Sulfur reduction in acid rock drainage environments

Microbiological suitability of acidophilic sulfur reduction for metal recovery was explored by enriching sulfur reducers from acidic sediments at low pH (from 2 to 5) with hydrogen, glycerol, methanol and acetate as electron donors at 30 °C. The highest levels of sulfide in the enrichments were detected at pH 3 with hydrogen and pH 4 with acetate. Cloning and sequencing of the 16S rRNA gene showed dominance of the deltaproteobacterial sulfur-reducing genus Desulfurella in all the enrichments and subsequently an acidophilic strain (TR1) was isolated. Strain TR1 grew at a broad range of pH (37) and temperature (2050 °C) and showed good metal tolerance (Pb2+, Zn2+, Cu2+, Ni2+), especially for Ni2+ and Pb2+, with maximal tolerated concentrations of 0.09 and 0.03 mM, respectively. Different sources of sulfur were tested in the enrichments, from which biosulfur showed fastest growth (doubling time of 1.9 days), followed by colloidal, chemical and sublimated sulfur (doubling times of 2.2, 2.5, and 3.6 days, respectively). Strain TR1?s physiological traits make it a good candidate to cope with low pH and high metal concentration in biotechnological processes for treatment of metal-laden acidic streams at low and moderately high temperature

Role of syntrophic microbial communities in high-rate methanogenic bioreactors

Anaerobic purification is a cost-effective way to treat high strength industrial wastewater. Through anaerobic treatment of wastewaters energy is conserved as methane, and less sludge is produced. For high-rate methanogenesis compact syntrophic communities of fatty acid-degrading bacteria and methanogenic archaea are essential. Here, we describe the microbiology of syntrophic communities in methanogenic reactor sludges and provide information on which microbiological factors are essential to obtain high volumetric methane production rates. Fatty-acid degrading bacteria have been isolated from bioreactor sludges, but also from other sources such as freshwater sediments. Despite the important role that fatty acid-degrading bacteria play in high-rate methanogenic bioreactors, their relative numbers are generally low. This finding indicates that the microbial community composition can be further optimized to achieve even higher rates.Our research is funded by grants from the division of Chemical Sciences (CW) and Earth and Life Sciences (ALW) of The Netherlands Organisation for Scientific Research (NWO) and by the Technology Foundation (STW), the applied science division of NWO

Syntrophic LCFA-degraders: “bacteria that can clean soap”

Wastewaters contain substantial amounts of long-chain fatty acids (LCFA) which, when in the form of sodium salts, are what we normally call soaps. These compounds, resulting from fats' hydrolysis, can be converted to high amounts of methane. Developing new technological solutions for LCFA methanation, but also understanding the physiology and microbiology of LCFA degradation is fundamental for the bioenergy valorization of fatty wastewaters. In this work we present an overview of our results on anaerobic LCFA microbial degradation. Molecular techniques were used to investigate the structure of microbial communities present in different LCFA-degrading communities, such as continuous oleate- and palmitate-fed bioreactors and several enrichment cultures degrading these two LCFA as well. Choice of oleate and palmitate as model substrates was due to their predominance in wastewaters and to the fact that they represent mono-unsaturated and saturated LCFA, respectively. DGGE fingerprinting and sequencing evidenced the importance of syntrophic bacteria, affiliated with the Syntrophomonas genus, in the degradation of these compounds. Enrichment on oleate or palmitate resulted in distinct bacterial communities, which might be related to LCFA chain-saturation differences. A new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture. The fact that S. zehnderi can degrade a wide range of fatty-acids with different chain length (C4-C18) and is also able to use unsaturated LCFA (e.g. oleate) makes it a destined and dedicated key for the anaerobic treatment of wastewaters, in which an assembly of different fatty-acids is normally present. Genome sequencing of S. zehnderi is currently undergoing

Enrichment and characterization of microorganisms capable of degrading various C1 compounds in the Black Sea

Background: Methylated compounds can be used as an energy source to drive interactions between sulfate reducing microorganisms and methanogens. This has potential impact on the current understanding of the global carbon and sulfur cycles. Objectives: The use of methylated compounds by anaerobic microorganisms present in the sulfidic permanently stratified Black Sea sediment and column water and the composition of these communities was investigated through enrichment studies. Methods: Black Sea sediment of three different depths between 5 and 30 centimeters below sea floor, as well as water at 105 meters deep were collected anoxically and used for enrichments, supplemented with 1 mM of either dimethylsulfide (DMS), dimethylsulfoniopropionate (DMSP), trimethylamine (TMA) and methanol as sole energy source. To promote methanogenesis, acetogenesis and sulfate reduction in the different enrichments, 20 mM molybdate, 20 mM bromoerhanosulfonate (BrES) and 20 mM BrES with 20 mM sulfate was added, respectively. Anoxic cultures were incubated at 20ºC in the dark. Uptake of substrate and product formation were monitored over 4 weeks. Active cultures were transferred to fresh medium to promote further enrichment. Analyses of 16s rRNA gene sequencing are ongoing to elucidate the inocula and culture communities. Results: All enrichments grew on the provided substrates. Over four weeks, utilization of substrate ranged between 20% and 100% for all enrichments. Subsequent transfers of the enrichments retained the decrease of substrate although utilization was slower. These results will be complemented with 16s rRNA gene sequencing data and community comparison developed n methanogenic, acetogenic and sulfate-reducing enrichments performed.info:eu-repo/semantics/publishedVersio

