Anaerobic degradation of citrate under sulfate reducing and methanogenic conditions

Citrate is an important component of metal processing effluents such as chemical mechanical planarization wastewaters of the semiconductor industry. Citrate can serve as an electron donor for sulfate reduction applied to promote the removal of metals, and it can also potentially be used by methanogens that coexist in anaerobic biofilms. The objective of this study was to evaluate the degradation of citrate with sulfate-reducing and methanogenic biofilms. During batch bioassays, the citrate, acetate, methane and sulfide concentrations were monitored. The results indicate that independent of the biofilm or incubation conditions used, citrate was rapidly fermented with specific rates ranging from 566 to 720 mg chemical oxygen demand (COD) consumed per gram volatile suspended solids per day. Acetate was found to be the main fermentation product of citrate degradation, which was later degraded completely under either methanogenic or sulfate reducing conditions. However, if either sulfate reduction or methanogenesis was infeasible due to specific inhibitors (2-bromoethane sulfonate), absence of sulfate or lack of adequate microorganisms in the biofilm, acetate accumulated to levels accounting for 90–100% of the citrate-COD consumed. Based on carbon balances measured in phosphate buffered bioassays, acetate, CO₂ and hydrogen are the main products of citrate fermentation, with a molar ratio of 2:2:1 per mol of citrate, respectively. In bicarbonate buffered bioassays, acetogenesis of H₂ and CO₂ increased the yield of acetate. The results taken as a whole suggest that in anaerobic biofilm systems, citrate is metabolized via the formation of acetate as the main metabolic intermediate prior to methanogenesis or sulfate reduction. Sulfate reducing consortia must be enriched to utilize acetate as an electron donor in order to utilize the majority of the electron-equivalents in citrate.     

The effect of incubation temperature on steady-state concentrations of hydrogen and volatile fatty acids during anaerobic degradation in slurries from wetland sediments

Abstract Increasing the incubation temperature of two swamp slurries from 2°C to37°C resulted in a 8- to 18-fold increase in the H₂ partial pressure. The concentration of volatile fatty acids remained fairly constant except for butyrate, which decreased with increasing temperature. Calculation of Gibbs free energies of syntrophic degradation of butyrate and propionate, and of methanogenesis from acetate and H₂ revealed that these reactions were exergonic after the slurries had stabilized at the incubation temperatures. The changes in H₂ partial pressure and butyrate concentration with temperature were found important to render the processes exergonic within the tested temperature range.     

Sulfide enhances methanogenesis in nitrate-containing methanogenic cultures

The effects of sulfide on nitrate reduction and methanogenesis using butyrate as a carbon source were investigated in a mixed mesophilic, methanogenic culture. In the sulfide-free medium, 25-75 mg l⁻¹ nitrate markedly inhibited the efficiencies of acetogenesis and methanogenesis processes. Adding 25 mg-S l⁻¹ increased methane production in nitrate-amended medium. Low sulfide levels shifted the nitrate reduction pathway from denitrification to dissimilatory nitrate reduction to ammonia (DNRA), thereby reducing the amounts of toxic nitric oxide and nitrous oxide produced that inhibit methanogenesis. The dose of 25 mg l⁻¹ sulfide was oxidized completely, during which heterotrophic DNRA predominated. The oxidized forms of sulfide reformed, limiting induction of the heterotrophic denitrification pathway. The actions of heterotrophic and autotrophic DNRA bacteria, denitrifiers, sulfate-reducing bacteria and methanogens mitigate nitrate toxicity during methanogenesis in an anaerobic process.     

Effect of different acetate:propionate ratios on the methanogenic community during thermophilic anaerobic digestion in batch experiments

The present study investigated the metabolism of different acetate:propionate ratios (0.25, 0.33, 0.5, 1.0, 2.0, 3.0, 4.0) in equimolar carbon concentration during an anaerobic decomposition process under defined laboratory conditions and evaluated the engaged methanogenic community. Significant differences on a metabolic level (gas production, gas composition, volatile fatty acid (VFA) concentration) were observed between acetate:propionate ratios ≤1 and ≥2. Generally ratios ≥2 resulted in a faster methane production and VFA decomposition compared to ratios ≤1. Regarding the composition of the methanogenic community as well as the abundance of Methanosarcinales these differences were not reflected in an appropriate manner when DNA based methods (dHPLC and qPCR) were applied. However, by a culture based approach these differences could be documented showing a significant difference in the number of cultivable methanogens between initial acetate:propionate ratios ≤1 and ≥2.     

