Unveiling the Occurrence and Non-Negligible Role of Amino Sugars in Waste Activated Sludge Fermentation by an Enriched Chitin-Degradation Consortium.

Polysaccharides in extracellular polymeric substances (EPS) can form a hybrid matrix network with proteins, impeding waste-activated sludge (WAS) fermentation. Amino sugars, such as N-acetyl-d-glucosamine (GlcNAc) polymers and sialic acid, are the non-negligible components in the EPS of aerobic granules or biofilm. However, the occurrence of amino sugars in WAS and their degradation remains unclear. Thus, amino sugars (∼6.0%) in WAS were revealed, and the genera of Lactococcus and Zoogloea were identified for the first time. Chitin was used as the substrate to enrich a chitin-degrading consortium (CDC). The COD balances for methane production ranged from 83.3 and 95.1%. Chitin was gradually converted to oligosaccharides and GlcNAc after dosing with the extracellular enzyme. After doing enriched CDC in WAS, the final methane production markedly increased to 60.4 ± 0.6 mL, reflecting an increase of ∼62%. Four model substrates of amino sugars (GlcNAc and sialic acid) and polysaccharides (cellulose and dextran) could be used by CDC. Treponema (34.3%) was identified as the core bacterium via excreting chitinases (EC 3.2.1.14) and N-acetyl-glucosaminidases (EC 3.2.1.52), especially the genetic abundance of chitinases in CDC was 2.5 times higher than that of WAS. Thus, this study provides an elegant method for the utilization of amino sugar-enriched organics.

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process.

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO3-, NO2-, and NH4+ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 108 copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

Simultaneous enrichment of denitrifying anaerobic methane-oxidizing microorganisms and anammox bacteria in a hollow-fiber membrane biofilm reactor.

In this study, the coculture system of denitrifying anaerobic methane oxidation (DAMO) microbes and anaerobic ammonium oxidation (anammox) bacteria was successfully enriched in a hollow-fiber membrane biofilm reactor (HfMBR) using freshwater sediment as the inoculum. The maximal removal rates of nitrate and ammonium were 78 mg N/L/day (131 mg N/m2/day) and 26 mg N/L/day (43 mg N/m2/day), respectively. Due to the high rate of methane mass transfer in HfMBR, the activity of DAMO archaea continued to increase during the enrichment period, indicating that HfMBR could be a powerful tool to enrich DAMO microorganisms. Effects of partial methane pressure, temperature, and pH on the cocultures were obvious. However, the microbial activity in HfMBR could be recovered quickly after the shock change of environmental factors. Furthermore, the result also found that DAMO bacteria likely had a stronger competitive advantage than anammox bacteria under the operating conditions in this study. High-throughput sequencing 16S rRNA genes illustrated that the dominant microbes were NC10, Euryarchaeota, Proteobacteria, Planctomycetes, and Chlorobi with relative abundance of 38.8, 26.2, 13.78, 6.2, and 3.6 %, respectively.

In-situ biogas sparging enhances the performance of an anaerobic membrane bioreactor (AnMBR) with mesh filter in low-strength wastewater treatment.

In the recent years, anaerobic membrane bioreactor (AnMBR) technology is being considered as a very attractive alternative for wastewater treatment due to the striking advantages such as upgraded effluent quality. However, fouling control is still a problem for the application of AnMBR. This study investigated the performance of an AnMBR using mesh filter as support material to treat low-strength wastewater via in-situ biogas sparging. It was found that mesh AnMBR exhibited high and stable chemical oxygen demand (COD) removal efficiencies with values of 95 ± 5 % and an average methane yield of 0.24 L CH4/g CODremoved. Variation of transmembrane pressure (TMP) during operation indicated that mesh fouling was mitigated by in-situ biogas sparging and the fouling rate was comparable to that of aerobic membrane bioreactor with mesh filter reported in previous researches. The fouling layer formed on the mesh exhibited non-uniform structure; the porosity became larger from bottom layer to top layer. Biogas sparging could not change the composition but make thinner thickness of cake layer, which might be benefit for reducing membrane fouling rate. It was also found that ultrasonic cleaning of fouled mesh was able to remove most foulants on the surface or pores. This study demonstrated that in-situ biogas sparging enhanced the performance of AnMBRs with mesh filter in low-strength wastewater treatment. Apparently, AnMBRs with mesh filter can be used as a promising and sustainable technology for wastewater treatment.

