100-
pH 5"0 Control ~' + BESA + Monensin
80.
65"2 1'4 3'2
0'4 244'7 213'6
2"5 77"6 77'8
pH 6.0 Control h + BESA + Monensin
_~ s0 ~"
74.6 0"9 2"3
0-2 136.4 136"9
0.04 51'9 62"8
aSewage sludge culture, Purdue chicken waste, static conditions, 37°C, 45 days incubation. BESA: 2-bromoethanesulfonic acid. Note: no alcohols were detected in these samples. hNo inhibitors.
-
40.
20. 0
-2500
Control 0.1 mM FeSO4 5 . 0 mM FeSO4
- 2000
111 Methane
Acetate
I
1000
X
5O0
Propionate
Fig. 3. Effect of ferrous sulfate on bioconversion of chicken waste. Sewage sludge culture; Purdue Farm chicken waste; static conditions; 37°C; 45 days incubation.
Bioconversion of chicken wastes to value-added products -2500 100-
Control m~l 0.1 mM NiCI2 1 5.0 rnM NiCI 2 10 mM NiCt 2
-2000
80-6
0 Methone
lml lID Acetote
1500
I
1000
500
Prooionote
Fig. 4. Effect of nickel chloride on bioconversion of chicken waste. Sewage sludge culture; Purdue Farm chicken waste; static conditions; 37°C; 45 days incubation.
propionic acid accumulation. The addition of sodium citrate into the fermentation medium did not enhance methane production (data not shown) nor the accumulation of propionic acid in the medium. However, accumulation of acetic acid in the medium was significantly greater when sodium citrate was present at 5 and 10 mmol liter-~. These data suggest that manipulation of the medium components as well as medium pH could provide selective pressures for accumulation of specific products during bioconversion of chicken wastes.
Experiment 5 To evaluate the efficacy of adding methanogens to the microbial consortium converting the chicken wastes to methane, the sewage sludge consortium was supplemented with an enrichment culture containing acetate-utilizing Methanothrix sp. and acetate- and H2-CO2-utilizing Methanosarcina sp. developed at ARCTECH. The results indicate that the addition of these methanogens to the sewage sludge consortium resulted in a 10% increase in methane production (Table 4) from Purdue chicken waste.
CONCLUSIONS This research confirms that a bioprocess for conversion of the carbon in chicken waste to methane is technically feasible. Chicken waste and the sewage sludge cultures used in these studies were able to convert the carbon in both the Purdue and Seaboard farm chicken waste to methane and organic acids; however, no alcohols were produced. Based on theoretical calculations, bioconversion of more than 60% of the carbon in the
233
Purdue chicken waste to methane and organic acids was demonstrated. The addition of chelators to the fermentation medium did not result in enhanced methane production, although a slightly higher concentration of methane was observed when 10 mM EDTA was added. Methane production increased with increasing nickel chloride concentration, confirming results reported by Williams et al. (1986). In the presence of 10 mM nickel chloride, propionate concentrations were decreased, while the acetic acid concentration increased by more than twofold. This organic acid accumulation was accompanied by only a slight increase in methane production, suggesting that acetate-cleaving microorganisms were inactive or present in insufficient numbers to efficiently produce methane from acetate. The addition of 0-1 mM ferrous sulfate resulted in a slight increase in methane production coupled with decreases in both acetate and propionate concentrations. The data obtained in these preliminary studies indicate that manipulation of medium components and the addition of methanogens could result in a consortium for use in a commercial process converting chicken waste to value-added products. Successful development of a biotechnology for economical conversion of chicken wastes to methane and other value-added products could significantly enhance the economics of poultry operations. Waste disposal costs could be dramatically decreased and environmental pollution reduced.
ACKNOWLEDGEMENTS We thank Dr Daman S. Walia for supporting this work through ARCTECH in-house Research and Development funding. We also thank Mr Fred Principe for his interest and encouragement.
REFERENCES Balch, W. E. & Wolfe, R. S. (1976). New approach to the cultivation of methanogenic bacteria: 2-mercaptoethane sulfonic acid (HS-COM) dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl. Environ. Microbiol., 32, 781-91. Barik, S., Prieto, S., Harrison, S. B., Clausen, E. C. & Gaddy, J. L. (1988). Biological production of alcohols from coal through indirect liquefaction. Appl. Biochem. Biotechnol., 18, 363-8. Bryant, M. P. (1972). Commentary on the Hungate techniques for culture of anaerobic bacteria. Am. J. Clin. Nutr., 25, 1324-8.
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Corder, R. E., Hook, L. A., Larkin, J. M. & Frea, J. 1. (1983). Isolation and characterization of two methane producing cocci: Methanogenium olentangyi, sp. nov., and Methanococcus deltae, sp. nov. Archiv. Microbiol., 134, 28-32. Jiang, Z. H., Steinsberger, S. C. & Shih, J. C. H. (1987). In situ utilization of biogas on a poultry farm: heating, drying and animal brooding. Biomass, 14, 269-81. Jones, H. B. & Ogden, E. A. (1984). Biomass energy potential from livestock and poultry wastes in the Southern United States. Biomass, 6, 25-35. McInerney, M. J., Bryant, M. P. & Pfennig, N. (1979). Anaerobic bacterium that degrades fatty acids in syntrophic association with methanogens. Archiv. Microbiol., 122,129-35. Patel, G. B., Khan, A. W. & Roth, L. A. (1978). Optimum levels of sulfate and iron for the cultivation of pure cultures of methanogens in synthetic media. J. Appl. Microbiol., 5, 347-56. Scherer, P. & Sahm, M. (1981a). Influence of sulfur containing compounds on the growth of Methanosarcina barkeri in a defined medium. Eur. J. Appl. Microbiol. Biotechnol., 12,28-35. Scherer, P. & Sabra, H. ( 1981 b). Effect of trace elements and vitamins on the growth of Methanosarcina barkeri. Acta Biotechnol., 1, 57-65. Schoneit, P., Moll, J. & Thauer, R. K. (1979). Nickel, cobalt and molybdenum requirement for growth of Methano-
bacterium thermoautotrophicum. Archiv. MicrobioL, 123, 105-7. Shih, J. C. H. (1984). Economical production and utilization of biogas and by-products from the NCSU poultry waste digester system. In Bioenergy 84, Proceedings of a Conf., Vol. 111Biomass Conversion, pp. 310-16. Speece, R. E. ( 1983). Anaerobic biotechnology for industrial wastewater treatment. Environ. Sci. Technol., 17, 416A-17A. USDA (1978). Estimating US Livestock, Poultry Manure and Nutrient Production, ESCS- 1,2. USDA (1983). The US Poultry Industry: Changing Economics and Structure, Report No. 502. US DOE-PETC (1989). Development of a novel approach for coal bioconversion to alcohol fuels. Topical Report, Contract No. DE-AC22-88PC88815, October. Washington Post (1988). Virginia's largest egg producer says it will cut its work force by half, 21 March. Williams, C. M., Shih, J. C. H. & Spears, J. W. (1986). Effect of nickel on biological methane generation from a laboratory poultry waste digester. Biotechnol. Bioeng., 28, 1608-10. Yang, P. Y. & Chandrasekharan, M. (1988). On-site hybrid anaerobic treatment of particulated poultry wastes. In Alternate Waste Treatment Systems, ed. R. J. Bhamidimarri, P. Y. Yang & M. Chandrasekharan. Elsevier Applied Science, London, pp. 154-65.