Effect of 2-bromo-ethane sulfonate, molybdate and chloroform on acetate consumption by methanogenic and sulfate-reducing populations in freshwater sediment

The relative importance of methanogenesis and sulfate reduction in freshwater sediment supplemented with acetate was investigated. Addition of acetate stimulated both methane formation and sulfate reduction, indicating that an active aceticlastic population of methanogens and sulfate reducers was present in the sediment. Sulfate reducers were most important in the consumption of acetate. However, when sulfate reducers were inhibited, acetate was metabolised at a similar rate by methanogens. Acetate, propionate and valerate accumulated only when both processes were inhibited by the combined addition of 2-bromo-ethane sulfonate and molybdate. The relative amounts of acetate, propionate and valerate were 93, 6 and 1 mol%, respectively. These results demonstrate the role of acetate as a key intermediate in the terminal step of organic matter mineralisation in the sediment. Addition of chloroform inhibited both methanogenesis and sulfate reduction. We studied the inhibitory effect of CHCl(3) on homoacetogenic bacteria, sulfate-reducing bacteria and methanogens. The results showed that inhibition by CHCl(3) correlates with microorganisms, which operate the acetyl-CoA cleavage pathway. We propose that chloroform can be used to elucidate the role of different metabolic types of sulfate reducers to sulfate reduction in natural environments.     

Propionate metabolism in a methanogenic enrichment culture. Direct reductive carboxylation and acetogenesis pathways

Abstract Serial dilutions of methanogenic sludges in propionate medium gave a methanogenic non-acetoclastic enrichment degrading 1 mol of propionate to 1.6 mol of acetate and 0.17 mol of methane, with a transient accumulation of butyrate. NMR recordings showed the conversion of [2-¹³C]- and [3-¹³C]-propionate to [3-¹³C]- and [4-¹³C]-butyrate, respectively, thus demonstrating a reductive carboxylation of propionate to butyrate. The labelling found in the accumulated acetate and fermentation balances also suggested that reductive carboxylation was the major pathway involved in propionate conversion to acetate.     

Biotechnological intensification of biogas production

The importance of syntrophic relationships among microorganisms participating in biogas formation has been emphasized, and the regulatory role of in situ hydrogen production has been recognized. It was assumed that the availability of hydrogen may be a limiting factor for hydrogenotrophic methanogens. This hypothesis was tested under laboratory and field conditions by adding a mesophilic (Enterobacter cloacae) or thermophilic hydrogen-producing (Caldicellulosyruptor saccharolyticus) strain to natural biogas-producing consortia. The substrates were waste water sludge, dried plant biomass from Jerusalem artichoke, and pig manure. In all cases, a significant intensification of biogas production was observed. The composition of the generated biogas did not noticeably change. In addition to being a good hydrogen producer, C. saccharolyticus has cellulolytic activity; hence, it is particularly suitable when cellulose-containing biomass is fermented. The process was tested in a 5-m³ thermophilic biogas digester using pig manure slurry as a substrate. Biogas formation increased at least 160–170% upon addition of the hydrogen-producing bacteria as compared to the biogas production of the spontaneously formed microbial consortium. Using the hydrogenase-minus control strain provided evidence that the observed enhancement was due to interspecies hydrogen transfer. The on-going presence of C. saccharolyticus was demonstrated after several months of semicontinuous operation.     

Sulfate reduction from phosphogypsum using a mixed culture of sulfate-reducing bacteria

Phosphogypsum (CaSO₄), a primary by-product of phosphoric acid production, is accumulated in large stockpiles and occupies vast areas of land. It poses a severe threat to the quality of water and land in countries producing phosphoric acid. In this study, the potential of sulfate-reducing bacteria for biodegradation of this sulfur-rich industrial solid waste was assessed. The effect of phosphogypsum concentration, carbon and nitrogen sources, temperature, pH and stirring on the growth of sulfate-reducing bacteria was investigated. Growth of sulfate-reducing bacteria was monitored by measuring sulfide production. Phosphogypsum was shown to be a good source of sulfate, albeit that the addition of organic carbon was necessary for bacterial growth. Biogenic sulfide production occurred with phosphogypsum up to a concentration of 40 g L⁻¹, above which no growth of sulfate-reducing bacteria was observed. Optimal growth was obtained at 10 g L⁻¹ phosphogypsum. Both the gas mixture H₂/CO₂ and lactate supported high amounts of H₂S formation (19 and 11 mM, respectively). The best source of nitrogen for sulfate-reducing bacteria was yeast extract, followed by ammonium chloride. The presence of nitrate had an inhibitory effect on the process of sulfate reduction. Stirring the culture at 150 rpm slightly stimulated H₂S formation, probably by improving sulfate solubility.     