The microbiology of conversion of long-chain fatty acids (LCFA) to biogas

Wastewaters, mainly the ones from food processing industries, contain considerable amounts of long-chain fatty acids (LCFA). These pollutant compounds, resulting from the hydrolysis of lipids, can be used as energetic resources for the production of biogas. A large amount of methane can be produced from LCFA; theoretically 1g of oleate, one of the most common LCFA found in wastewaters, can be converted to 1.01 L of methane (at standard temperature and pressure), while 1 g of glucose yields only 0.37 L methane. In its core this is a biological process, thus strongly linked to the performance and efficiency of the different microorganisms interacting in the process. Insight into the phylogenetic and functional communities involved in LCFA degradation is necessary to understand and enable the effective performance of bioreactors treating these compounds. In this work we describe the application of culture-dependent and culture-independent strategies to study microbiological and physiological aspects of the degradation of LCFA in anaerobic environments. Two LCFA were used as model substrates: oleate, a mono-unsaturated LCFA (C18:0), and palmitate, a saturated LCFA (C16:0), both abundant in LCFA-rich wastewaters. LCFA-degrading communities were developed by selective enrichments growing on oleate and palmitate. Changes in the microbial composition during enrichment were analyzed by DGGE profiling of PCR-amplified 16S rRNA gene fragments. Predominant DGGE-bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. 16S rRNA gene sequences clustering within the Syntrophomonadaceae family were identified as corresponding to predominant DGGE-bands in the oleate- and palmitateenrichment cultures. In stable palmitate-enrichment cultures members of the Syntrophobacteraceae family were also present. Further on, a new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture. This mesophilic, syntrophic, fatty acid oxidizing bacterium degrades straight-chain fatty acids with 4 to 18 carbon atoms but, also, unsaturated LCFA, such as oleate. The presence of Syntrophomonas zehnderi related bacteria in several sludges after contact with oleate was, subsequently, verified by DGGE-fingerprinting analysis and suggests its important role in anaerobic oleate degradation in bioreactor sludge. Future work on the performance of bioaugmented reactors with this versatile LCFA-degrading bacterium promise new results on the efficient conversion of LCFA to methane

Enrichment of carbon monoxide utilising microorganisms from methanogenic bioreactor sludge

Conversion of CO is the rate limiting step during anaerobic conversion of syngas (a gaseous mixture mainly composed of CO, CO2 and H2). In this work we study the microbial diversity in anaerobic sludge submitted to extended contact to syngas in a multi-orifice baffled bioreactor (MOBB). Methane was the main product resulting from syngas conversion in the MOBB. Enrichment cultures started with this sludge produced methane as final product, but also acetate. 16S rRNA gene analysis revealed a predominance of Acetobacterium and Sporomusa species in the enrichments. These are homoacetogenic bacteria that might be involved in CO conversion to acetate. Hydrogen was formed as intermediary from CO conversion and likely used by hydrogenotrophs with the formation of methane. Pasteurisation and serial dilutions of stable CO-converting enrichments resulted in a microbial culture dominated by two Sporomusa species that are able to use CO as sole substrate

Anaerobic LCFA degradation: a role for non-syntrophic conversions?

For many years the focus of lipids/long-chain fatty-acids (LCFA) wastewater treatment was on technological and process developments. More recently, promising results on the anaerobic treatment of LCFA-containing wastewaters[1] widened the attention to the microbiology aspects as well. In anaerobic bioreactors, LCFA can be β-oxidized to acetate and H2 by acetogenic bacteria, in obligatory syntrophy with methanogens. Presently, 14 species have been described that grow on fatty-acids in syntrophy with methanogens, all belonging to the families Syntrophomonadaceae and Syntrophaceae[2]. Among these, only 4 species are able to degrade mono- and/or polyunsaturated LCFA. The reason why the degradation of unsaturated LCFA is not more widespread remains unknown. Early studies suggested that degradation of unsaturated LCFA requires complete chain saturation prior to β-oxidation[2]. Unsaturated LCFA, such as linoleate (C18:2) and oleate (C18:1), would be metabolized through a hydrogenation step yielding stearate (C18:0), then entering the β-oxidation cycle. However, this theory is inconsistent with the observed accumulation of palmitate (C16:0) in continuous bioreactors fed with oleate[1]. We hypothesize that LCFA chain saturation might be a non-syntrophic process, i.e. unsaturated LCFA can function as electron donors and acceptors, as protons released in a first β-oxidation step can be used to hydrogenate the unsaturated hydrocarbon. To test this, linoleate (C18:2), oleate (C18:1) and a mixture of stearate (C18:0) and palmitate (C16:0) were continuously fed to bioreactors with methanogenesis-active or -inhibited anaerobic sludge. In the reactors fed with linoleate and oleate, palmitate accumulated in methanogenesis-active and -inhibited bioreactors up to concentrations of approximately 2 mM and 8 mM, respectively. In methanogenesis-inhibited bioreactors fed with a mixture of saturated LCFA (stearate and palmitate) no biological activity occurred. These results suggest the occurrence of a non-syntrophic step during the degradation of unsaturated LCFA in anaerobic bioreactors. The identification of microbial communities involved in non-syntrophic linoleate/oleate to palmitate conversion will give more insights into this novel biochemical mechanism

