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Applied and Environmental Microbiology, August 2005, p. 4728-4735, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4728-4735.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Biological Control of Hog Waste Odor through Stimulated Microbial Fe(III) Reduction

John D. Coates,1* Kimberly A. Cole,2,{dagger} Urania Michaelidou,2 Jennifer Patrick,2 Michael J. McInerney,3 and Laurie A. Achenbach2

Department of Plant and Microbial Biology, University of California, Berkeley, California 94720,1 Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901,2 Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 730193

Received 7 January 2005/ Accepted 15 March 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Odor control and disposal of swine waste have inhibited expansion of swine production facilities throughout the United States. Swine waste odor is associated primarily with high concentrations of volatile fatty acids (VFAs). Here, we demonstrate that stimulated Fe(III) reduction in hog manure can rapidly remove the malodorous compounds and enhance methane production by 200%. As part of these studies, we enumerated the indigenous Fe(III)-reducing population in swine waste and identified members of the family Geobacteraceae as the dominant species. These organisms were present at concentrations as high as 2 x 105 cells g–1. Several pure cultures of Fe(III) reducers, including Geobacter metallireducens, Geobacter humireducens, Geobacter sulfurreducens, Geobacter grbiciae, Geothrix fermentans, and Geovibrio ferrireducens, readily degraded some or all of the malodorous VFAs found in swine manure. In contrast, Shewanella algae did not degrade any of these compounds. We isolated an Fe(III) reducer, Geobacter strain NU, from materials collected from primary swine waste lagoons. This organism degraded all of the malodorous VFAs tested and readily grew in swine waste amended with Fe(III). When raw waste amended with Fe(III) was inoculated with strain NU, the VFA content rapidly decreased, corresponding with an almost complete removal of the odor. In contrast, the raw waste without Fe(III) or strain NU showed a marked increase in VFA content and a rapid pH drop. This study showed that Fe(III) supplementation combined with appropriate bioaugmentation provides a simple, cost-effective approach to deodorize and treat swine waste, removing a significant impediment to the expansion of pork production facilities.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Swine production is an important industry in the United States, with an annual value of more than 12 billion dollars. In 1997, the annual nationwide production was more than 100 million pigs, with a resultant production of 8.5 x 106 tons (dry weight) of manure per year requiring treatment and disposal (21, 28a). The low profit margins associated with swine production and the difficulties associated with poor public acceptance of hog facilities in many regions have resulted in a significant increase in the number of high-density-confinement rearing facilities in an attempt to improve efficiency based on economies of scale. Although hog wastes can be used as a good nutritional source of nitrogen and phosphorous for agronomic crops, the practice of land spreading has come under intensive scrutiny in the last decade because of runoff contamination of water resources and the odor of the untreated slurry. In addition, swine manures contain high concentrations of metals such as iron, copper, zinc, cobalt, and cadmium that are present as both soluble organic complexes and insoluble oxides and can accumulate in a soil environment as a result of land spreading (34). Thus, major air, soil, and water pollution problems are associated with storage and disposal of swine waste.

Odor management has become a crucial issue for the swine industry, and the sustainability, productivity, and profitability of a producer depend on the extent to which odor emissions can be controlled (62). Malodorous components of swine waste can be divided into four classes: volatile fatty acids (VFAs), indoles and phenols, ammonia and volatile amines, and volatile sulfur compounds (63). Each of these components is primarily microbially formed through the activity of fermentative bacteria that degrade the complex organics present in the waste (56, 62). These compounds accumulate in storage facilities, where the mixtures of feces and urine collected from under-floor collection pits decompose under the prevailing anaerobic conditions. Although these pits are left exposed to the atmosphere, the high organic content of swine waste ensures that oxygen diffusing from the atmosphere is rapidly removed biologically (10). As such, although the malodorous components are readily biodegraded by many respiratory microbial species, the activity of these organisms is inhibited by the limiting availability of suitable electron acceptors or by the organisms' low rate of metabolism under methanogenic conditions (29, 48).

