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

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-m-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.

Exploring long-term retention and reactivation of micropollutant biodegradation capacity.

The factors limiting micropollutant biodegradation in the environment and how to stimulate this process have often been investigated. However, little information is available on the capacity of microbial communities to retain micropollutant biodegradation capacity in the absence of micropollutants or to reactivate micropollutant biodegradation in systems with fluctuating micropollutant concentrations. This study investigated how a period of 2 months without the addition of micropollutants and other organic carbon affected micropollutant biodegradation by a micropollutant-degrading microbial community. Stimulation of micropollutant biodegradation was performed by adding different types of dissolved organic carbon (DOC)-extracted from natural sources and acetate-increasing 10 × the micropollutant concentration, and inoculating with activated sludge. The results show that the capacity to biodegrade 3 micropollutants was permanently lost. However, the biodegradation activity of 2,4-D, antipyrine, chloridazon, and its metabolites restarted when these micropollutants were re-added to the community. Threshold concentrations similar to those obtained before the period of no substrate addition were achieved, but biodegradation rates were lower for some compounds. Through the addition of high acetate concentrations (108 mg-C/L), gabapentin biodegradation activity was regained, but 2,4-D biodegradation capacity was lost. An increase of bentazon concentration from 50 to 500 µg/L was necessary for biodegradation to be reactivated. These results provide initial insights into the longevity of micropollutant biodegradation capacity in the absence of the substance and strategies for reactivating micropollutant biodegrading communities.

Biostimulation with oxygen and electron donors supports micropollutant biodegradation in an experimentally simulated nitrate-reducing aquifer.

The availability of suitable electron donors and acceptors limits micropollutant natural attenuation in oligotrophic groundwater. This study investigated how electron donors with different biodegradability (humics, dextran, acetate, and ammonium), and different oxygen concentrations affect the biodegradation of 15 micropollutants (initial concentration of each micropollutant = 50 µg/L) in simulated nitrate reducing aquifers. Tests mimicking nitrate reducing field conditions showed no micropollutant biodegradation, even with electron donor amendment. However, 2,4-dichlorophenoxyacetic acid and mecoprop were biodegraded under (micro)aerobic conditions with and without electron donor addition. The highest 2,4-dichlorophenoxyacetic acid and mecoprop biodegradation rates and removal efficiencies were obtained under fully aerobic conditions with amendment of an easily biodegradable electron donor. Under microaerobic conditions, however, amendment with easily biodegradable dissolved organic carbon (DOC) inhibited micropollutant biodegradation due to competition between micropollutants and DOC for the limited oxygen available. Microbial community composition was dictated by electron acceptor availability and electron donor amendment, not by micropollutant biodegradation. Low microbial community richness and diversity led to the absence of biodegradation of the other 13 micropollutants (such as bentazon, chloridazon, and carbamazepine). Finally, adaptation and potential growth of biofilms interactively determined the location of the micropollutant removal zone relative to the point of amendment. This study provides new insight on how to stimulate in situ micropollutant biodegradation to remediate oligotrophic groundwaters as well as possible limitations of this process.

Influence of redox condition and inoculum on micropollutant biodegradation by soil and activated sludge communities.

Micropollutant biodegradation is selected by the interplay among environmental conditions and microbial community composition. This study investigated how different electron acceptors, and different inocula with varying microbial diversity, pre-exposed to distinct redox conditions and micropollutants, affect micropollutant biodegradation. Four tested inocula comprised of agricultural soil (Soil), sediment from a ditch in an agricultural field (Ditch), activated sludge from a municipal WWTP (Mun AS), and activated sludge from an industrial WWTP (Ind AS). Removal of 16 micropollutants was investigated for each inoculum under aerobic, nitrate reducing, iron reducing, sulfate reducing, and methanogenic conditions. Micropollutant biodegradation was highest under aerobic conditions with removal of 12 micropollutants. Most micropollutants were biodegraded by Soil (n = 11) and Mun AS inocula (n = 10). A positive correlation was observed between inoculum community richness and the number of different micropollutants a microbial community initially degraded. The redox conditions to which a microbial community had been exposed appeared to positively affect micropollutant biodegradation performance more than pre-exposure to micropollutants. Additionally, depletion of the organic carbon present in the inocula resulted in lower micropollutant biodegradation and overall microbial activities, suggesting that i) an additional carbon source is needed to promote micropollutant biodegradation; and ii) overall microbial activity can be a good indirect indicator for micropollutant biodegradation activity. These results could help to develop novel micropollutant removal strategies.

Kinetics modelling of Cu(II) biosorption on to coconut shell and Moringa oleifera seeds from tropical regions.

Adsorption kinetic studies are of great significance in evaluating the performance of a given adsorbent and gaining insight into the underlying mechanism. This work investigated the sorption kinetics of Cu(II) on to coconut shell and Moringa oleifera seeds using batch techniques. To understand the mechanisms of the biosorption process and the potential rate-controlling steps, kinetic models were used to fit the experimental data. The results indicate that kinetic data were best described by the pseudo-second-order model with correlation coefficients (R2) of 0.9974 and 0.9958 for the coconut shell and Moringa oleifera seeds, respectively. The initial sorption rates obtained for coconut shell and Moringa oleifera seeds were 9.6395 x 10(-3) and 8.3292 x 10(-2) mg g(-1) min(-1), respectively. The values of the mass transfer coefficients obtained for coconut shell (1.2106 x 10(-3) cm s(-1)) and Moringa oleifera seeds (8.965 x 10(-4) cm s(-1)) indicate that the transport of Cu(II) from the bulk liquid to the solid phase was quite fast for both materials investigated. The results indicate that intraparticle diffusion controls the rate of sorption in this study; however, film diffusion cannot be neglected, especially at the initial stage of sorption.

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.

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.

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 4.8 L L(-1) day(-1) (196 mmol L(-1) day(-1)) and 3.0 mmol L(-1) day(-1). Two bioreactors were controlled at 15 degrees C and two at 30 degrees C, one reactor at 30 degrees C contained also anaerobic granular sludge. At 15 degrees 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 1.0 mmol g(volatile suspended solids) (-1) day(-1) (286 micromol g(dry weight) (-1) day(-1)). No increase in AOM and SR was observed in the two bioreactors operated at 30 degrees C. The microbial community of one of the 15 degrees 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.

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.

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.

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)).

Effect of environmental conditions on sulfate reduction with methane as electron donor by an Eckemförde Bay enrichment.

Sulfate reduction (SR) coupled to anaerobic oxidation of methane (AOM) is meditated by marine microorganisms and forms an important process in the global sulfur and carbon cycle. In this research, the possibility to use this process for the removal and recovery of sulfur and metal compounds from waste streams was investigated. A membrane bioreactor was used to enrich for a community of methane-oxidizing sulfate-reducing microorganisms from Eckernförde Bay sediment The AOM and SR rate of the obtained enrichment were 1.0 mmol gvss(-1) d(-1). The operational window and optimal environmental conditions for SR with methane as electron donor were assessed. The optimum pH, salinity, and temperature were 7.5, 30% per hundred and 20 degrees C, respectively. The enrichment had a good affinity for sulfate (Km < 0.5 mM) and a low affinity for methane (Kn > 0.075 MPa). A0M coupled to SR was completely inhibited at 2.4 (L0.1) mM sulfide. AOM occurred with sulfate, thiosulfate, and sulfite as electron accepters. Sulfate reduction with methane as electron donor can be applied for the removal of sulfate or for the production of sulfide,for metal precipitation. However, the low optimal temperature and the high salt requirement limit the operational window of the process.