Roel J. W. Meulepas

Enrichment of anaerobic methanotrophs in sulfate‐reducing membrane bioreactors

Anaerobic oxidation of methane (AOM) in marine sediments is coupled to sulfate reduction (SR). AOM is mediated by distinct groups of archaea, called anaerobic methanotrophs (ANME). ANME co‐exist with sulfate‐reducing bacteria, which are also involved in AOM coupled SR. The microorganisms involved in AOM coupled to SR are extremely difficult to grow in vitro. Here, a novel well‐mixed submerged‐membrane bioreactor system is used to grow and enrich the microorganisms mediating AOM coupled to SR. Four reactors were inoculated with sediment sampled in the Eckernförde Bay (Baltic Sea) and operated at a methane and sulfate loading rate of 4.8u2009Lu2009L−1u2009day−1 (196u2009mmolu2009L−1u2009day−1) and 3.0u2009mmolu2009L−1u2009day−1. Two bioreactors were controlled at 15°C and two at 30°C, one reactor at 30°C contained also anaerobic granular sludge. At 15°C, the volumetric AOM and SR rates doubled approximately every 3.8 months. After 884 days, an enrichment culture was obtained with an AOM and SR rate of 1.0u2009mmolu2009g u2009volatileu2009suspendedu2009solids−1 u2009day−1 (286u2009µmolu2009g u2009dryu2009weight−1 u2009day−1). No increase in AOM and SR was observed in the two bioreactors operated at 30°C. The microbial community of one of the 15°C reactors was analyzed. ANME‐2a became the dominant archaea. This study showed that sulfate reduction with methane as electron donor is possible in well‐mixed bioreactors and that the submerged‐membrane bioreactor system is an excellent system to enrich slow‐growing microorganisms, like methanotrophic archaea. Biotechnol. Bioeng. 2009; 104: 458–470

Effect of carbon monoxide, hydrogen and sulfate on thermophilic (55°C) hydrogenogenic carbon monoxide conversion in two anaerobic bioreactor sludges

The conversion routes of carbon monoxide (CO) at 55°C by full-scale grown anaerobic sludges treating paper mill and distillery wastewater were elucidated. Inhibition experiments with 2-bromoethanesulfonate (BES) and vancomycin showed that CO conversion was performed by a hydrogenogenic population and that its products, i.e. hydrogen and CO2, were subsequently used by methanogens, homo-acetogens or sulfate reducers depending on the sludge source and inhibitors supplied. Direct methanogenic CO conversion occurred only at low CO concentrations [partial pressure of CO (PCO) <0.5xa0bar (1xa0bar=105xa0Pa)] with the paper mill sludge. The presence of hydrogen decreased the CO conversion rates, but did not prevent the depletion of CO to undetectable levels (<400xa0ppm). Both sludges showed interesting potential for hydrogen production from CO, especially since after 30xa0min exposure to 95°C, the production of CH4 at 55°C was negligible. The paper mill sludge was capable of sulfate reduction with hydrogen, tolerating and using high CO concentrations (PCO>1.6xa0bar), indicating that CO-rich synthesis gas can be used efficiently as an electron donor for biological sulfate reduction.

Microbial diversity and community structure of a highly active anaerobic methane-oxidizing sulfate-reducing enrichment

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged-membrane bioreactor system and capable of high-rate AOM (286 mumol g(dry weight)(-1) day(-1)) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME-2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate-reducing bacteria commonly found in association with other ANME-related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME-2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with (13)C-labelled methane showed substantial incorporation of (13)C label in the bacterial C(16) fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl-archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.