Swine waste can be treated microbially in aerobic activated sludge systems; however, these systems are energy intensive, and there is a large production of microbial biomass (1.0 to 1.5 mol mol–1 waste treated) (33) that also requires treatment and disposal. Alternatively, anaerobic treatment processes, such as the use of methanogenic bioreactors, can be applied for the removal of odor and the generation of combustible biogases. This type of treatment has the advantages over aerobic systems of lower energy input and of much lower yield of biomass per liter of treated waste (0.032 mol mol–1 waste treated) (25). However, traditional methanogenic systems are slow due to the long doubling times of the fatty acid-degrading, syntrophic bacteria whose activity is central to the process (46). Alternative treatment systems based on sulfate- or nitrate-reducing bacteria could potentially be faster, due to the favorable thermodynamics of these metabolisms (46, 58). However, both of these systems can produce noxious and toxic products (e.g., sulfide, nitrite, and nitrogen oxides).

Here, we describe an alternative approach for treatment of the malodorous compounds associated with hog waste by stimulating their removal through Fe(III) supplementation and bioaugmentation with a novel dissimilatory Fe(III)-reducing organism. This process can be used to treat hog waste directly in primary and secondary lagoons without the need to construct dedicated bioreactors. Microbial Fe(III) reduction is an energetically favorable process, and in the natural environment, Fe(III)-reducing bacteria (FeRB) can outcompete and inhibit both sulfate-reducing and methanogenic bacteria (10, 38, 44, 60). FeRB also have diverse metabolisms, and many pure-culture examples exist that can completely oxidize straight- and branched-chain fatty acids and aromatic organics without the need for the activity of the rate-limiting syntrophic bacteria (14, 37). The respiratory end product of microbial Fe(III) reduction, Fe(II), is nontoxic and can be recycled after abiotic reoxidation through its reaction with O2. In addition, added iron will abiotically react with malodorous HS– ions, forming non-odor-causing metal sulfide precipitates. This approach has the potential for the long-term and sustainable removal of the major odor-causing components of hog manure.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Medium and culture conditions.
All FeRB were maintained in an anoxic, defined freshwater medium previously described (11, 16), with 10 mM acetate as the sole electron donor and 10 mM Fe(III) chelated with nitrilotriacetic acid [Fe(III)-NTA] (52) as the sole electron acceptor, using standard anaerobic culturing techniques (3, 28, 49). Anoxic medium was prepared under a headspace of N2-CO2 (80:20, vol/vol) by boiling to remove dissolved O2 prior to dispensing the medium under an N2-CO2 (80:20, vol/vol) gas phase into anaerobic pressure tubes or serum bottles and sealing them with thick butyl rubber stoppers. Freshly prepared medium was sterilized by autoclaving at 121°C for 15 min, and culture incubations were carried out at 30°C in the dark.

Swine waste.
When needed, swine waste was graciously collected by Tom Rosenthal of the Southern Illinois University Carbondale (SIUC) campus Agricultural Research Center (Swine Center) from the primary waste treatment lagoons. The waste was collected from below the surface at the sediment interface into clean canning jars, which were filled to capacity and sealed with airtight screw caps. The collected materials were brought back to the laboratory and used immediately.

MPN counts and PCR analysis.
Freshly collected swine waste was used to inoculate the previously described (8) basal medium in triplicate, amended with 2,6-anthraquinone disulfonate (AQDS) (5 mM) as the electron acceptor and hydrogen (101 kPa), acetate (2 mM), lactate (2 mM), or palmitate (1 mM) as the sole electron donor. Most-probable-number (MPN) series, with hydrogen as the sole electron donor, were also amended with 0.1 mM acetate as an appropriate carbon source. Sodium pyrophosphate (1%, wt/vol) was added to the first dilution tubes in the MPN series to detach the cells from the sediment particles. All MPN tubes were incubated at room temperature in the dark for 60 days prior to analysis. Positives in the MPN series were identified visually by a color change of the medium from tan to red as the AQDS was reduced to its hydroquinone form 2,6-anthrahydroquinone disulfonate.

To identify the dominant AQDS/Fe(III)-reducing organisms in the swine waste, DNA was extracted from the highest-dilution tubes of the MPN series showing positive growth. Cell pellets harvested from 1.5 ml of the respective culture broths were prepared for PCR by adding 40 µl sterile H2O and 5 µl chloroform and lysing the cells by heating at 95°C for 10 min. PCR analysis to detect members of the Geobacteraceae and Geothrix and Shewanella species was performed using primer sets specific for each of these species, as previously described (10).