The energetic consequences of hydrogen gradients in methanogenic ecosystems

Abstract In the dense microbial aggregates usually found in methanogenic waste water treatment systems, hydrogen has to diffuse from producers to consumers at considerable rates. The ensuing hydrogen gradients dissipate part of the potential energy that would otherwise be available to the hydrogen-consuming organisms. The present paper evaluates the energetic consequences of this phenomenon.     

Bicarbonate-dependent production and methanogenic consumption of acetate in anoxic paddy soil

Abstract Acetate turnover was measured in slurries of anoxic methanogenic paddy soil after addition of carrier-free [2-¹⁴C]-acetate. Acetate concentrations stayed fairly constant for about 1–2 days indicating steady state between production and consumption reactions. Depending on the experiment, acetate concentrations were between 100 and 3000 μM. Turnover rates were determined from the logarithmic decrease of [2-¹⁴C]-acetate or from the accumulation of acetate in the presence of chloroform resulting in similar values, i.e. 12–13 nmol h⁻¹g⁻¹d.w. soil at 17°C and 36–88 nmol h⁻¹g⁻¹d.w. at 30°C. Acetate consumption was completely inhibited by chloroform. The respiratory index (RI) was < 0.27. Hence, acetate was apparently consumed by methanogenic bacteria. About 80–90% of the CH₄ produced originated from the methyl group of acetate. The role of homoacetogenesis for acetate production was studied by measuring the incorporation of radioactive bicarbonate into acetate. In different experiments, CO₂ incorporation accounted for fractions of 1–60% of the acetate produced, about 10% being the most likely value for steady-state conditions. The fraction increased at high H₂ concentrations and decreased at high acetate concentrations. The rate of H₂ production that was required for chemolithotrophic acetate production from CO₂ was two orders of magnitude higher than the actually measured rate. Hence, most of the CO₂ incorporation into acetate was caused by electron donors other than H₂ (e.g., carbohydrates) and/or by exchange reactions.     

Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction

Abstract Enrichment cultures from marine sediments mineralized benzene while using sulfate as the terminal electron acceptor. Parallel cultures using river marsh sediment displayed no activity. Mineralization was confirmed by release of ¹⁴CO₂ from radiolabeled benzene. The dependence on sulfate reduction was demonstrated by stoichiometric balances and the use of specific inhibitors. This work supports recent observations that anaerobic benzene degradation takes place coupled to sulfate reduction.     

Sulfate reduction by a syntrophic propionate-oxidizing bacterium

The syntrophic propionate-oxidizing bacterium MPOB was able to grow in the absence of methanogens by coupling the oxidation of propionate to the reduction of sulfate. Growth on propionate plus sulfate was very slow (=0.024 day^–1). An average growth yield was found of 1.5 g (dry weight) per mol of propionate. MPOB grew even slower than other sulfate-reducing syntrophic propionate-oxidizing bacteria. The growth rates and yields of strict sulfate-reducing bacteria (Desulfobulbus sp.) grown on propionate plus sulfate are considerably higher.     

End products of metabolism in the anaerobic ciliate Trimyema compressum

Abstract The products of anaerobic and micro-aerobic (0.8% O₂) metabolism of the sapropelic ciliate Trimyema compressum strain N were studied. Under anaerobic conditions ethanol was formed in large amounts representing 44% of the total carbon excreted. Acetate, lactate, formate, CO₂ and H₂ were minor products, while succinate was formed in hardly detectable amounts. Under micro-aerobic conditions O₂ was consumed, CO₂ and formate were produced as major end products and no H₂, ethanol and succinate were formed.     

Microautoradiographic studies on host-parasite interactions II. The exchange of 3H-lysine between Uromyces phaseoli and Phaseolus vulgaris

A new species of anaerobic bacterium that degrades the even-numbered carbon fatty acids, butyrate, caproate and caprylate, to acetate and H₂ and the odd-numbered carbon fatty acids, valerate and heptanoate, to acetate, propionate and H₂ was obtained in coculture with either an H₂-utilizing methanogen or H₂-utilizing desulfovibrio. The organism could be grown only in syntrophic association with the H₂-utilizer and no other energy sources or combination of electron donor and acceptors were utilized. It was a Gram-negative helical rod with 2 to 8 flagella, about 20 nm in diameter, inserted in a linear fashion about 130 nm or more apart along the concave side of the cell. It grew with a generation time of 84 h in co-culture with Methanospirillum hungatii and was present in numbers of at least 4.5×10^-6 per g of anaerobic digestor sludge.