Effect of methanogenic substrates on anaerobic oxidation of methane and sulfate reduction by an anaerobic methanotrophic enrichment

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) is assumed to be a syntrophic process, in which methanotrophic archaea produce an interspecies electron carrier (IEC), which is subsequently utilized by sulfate-reducing bacteria. In this paper, six methanogenic substrates are tested as candidate-IECs by assessing their effect on AOM and SR by an anaerobic methanotrophic enrichment. The presence of acetate, formate or hydrogen enhanced SR, but did not inhibit AOM, nor did these substrates trigger methanogenesis. Carbon monoxide also enhanced SR but slightly inhibited AOM. Methanol did not enhance SR nor did it inhibit AOM, and methanethiol inhibited both SR and AOM completely. Subsequently, it was calculated at which candidate-IEC concentrations no more Gibbs free energy can be conserved from their production from methane at the applied conditions. These concentrations were at least 1,000 times lower can the final candidate-IEC concentration in the bulk liquid. Therefore, the tested candidate-IECs could not have been produced from methane during the incubations. Hence, acetate, formate, methanol, carbon monoxide, and hydrogen can be excluded as sole IEC in AOM coupled to SR. Methanethiol did inhibit AOM and can therefore not be excluded as IEC by this study.

Growth of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a high-pressure membrane capsule bioreactor.

ABSTRACT Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-μm-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the Desulfuromonadales and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.

Trace methane oxidation and the methane dependency of sulfate reduction in anaerobic granular sludge.

This study investigates the oxidation of labeled methane (CH(4)) and the CH(4) dependence of sulfate reduction in three types of anaerobic granular sludge. In all samples, (13)C-labeled CH(4) was anaerobically oxidized to (13)C-labeled CO(2), while net endogenous CH(4) production was observed. Labeled-CH(4) oxidation rates followed CH(4) production rates, and the presence of sulfate hampered both labeled-CH(4) oxidation and methanogenesis. Labeled-CH(4) oxidation was therefore linked to methanogenesis. This process is referred to as trace CH(4) oxidation and has been demonstrated in methanogenic pure cultures. This study shows that the ratio between labeled-CH(4) oxidation and methanogenesis is positively affected by the CH(4) partial pressure and that this ratio is in methanogenic granular sludge more than 40 times higher than that in pure cultures of methanogens. The CH(4) partial pressure also positively affected sulfate reduction and negatively affected methanogenesis: a repression of methanogenesis at elevated CH(4) partial pressures confers an advantage to sulfate reducers that compete with methanogens for common substrates, formed from endogenous material. The oxidation of labeled CH(4) and the CH(4) dependence of sulfate reduction are thus not necessarily evidence of anaerobic oxidation of CH(4) coupled to sulfate reduction.

Anaerobic bioleaching of metals from waste activated sludge.

Heavy metal contamination of anaerobically digested waste activated sludge hampers its reuse as fertilizer or soil conditioner. Conventional methods to leach metals require aeration or the addition of leaching agents. This paper investigates whether metals can be leached from waste activated sludge during the first, acidifying stage of two-stage anaerobic digestion without the supply of leaching agents. These leaching experiments were done with waste activated sludge from the Hoek van Holland municipal wastewater treatment plant (The Netherlands), which contained 342 μg g(-1) of copper, 487 μg g(-1) of lead, 793 μg g(-1) of zinc, 27 μg g(-1) of nickel and 2.3 μg g(-1) of cadmium. During the anaerobic acidification of 3 gdry weight L(-1) waste activated sludge, 80-85% of the copper, 66-69% of the lead, 87% of the zinc, 94-99% of the nickel and 73-83% of the cadmium were leached. The first stage of two-stage anaerobic digestion can thus be optimized as an anaerobic bioleaching process and produce a treated sludge (i.e., digestate) that meets the land-use standards in The Netherlands for copper, zinc, nickel and cadmium, but not for lead.