Isolation of strain NU.
Enrichments for FeRB were established with freshly collected swine waste from the swine lagoons at the SIUC Agricultural Research Center (Swine Center). Acetate (10 mM) was used as the sole electron donor, with Fe(III)-NTA (10 mM) as the sole electron acceptor. After 2 weeks of incubation at 30°C, several of the enrichments were visually positive for Fe(III) reduction (indicated by a color change from translucent orange to colorless with the presence of a white precipitate). One highly enriched culture was obtained by continual transfer over several weeks (using a 10% inoculum) into fresh medium with acetate (10 mM) and Fe(III)-NTA (10 mM). A new Fe(III)-reducing organism was isolated by plating the active culture on medium solidified with 2% (wt/vol) Noble agar and incubating it at 30°C in the dark under anaerobic conditions. The organism was designated strain NU.

16S rRNA gene sequencing and analysis.
16S rRNA gene sequences were generated as previously described (1, 15). Sequence entry and manipulation were performed with the MacVector 7.2.2 sequence analysis software program for the Macintosh (Oxford Molecular). Sequences of select 16S rRNAs were downloaded from the Ribosomal Database Project (45) and GenBank (6) into the computer program SeqApp (24). FeRB 16S rRNA gene sequences were manually added to the alignment by using secondary-structure information for accurate sequence alignment. Distance, parsimony, and maximum-likelihood analysis of the aligned sequences were based on analysis of 636 base pairs and were performed using PAUP 4.0b10 (57). Bootstrap analysis of 100 replications was conducted using a heuristic search strategy to assess the confidence levels for various clades. GenBank accession numbers for the sequences shown below (see Fig. 3) are as follows: Trichlorobacter thiogenes, AF223382; Geobacter sp. strain CdA-2, Y19190; Geobacter sp. strain CdA-3, Y19191; Geobacter chapellei, U41561; Pelobacter propionicus, X70954; Geobacter sulfurreducens, U13928; Geobacter hydrogenophilus H2, U28173; Geobacter metallireducens, L07834; Geobacter pelophilus, U96918; Geobacter humireducens, AY187306; and Desulfomonile tiedjei, M26635.



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  		FIG. 3. Phylogenetic tree of the 16S rRNA gene sequence data set resulting from distance analysis using the Jukes-Cantor correction. The same topology was obtained using either parsimony or maximum likelihood and was based on 636 sequence characters.

 
Swine waste treatment.
In order to determine the potential applicability of strain NU, or FeRB in general, to the treatment of swine manure odor, freshly collected waste from the SIUC primary lagoon was dispensed in 1-liter aliquots into three 2-liter bottles under an aerobic headspace and sealed with thick butyl rubber stoppers. One of the prepared bottles was inoculated (10% by volume) with an active acetate-oxidizing Fe(III)-reducing culture of strain NU and amended with approximately 100 mM amorphous Fe(III) oxide, one bottle was merely amended with approximately 100 mM amorphous Fe(III) oxide, and the third bottle was unamended. All bottles were incubated in the dark at 30°C. Headspace samples were collected at various intervals for methane analysis. Liquid samples were also collected at various intervals for analyses of VFA, pH, and Fe(II) and total iron content.

Analytical techniques.
Fe(III) concentrations were determined colorimetrically by the ferrozine assay after HCl extraction as previously described (43). Concentrations of 2,6-anthrahydroquinone, the reduced form of 2,6-anthraquinone, were determined colorimetrically at 450 nm as described previously (12, 39, 41). Cell growth was determined by direct microscopic cell counts or by protein assay as previously described (8, 15). Nitrate concentrations were determined by ion chromatography of aqueous samples, using a Dionex DX500 equipped with an AS9-SC column with a 2 mM sodium carbonate-7.5 mM sodium bicarbonate mobile phase at a flow rate of 2 ml/min. Organic acid concentrations were analyzed by high-pressure liquid chromatography with UV detection (Shimadzu model SPD-10A) using an HL-75H+ cation-exchange column (Hamilton no. 79476). The eluent was 0.016 N H2SO4 at a flow rate of 0.4 ml min–1. Biogas analysis was performed on 1-ml aliquots of headspace gas collected with an N2-flushed airtight syringe. The biogas samples were injected into a gas chromatograph equipped with a Porapak N 80/100-mesh column (12 ft by 1/8 in. [diameter]; stainless steel) and a thermal conductivity detector. Chromatography was performed with an N2 mobile phase at a flow rate of 20 ml min–1 and a column temperature of 65°C. The injector and detector temperatures were 180 and 200°C, respectively.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Swine waste and microbial Fe(III) reduction.
FeRB represent a very diverse group, both phenotypically and taxonomically, and demonstrate a broad degradative capacity (14, 37, 40). In addition to the oxidation of simple fatty acids and alcohols, many important environmental contaminants, such as aromatic hydrocarbons, halogenated solvents, and chlorinated benzenes, are degraded under Fe(III)-reducing conditions (7, 11, 14, 31, 39). Several pure-culture isolates of Fe(III)-reducing bacteria are known to oxidize long-chain fatty acids (13, 16) and aromatics such as toluene and benzoate (17, 18) and to dehalogenate chlorinated solvents such as tetrachloromethane and tetrachloroethylene (32, 51).