Advanced phosphorus recovery using a novel SBR system with granular sludge in simultaneous nitrification, denitrification and phosphorus removal process.

In this study, a novel process for phosphorus (P) recovery without excess sludge production from granular sludge in simultaneous nitrification-denitrification and P removal (SNDPR) system is presented. Aerobic microbial granules were successfully cultivated in an alternating aerobic-anaerobic sequencing batch reactor (SBR) for removing P and nitrogen (N). Dense and stable granular sludge was created, and the SBR system showed good performance in terms of P and N removal. The removal efficiency was approximately 65.22 % for N, and P was completely removed under stable operating conditions. Afterward, new operating conditions were applied in order to enhance P recovering without excess sludge production. The initial SBR system was equipped with a batch reactor and a non-woven cloth filter, and 1.37 g of CH3COONa·3H2O was added to the batch reactor after mixing it with 1 L of sludge derived from the SBR reactor to enhance P release in the liquid fraction, this comprises the new system configuration. Under the new operating conditions, 93.19 % of the P contained in wastewater was released in the liquid fraction as concentrated orthophosphate from part of granular sludge. This amount of P could be efficiently recovered in the form of struvite. Meanwhile, a deterioration of the denitrification efficiency was observed and the granules were disintegrated into smaller particles. The biomass concentration in the system increased firstly and then maintained at 4.0 ± 0.15 gVSS/L afterward. These results indicate that this P recovery operating (PRO) mode is a promising method to recover P in a SNDPR system with granular sludge. In addition, new insights into the granule transformation when confronted with high chemical oxygen demand (COD) load were provided.

Experimental evaluation of the metabolic reversibility of ANME-2d between anaerobic methane oxidation and methanogenesis.

The "reverse methanogenesis" hypothesis as the metabolic pathway of AOM has recently been supported in the novel ANME lineage ANME-2d in denitrifying anaerobic methane oxidation (DAMO). However, no previous studies have experimentally evaluated the reversal of methane oxidation and methane production in this archaea. In the present study, the metabolic reversibility of ANME-2d from AOM to methanogenesis was evaluated using H2/CO2 and acetate as substrates. The results showed that the system produced methane from H2/CO2 but not from acetate. However, the clone library and real-time PCR analysis of the culture showed that both the percentage and quantity of ANME-2d decreased significantly under this condition, while methanogen abundance increased. Further high-throughput sequencing results showed that the archaea community did not change at the fourth day after H2/CO2 was supplied, but changed profoundly after methanogenesis took place for 3 days. The percentage of DAMO archaea in the total archaea decreased obviously, while more methanogens grew up during this period. Comparatively, the bacteria community changed profoundly at the fourth day. These results indicated that ANME-2d might not reverse its metabolism to produce methane from H2/CO2 or acetate. After archaea were returned to DAMO conditions, DAMO activity decreased and the amount of ANME-2d continued to fall, implying that the lineage had suffered from severe injury and required a long recovery time.

Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field.

The nitrate-dependent denitrifying anaerobic methane oxidation (DAMO) process, which is metabolized together by anaerobic methanotrophic archaea and NC10 phylum bacteria, is expected to be important for the global carbon and nitrogen cycles. However, there are little studies about the existence of this process and the functional microbes in environments. Therefore, the coexistence of DAMO archaea and bacteria in a paddy field was evaluated in this study. Next-generation sequencing showed that the two orders, Methanosarcinales and Nitrospirales, to which DAMO archaea and DAMO bacteria belong, were detected in the four soil samples. Then the in vitro experiments demonstrated both of nitrite- and nitrate-dependent DAMO activities, which confirmed the coexistence of DAMO archaea and DAMO bacteria. It was the first report about the coexistence of DAMO archaea and bacteria in a paddy field. Furthermore, anammox bacteria were detected in two of the four samples. The in vitro experiments did not show anammox activity in the initial period but showed low anammox activity after 20 days' enrichment. These results implicated that anammox bacteria may coexist with DAMO microorganisms in this field, but at a very low percentage.

New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches.