Enrichment of ANME-1 from Eckernförde Bay sediment on thiosulfate, methane and short-chain fatty acids

The microorganisms involved in sulfate-dependent anaerobic oxidation of methane (AOM) have not yet been isolated. In an attempt to stimulate the growth of anaerobic methanotrophs and associated sulfate reducing bacteria (SRB), Eckernförde Bay sediment was incubated with different combinations of electron donors and acceptors. The organisms involved in AOM coupled to sulfate reduction (ANME-1, ANME-2, and Desulfosarcina/Desulfococcus) were monitored using specific primers and probes. With thiosulfate as sole electron acceptor and acetate, pyruvate or butyrate as the sole electron donor, ANME-1 became the dominant archaeal species. This finding suggests that ANME-1 archaea are not obligate methanotrophs and that ANME-1 can grow on acetate, pyruvate or butyrate.

Desulfovibrio paquesii sp. nov., a hydrogenotrophic sulfate-reducing bacterium isolated from a synthesis-gas-fed bioreactor treating zinc-and sulfate-rich wastewater

A hydrogenotrophic, sulfate-reducing bacterium, designated strain SB1(T), was isolated from sulfidogenic sludge of a full-scale synthesis-gas-fed bioreactor used to remediate wastewater from a zinc smelter. Strain SB1(T) was found to be an abundant micro-organism in the sludge at the time of isolation. Hydrogen, formate, pyruvate, lactate, malate, fumarate, succinate, ethanol and glycerol served as electron donors for sulfate reduction. Organic substrates were incompletely oxidized to acetate. 16S rRNA gene sequence analysis showed that the closest recognized relative to strain SB1(T) was Desulfovibrio gigas DSM 1382(T) (97.5 % similarity). The G+C content of the genomic DNA of strain SB1(T) was 62.2 mol%, comparable with that of Desulfovibrio gigas DSM 1382(T) (60.2 mol%). However, the level of DNA-DNA relatedness between strain SB1(T) and Desulfovibrio gigas DSM 1382(T) was only 56.0 %, indicating that the two strains are not related at the species level. Strain SB1(T) could also be differentiated from Desulfovibrio gigas based on phenotypic characteristics, such as major cellular fatty acid composition (anteiso-C(15 : 0), iso-C(14 : 0) and C(18 : 1) cis 9) and substrate utilization. Strain SB1(T) is therefore considered to represent a novel species of the genus Desulfovibrio, for which the name Desulfovibrio paquesii sp. nov. is proposed. The type strain is SB1(T) (=DSM 16681(T)=JCM 14635(T)).

Bioprocess engineering of sulfate reduction for environmental technology

INTRODUCTION The microbiota present in the sulphur cycle have been studied since the end of the nineteenth century when the pioneering work of the famous microbiologists Winogradsky and Beijerinck took place. Sulphur conversions involve the metabolism of several different specific groups of bacteria, e.g. sulphate-reducing bacteria (SRB), phototrophic sulphur bacteria and thiobacilli, specialized to use these sulphur compounds in their different redox states (Lens and Kuenen, 2001). Many of these microorganisms possess unique metabolic and ecophysiological features, and to date there are still regular reports of novel microorganisms with extraordinary properties. Several of the microbial conversions of the sulphur cycle can be implemented for pollution control (Table 13.1). This chapter overviews the applications in environmental technology, which utilize the metabolism of SRB as the key process. Technological utilization of SRB sounds at first somewhat controversial, as sulphate reduction has been considered unwanted for many years in anaerobic wastewater treatment (Hulshoff Pol et al. , 1998). Emphasis of the research in the 1970s–1980s was mainly on the prevention or minimalization of sulphate reduction during methanogenic wastewater treatment (Colleran et al. , 1995). From the 1990s onwards, interest has grown in applying sulphate reduction for the treatment of specific wastestreams, e.g. inorganic sulphate-rich wastewaters such as acid mine drainage, metal polluted groundwater and flue-gas scrubbing waters. Nowadays, sulphur-cycle-based technologies are not solely considered as “end-of-pipe” applications, but their potential for pollution prevention as well as for sulphur, metal or water recovery and re-use are now fully recognized.