Hog slurry contains high concentrations of soluble branched- and straight-chain VFAs and monoaromatics as well as sulfur-containing compounds released as a result of the hydrolytic activity of bacteria (63). Thirteen compounds (Table 1) have been identified as the key causative agents of swine waste odor (G. Riskowski, University of Illinois, personal communication). The characteristic odor is associated primarily with the VFA content, especially butyrate, isobutyrate, valerate, and isovalerate (63). To assess the potential for Fe(III)-reducing bacteria to biodegrade the dominant VFAs present in swine waste, we screened active pure cultures of Geobacter metallireducens, G. humireducens, G. sulfurreducens, Geobacter grbiciae, Geothrix fermentans, Shewanella algae, and Geovibrio ferrireducens for their ability to degrade the individual VFAs (Table 2). All of the Geobacter species tested except G. sulfurreducens were capable of oxidizing some or all of the compounds tested (Table 2). Several of the Geobacter species could utilize the VFAs individually (data not shown) or as a mixture of all 13 of the components listed in Table 1, as shown for G. metallireducens (Fig. 1). Chromatographic analysis of the VFAs of the G. metallireducens culture revealed complete degradation of acetate, propionate, butyrate, and isobutyrate and the partial removal of valerate and isovalerate (data not shown). The complete removal of valerate and isovalerate was limited by the depletion of the available Fe(III) in these experimental bottles.


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  		TABLE 1. Primary malodorous components of swine waste and their respective concentrationsa

 

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  		TABLE 2. Degradation of the prominent malodorous VFAs associated with swine waste by various phylogenetically diverse Fe(III)-reducing bacteria

 


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  		FIG. 1. Growth and Fe(II) production from Fe(III) by G. metallireducens with the simulated swine waste outlined in Table 2 as the sole electron donor.

 
In addition to the Geobacter species, members of other genera of known Fe(III) reducers, including Geovibrio ferrireducens and Geothrix fermentens, also degraded the VFAs (Table 2), implying that phylogenetically diverse Fe(III) reducers can utilize these compounds and that their activity could potentially be stimulated in swine waste. In contrast, Shewanella algae did not oxidize any of the compounds tested, which is consistent with the fact that Shewanella species are incomplete oxidizers and use a relatively limited range of organic electron donors (40).

Enumeration and isolation of FeRB from the swine lagoon.
FeRB in samples freshly collected from the swine waste lagoons at the Southern Illinois University swine facility were enumerated by MPN. Enumerations were performed using four different carbon and energy sources (H2, acetate, lactate, and palmitate) to account for the different metabolic capabilities of the organisms. AQDS was used as the electron acceptor for these studies to allow easy identification of positives by the change in color from light tan in the oxidized form to bright red in the reduced form (12). Previous studies demonstrated that all tested AQDS-reducing bacteria were also capable of dissimilatory Fe(III) reduction (12, 39). The MPN studies showed the presence of a significant microbial community indigenous to the swine waste lagoon sediments capable of reducing AQDS (Table 3). The microbial counts were similar regardless of the electron donor used, although the hydrogenotrophic population [(2.31 ± 1.33) x 105] was slightly larger than that of the organotrophic acetate-oxidizing FeRB [(9.33 ± 4.17) x 104]. Previous studies demonstrated that anaerobic trophic groups of respiratory bacteria, such as sulfate-reducing bacteria and FeRB, generally fall into two categories: those that completely oxidize multicarbon compounds to carbon dioxide and those that incompletely oxidize multicarbon organics to acetate (17, 61). In general, all of the incomplete oxidizers also use H2 or lactate as suitable electron donors (17, 37, 61). H2 and acetate are the primary end products of the biodegradation of complex organics in anoxic environments and as such are considered to be the most important electron donors for anaerobic microbial respiration (42, 43, 46-48). The different electron donors in the present study were selected to reflect the dominant electron donors available in natural environments and to ensure that both complete and incomplete oxidizers were represented. The MPN results suggest that FeRB capable of using diverse substrates were present in significant numbers.