The significance of ANME-2d in methane sink in the environment has been overlooked, and there was no any study evaluating the distribution of ANME-2d in the environment. New primers were thus needed to be designed for following research. In this paper, a pair of primers (DP397F and DP569R) was designed to quantify ANME-2d. The specificity and amplification efficiency of this primer pair were acceptable. PCR amplification of another pair of primers (DP142F and DP779R) generated a single, bright targeted band from the enrichment sample, but yielded faint, multiple bands from the environmental samples. Nested PCR was conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the copies of the 16s ribosomal RNA gene of ANME-2d in these samples ranged from 3.72 × 10(4) to 2.30 × 10(5) copies µg(-1) DNA, indicating that the percentage of ANME-2d was greatest in a polluted river sample and least in a rice paddy sample. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples; the latter finding suggests that ANME-2d may spread in environments.

Identification and degradation of structural extracellular polymeric substances in waste activated sludge via a polygalacturonate-degrading consortium.

By maintaining the cell integrity of waste activated sludge (WAS), structural extracellular polymeric substances (St-EPS) resist WAS anaerobic fermentation. This study investigates the occurrence of polygalacturonate in WAS St-EPS by combining chemical and metagenomic analyses that identify ∼22% of the bacteria, including Ferruginibacter and Zoogloea, that are associated with polygalacturonate production using the key enzyme EC 5.1.3.6. A highly active polygalacturonate-degrading consortium (GDC) was enriched and the potential of this GDC for degrading St-EPS and promoting methane production from WAS was investigated. The percentage of St-EPS degradation increased from 47.6% to 85.2% after inoculation with the GDC. Methane production was also increased by up to 2.3 times over a control group, with WAS destruction increasing from 11.5% to 28.4%. Zeta potential and rheological behavior confirmed the positive effect which GDC has on WAS fermentation. The major genus in the GDC was identified as Clostridium (17.1%). Extracellular pectate lyases (EC 4.2.2.2 and 4.2.2.9), excluding polygalacturonase (EC 3.2.1.15), were observed in the metagenome of the GDC and most likely play a core role in St-EPS hydrolysis. Dosing with GDC provides a good biological method for St-EPS degradation and thereby enhances the conversion of WAS to methane.

Influence of low air pressure on combined nitritation and anaerobic ammonium oxidation process.

At high altitude, wastewater aeration efficiency is low, which is detrimental to nitrification in conventional biological nitrogen removal. The combined partial nitritation and anaerobic ammonium oxidation (CPNA) process requires little oxygen and can be appropriate in low-pressure conditions. As such, in this study, we investigated the effect of air pressure on CPNA using a laboratory-scale reactor. We found that low air pressure promoted the removal of total inorganic nitrogen (TIN), achieving a TIN removal rate of 43,000 mg·N/(kg·VSS·d). The secretion of extracellular polymeric substances under low air pressure was not significantly different from that under ordinary air pressure, indicating no adverse effects on microbial aggregation ability, stability, or settleability. The abundance of aerobic ammonia-oxidizing bacteria (AeAOB) increased from 0.2% to 5.6%, and the activity of anaerobic ammonia-oxidizing bacteria (AnAOB) enhanced, giving AeAOB and AnAOB a competitive advantage over nitrite-oxidizing bacteria, thus forming a microbial community structure favorable to the CPNA process. Our further analysis of the results of batch tests in serum bottles confirmed the positive effect of low air pressure on the anaerobic ammonium oxidation (anammox) process, with a 28.5% ± 1.9% improvement in the specific anammox rate at 70 kPa compared with 100 kPa. AnAOB activity increased, which was reflected in the intracellular heme content increasing from 0.56 ± 0.18 µmol/(g·VSS) at 100 kPa to 2.56 ± 0.20 µmol/(g·VSS) at 70 kPa. We clarified the CPNA-process-promoting effect of low air pressure, which shows potential for nitrogen removal in high-altitude regions.

Effects of the carbon/nitrogen (C/N) ratio on a system coupling simultaneous nitrification and denitrification (SND) and denitrifying phosphorus removal (DPR).