Hydrogen producing microbial communities of the biocathode in a microbial electrolysis cell

In the search for alternatives for fossil fuels and the reuse of the energy from waste streams, the microbial electrolysis cell is a promising technique. The microbial electrolysis cell is a two electrode system in which at the anode organic substances, including waste water, are used by microorganisms that release the terminal electrons to the electrode. These electrons are subsequently used at the cathode resulting in the production of a current. By addition of a small voltage, hydrogen gas can be produced by combining electrons and protons at the cathode. To catalyse the hydrogen evolution reaction at the cathode, expensive catalysts such as platinum are required. Recently, the use of biocathodes has shown great potential as an alternative for platinum. The microbial community responsible for the hydrogen evolution in such systems is, however, not well understood. In this study we focused on the characterization of the microbial communities of the microbial electrolysis cell biocathode using molecular techniques. The results show that the microbial community consists of 44% Proteobacteria, 27% Firmicutes, 18% Bacteriodetes and 12% related to other phyla. Within the major phylogenetic groups we found several clusters of uncultured species belonging to novel taxonomic groups at genus level. These novel taxonomic groups developed under environmentally unusual conditions and might have properties that have not been described before. Therefore it is of great interest to study those novel groups further. Within the Proteobacteria a major cluster belonged to the Deltaproteobacteria and based on the known characteristics of the closest related cultured species, we suggest a mechanism for microbial electron transfer for the production of hydrogen at the cathode

Characterization of an anaerobic thermophilic glycerol-degrading enrichment culture

Background: The glycerol market was totally changed by the biodiesel industry, which resulted in the production of an excess of this compound as an industrial by-product. As a consequence, the price of glycerol dropped and a huge interest in alternatives for its valorisation emerged since then. In the field of Biotechnology research, glycerol is an attractive compound for the microbial production of chemical building blocks. Objectives: The aim of this work was to investigate thermophilic anaerobic communities capable of conversion of glycerol. Methods: Thermophilic sludge from a lab-scale anaerobic reactor fed with skim milk and sodium oleate (50:50% chemical oxygen demand) was incubated at 55°C in closed bottles containing bicarbonate-buffered medium supplemented with 10mM glycerol. Periodic successive transfers of the glycerol-converting enrichment culture, combined with serial dilutions were performed. After eight generations a highly enriched, low diversity (microscopic observations and 16s rRNA DGGE profiling) microbial culture was obtained. Conclusions: The enriched culture converted glycerol mainly to methane (6mM) and acetate (7mM) within 6 days of incubation. A yet unknown organic compound was also produced. Sequencing results obtained on the Illumina platform showed the bacterial predominance of an uncultured Thermotoga species (75 % of the retrieved sequences), an uncultured Anaerobaculum species (13 %) and a close relative to Thermoanaerobacter pseudethanolicus (5 %). Isolation of the new uncultured Thermotoga and Anaerobaculum species is ongoing and their role in glycerol degradation will be assessed

Chain elongation in anaerobic reactor microbiomes to recover resources from waste

Different microbial pathways can elongate the carbon chains of molecules in open cultures of microbial populations (i.e. reactor microbiomes) under anaerobic conditions. Here, we discuss three such pathways: 1. homoacetogenesis to combine two carbon dioxide molecules into acetate; 2. succinate formation to elongate glycerol with one carbon from carbon dioxide; and 3. reverse β oxidation to elongate short-chain carboxylates with two carbons into medium-chain carboxylates, leading to more energy-dense and insoluble products (e.g. easier to separate from solution). The ability to use reactor microbiomes to treat complex substrates can simultaneously address two pressing issues: 1. providing proper waste management; and 2. producing renewable chemicals and fuels.The authors thank Wolfgang Bucket (MPI Marburg) for assistance with Figure 1. C.M.S. and L.T.A. were supported by the U. S. Army Research Laboratory and the U. S. Army Research Office under contract/grant number W911NF-12-1-0555. H.R. was supported for this work by the Cornell University Agricultural Experiment Station federal formula funds, Project No. NYC-123452 received from the National Institutes for Food and Agriculture (NIFA), U.S. Department of Agriculture. K.R. was supported by the European Research Council Starter Grant Electrotalk and the Multidisciplinary Research Partnership Ghent Bio-Economy. A.J.M.S. was supported by the Chemical Sciences division of the Netherlands Science Foundation (CW-TOP 700.55.343) and the European Research Council (ERC grant 323009)