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  		TABLE 3. Counts of dissimilatory Fe(III)-reducing bacteria in freshly collected swine waste

 
FeRB that represent the alpha, beta, gamma, and epsilon subclasses of the Proteobacteria (reference 40 and references therein) as well as those forming novel lines of descent in the bacterial domain (9, 16, 35, 40) have been isolated. Although there is a large diversity of mesophilic organisms capable of growth by dissimilatory Fe(III) reduction, previous studies have demonstrated that in most environments, the predominant species and most readily isolated strains belong to the family Geobacteraceae in the delta subclass of the Proteobacteria (35) and usually belong to the Geobacter genus (17, 36). In an attempt to identify which organisms may be dominant in the swine waste lagoons, DNA was extracted from the highest dilution of the MPN series that showed growth and was amplified with 16S rRNA gene primer sets specific for members of the Geobacteraceae or for Shewanella or Geothrix species. The results revealed that members of the family Geobacteraceae were the dominant FeRB present, regardless of the electron donor (Fig. 2). No PCR products were observed with primer sets specific for Shewanella or Geothrix species. These results further demonstrate the importance of the family Geobacteraceae in Fe(III)-reducing environments and are consistent with the findings of several previous studies (27, 36, 53, 55).



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  		FIG. 2. Gel electrophoresis of PCR products obtained through amplification of the DNA extracted from the highest-positive-dilution tubes of the swine waste MPN series, using primer sets specific for members of the Geobacteraceae and Geothrix and Shewanella species. Lanes are identified in each quadrant. Lanes 1 and 14, molecular weight markers; lanes 2, 3, and 4, pure-culture controls of Geobacter metallireducens, Geothrix fermentans, and Shewanella algae, respectively; lanes 5, 6, and 7, PCR products obtained from amplification of 16S rRNA genes from MPN tubes incubated with H2 as the electron donor; lanes 8, 9, and 10, PCR products obtained from amplification of 16S rRNA genes from MPN tubes incubated with lactate as the electron donor; lanes 11, 12, and 13, PCR products obtained from amplification of 16S rRNA genes from MPN tubes incubated with acetate as the electron donor.

 
Analysis of the Fe(III) and total iron content of freshly collected samples from the swine waste lagoons indicated that all of the iron (1.4 mmol/liter) was in the reduced form [Fe(II)] and was thus not available to the FeRB for growth. Thus, while swine waste contains FeRB, their activity appears to be limited by availability of Fe(III).

Isolation of strain NU.
Small colonies were apparent on the surface of the agar plates after 1 week of incubation. The visible colonies ranged from 1 to 2 mm in diameter and were pink in color and surrounded by a clear halo in the orange-colored agar. Several of the pink colonies were selected for isolation and were transferred into fresh media amended with Fe(III)-NTA (10 mM) and acetate (10 mM). Several identical isolates were obtained, and one, strain NU, was selected for further characterization.

Strain NU was a completely oxidizing, nonfermentative, gram-negative, obligate anaerobe (data not shown). Analysis of the partial sequence of the 16S rRNA gene placed strain NU in the Geobacteraceae family in the delta subclass of the Proteobacteria (Fig. 3), with its closest relative being Trichlorobacter thiogenes. Physiological characterization of this organism demonstrated that it could oxidize the individual VFAs listed in Table 1, coupled to dissimilatory Fe(III) reduction (data not shown). Strain NU grew and reduced Fe(III) quite rapidly in the raw swine waste without any dilution (data not shown). Interestingly, this organism grew optimally in the raw swine waste amended with 100 mM Fe(III). Any dilution of the swine waste or increase in the Fe(III) concentration resulted in a significant decrease in the rate of Fe(III) reduction. Analysis of the VFA concentration of the inoculated waste indicated that strain NU utilized the VFAs in order of molecular size, starting with the least complex VFA, acetate (data not shown). After 6 days of incubation, in excess of 65% of the initial acetate and 28% of the initial propionate had been removed, at which point the organism became limited for an electron acceptor as it had reduced all of the available Fe(III) sources. These results indicate that strain NU has good potential for the biological removal of the odor-causing components of swine waste from undiluted swine waste lagoons.

Treatment of swine waste with strain NU.
In order to determine the potential applicability of strain NU, or FeRB in general, to the treatment of swine manure odor, freshly collected waste from the SIUC primary lagoon was inoculated with an active acetate-oxidizing, Fe(III)-reducing culture of strain NU and amended with Fe(III). The results obtained were compared with uninoculated controls, with and without Fe(III) amendments. The added Fe(III) was rapidly reduced within the first 3 weeks of the 5-week incubation in both the inoculated and uninoculated samples (Fig. 4a). High-pressure liquid chromatography analysis of the swine waste throughout the incubation indicated that strain NU with Fe(III) supplementation had a significant effect on the VFA content (Fig. 4b). At the initiation of the experiment, the VFA content in the swine waste of each bottle was dominated by acetate, which represented an average of almost 42% of the total VFA content (Table 4). During the first week of incubation, the total VFA content in all samples increased from an initial average concentration of 33 mM (Fig. 4b). The total VFA content in the untreated controls rapidly and continuously increased throughout the 5 weeks of the incubation, to achieve a maximum total VFA concentration of greater than 100 mM, with a net increase of greater than 68 mmol liter–1 VFA. This was likely due to the activity of fermentative bacteria, degrading the complex organics present in the waste that exceeded the ability of the indigenous syntrophic and methanogenic populations to remove the products of fermentative metabolism (Fig. 4b) (56, 62). The VFA content after the 5-week incubation was dominated by acetate and propionate, which represented 47% and 35% of the total VFA content, respectively (Table 4).



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  		FIG. 4. Treatment of raw swine waste through amendment with Fe(III) alone or Fe(III) and strain NU. Results for unamended control sample are shown in panel b. (a) Fe(II) and total iron content; (b) total VFA (acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate) concentration; (c) methane production; and (d) pH values in treated and untreated swine waste.

 

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  		TABLE 4. Concentration of individual VFAs throughout the 5-week treatment of swine waste

 
In contrast to the untreated samples, both of the treated samples showed a net decrease in total VFA content after the 5-week incubation (Fig. 4b). The uninoculated samples amended with Fe(III) showed the largest increase in total VFA content after the first week, reaching a maximum of almost 66 mM, which was dominated by acetate and propionate (representing 50% and 33% of the total VFA content, respectively) (Fig. 4b and Table 4). During the following 4 weeks, the total VFA content in these samples continuously decreased to 32 mM and was dominated by propionate (70% of the total VFA content) (Table 4). In the case of the samples inoculated with strain NU and amended with Fe(III), there was an initial increase in total VFA during the first week, which was primarily the result of a rapid increase in the propionate concentration (Table 4). This was followed by a rapid and continuous removal of VFAs during the next several weeks to achieve a total VFA concentration of less than 5.5 mM after 5 weeks of incubation. The final VFA content was composed of almost equimolar amounts of acetate (1.40 mM), propionate (1.57 mM), and isobutyrate (1.64 mM) (Table 4). After 5 weeks of incubation, no significant unpleasant odor could be detected in these samples, while a pungent odor was still obvious in the Fe(III)-amended and untreated control samples.

Interestingly, methane production in the Fe(III)-supplemented samples inoculated with strain NU was significantly greater (twofold) than in the untreated samples (Fig. 4c). This effect was particularly evident once the Fe(III) in the treated samples was depleted after the initial 3 weeks of incubation (Fig. 4a and c). Although microbial Fe(III) reduction is thermodynamically more favorable than methanogenesis (10, 38, 42) and can inhibit methanogenesis (44, 60), this effect occurs only in environments where the electron donor is limited, such as aquifer systems (10, 38, 42). In swine waste, there is an abundant supply of suitable electron donors to support different microbial respiratory processes simultaneously. Previous studies demonstrated that in the absence of a suitable electron acceptor, some Fe(III)-reducing bacteria can grow syntrophically with H2-using bacteria (20, 30). In the treated swine waste, strain NU and the indigenous Fe(III)-reducing populations were probably metabolizing the VFAs coupled to Fe(III) reduction during the first 3 weeks of incubation. Once the Fe(III) was depleted, it is quite possible that these organisms switched to syntrophic metabolism, which would explain the continued metabolism of VFAs and methane production after Fe(III) was used up (Fig. 4a, b, and c).

The pH of the untreated samples decreased, indicating acidity, during the first week of incubation as a result of the rapid buildup of VFAs (Fig. 4d). In contrast, the pH in the treated samples remained relatively constant at circum-neutral values throughout the 5-week incubation (Fig. 4d). The degradation of complex organic material under methanogenic conditions is dependent on stable environmental conditions, such as pH, to sustain the activity of methanogens and slow-growing syntrophic populations (19, 46-48, 50, 54). The inhibitory effect of decreasing pH is known to be enhanced by VFAs. As the pH decreases, the concentration of the undissociated form of the acid (HA) increases relative to the ionized form (A–). Undissociated short-chain organic acids can readily diffuse across biological membranes and dissipate the proton motive force (4). Fe(III) supplementation, with or without inoculation with strain NU, kept total VFA concentrations much lower than those observed in the untreated samples (Fig. 4b and Table 4). The concentrations of the undissociated forms of VFAs in the treated samples, with and without inoculation, peaked during the first 3 weeks of incubation (2.3 and 1.9 mM, respectively) and then declined to less than 1 mM after 7 weeks. Most of the time, these values were higher than those shown to inhibit acetoclastic methanogenesis and propionate degradation in acclimated sludge (22, 23) and to cause unstable operating conditions in sludge digestors (2). However, the concentration of undissociated acids was lower than that in the untreated samples, which steadily increased from an initial value of about 1.3 mM to a final value of 8.7 mM after 7 weeks. The continued degradation of VFAs, which prevented large changes in the pH, plus the increased population levels of fatty acid degraders due to Fe(III) supplementation and inoculation with strain NU may explain the large and continued production of methane after Fe(III) was depleted (Fig. 4c).

Fe(III)-reducing systems offer a unique solution for the biological treatment and odor control of swine waste for a number of reasons: the broad range of compounds that can be degraded by FeRB; the energetically favorable redox process, which should alleviate the thermodynamic limitations on VFA degradation (26); the cheap recycling of the terminal electron acceptor by air oxidation; the binding of volatile sulfides as nonsoluble metal sulfides, which may be collected and used as a potential nutritional sulfur source; and the potential high value of the biomass produced, which may be used as inocula for bioremediative technologies involving metal reduction or may alternatively have nutritional value as a source of single-cell protein. FeRB systems could probably be developed without much modification of existing lagoon storage systems in swine production facilities, through inoculation with appropriate FeRB such as Geobacter strain NU and seeding with an appropriate form of Fe(III) as the terminal electron acceptor. Our studies indicate that FeRB communities are present in swine wastes and are dominated by members of the Geobacteraceae but are limited by the supply of Fe(III) as the electron acceptor. It is interesting that the production of methane was significantly enhanced in the swine waste samples treated with Fe(III) and inoculated with an active culture of strain NU. This is an added bonus to the proposed odor treatment strategy as it provides a recoverable energy source. Whether enhancement of methane production is due only to the effective maintenance of a neutral pH in the wastes, providing a more ideal growth environment for the methanogenic and syntrophic populations, or is further complemented by strain NU, providing a more rapid and effective catabolism of complex organic materials through syntrophic cooperation, will require further study. On the other hand, microbial terminal-electron-accepting processes are not mutually exclusive, and previous studies have demonstrated that mixed respiratory processes can occur in heavily contaminated environments (5).


   ACKNOWLEDGMENTS
 
Support to J.D.C. for this research was provided by grant 98I-13 from the Illinois Council on Food and Agriculture Research.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720. Phone: (510) 643-8455. Fax: (510) 642-4995. E-mail: jcoates{at}nature.berkeley.edu. Back

{dagger} Present address: BioInsite LLC, Southern Illinois Research Park, Carbondale, IL 62903. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Applied and Environmental Microbiology, August 2005, p. 4728-4735, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4728-4735.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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