Simultaneous nitrification and denitrification (SND) were coupled with a denitrifying phosphorus removal (DPR) to achieve simultaneous nutrient and carbon removal. With influent chemical oxygen demand (COD), ammonia-N (NH4+-N), and total phosphorus (TP) concentrations of 250, 50, and 8 mg/L, the SND-DPR coupled system achieved stable nutrient removal efficiency of COD, NH4+-N, TN and TP were 91.8 ± 1.7%, 88.4 ± 1.8%, 64 ± 3.3% and 99.2 ± 0.6%, respectively. Enhancing the C/N ratio strengthened the storage of intracellular polymers and provided sufficient intracellular carbon sources for phosphorus uptake. The nutrient removal efficiency reached the highest level at a C/N ratio of 5, and no advantage was observed after increasing the C/N ratio to 7. Nutrients were mainly removed during the aerobic stage at a low DO concentration as well during the anoxic stage, which helped achieve concurrent nitrification and denitrification by ordinary heterotrophic organisms (OHOs), promote denitrifying and aerobic phosphorus removal, and conserve organic carbon demand and energy consumption for aeration. The system was limited for DO in the aerobic stage at a low DO concentration, resulting in a deficiency in electron acceptors (O2 and NO3-N) and limiting the subsequent promotion of phosphorus uptake and TN removal. The limited DO content in the low DO stage was the key factor involved in enhancing the nutrient removal efficiency along with the increasing influent C/N ratio.

Acute toxicity and ecological risk assessment of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC in ultraviolet (UV)-filters.

Ultraviolet (UV) filters are used in cosmetics, personal care products and packaging materials to provide sun protection for human skin and other substances. Little is known about these substances, but they continue to be released into the environment. The acute toxicity of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC to Chlorella vulgaris and Daphnia magna were analyzed in this study. The 96 h-EC50 values of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC on C. vulgaris were 183.60, 3.50 and 0.16874 mg/L, respectively. The 48 h-LC50 of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC on D. magna were 12.50, 3.74 and 0.54445 mg/L, respectively. The toxicity of a mixture of 4,4'-dihydroxybenzophenone and 4-MBC showed addictive effect on C. vulgaris, while the toxicity of mixtures of 4,4'-dihydroxybenzophenone and 2,4,4'-trihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC as well as 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC all showed antagonistic effect on C. vulgaris. The induced no-effect concentrations of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC by the assessment factor (AF) method were 0.0125, 0.00350 and 0.000169 mg/L, respectively.

High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor.

Anaerobic biological techniques are widely used in the reductive decolorization of textile wastewater. However, the decolorization efficiency of textile wastewater by conventional anaerobic biological techniques is generally limited due to the low biomass retention capacity and short hydraulic retention time (HRT). In this study, a methane-based hollow fiber membrane bioreactor (HfMBR) was initially inoculated with an enriched anaerobic methane oxidation (AOM) culture to rapidly form an anaerobic biofilm. Then, synthetic azo dye wastewater containing methyl orange (MO) was fed into the HfMBR. MO decolorization efficiency of ∼ 100 % (HRT = 2 to 1.5 days) and maximum decolorization rate of 883 mg/L/day (HRT = 0.5 day) were obtained by the stepwise increase of the MO loading rate into the methane-based HfMBR. Scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) analysis visually revealed that archaea clusters formed synergistic consortia with adjacent bacteria. Quantitative PCR (qPCR), phylogenetic and high-throughput sequencing analysis results further confirmed the biological consortia formation of methane-related archaea and partner bacteria, which played a synergistic role in MO decolorization. The high removal efficiency and stable microbial structure in HfMBR suggest it is a potentially effective technique for high-toxic azo dyes removal from textile wastewater.

Microbial selenite reduction coupled to anaerobic oxidation of methane.

Denitrifying anaerobic methane oxidation (DAMO) is the process of coupling the anaerobic oxidation of methane (AOM) with denitrification, which plays an important part in controlling the flow of methane in anoxic niches. In this study, we explored the feasibility of microbial selenite reduction using methane by DAMO culture. Isotopic 13CH4 and long-term experiments showed that selenite reduction was coupled to methane oxidation, and selenite was ultimately reduced to Se (0) by the analyses of scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The introduction of nitrate, the original electron acceptor in the DAMO culture, inhibited selenite reduction. Meanwhile, the microbial community of DAMO culture was significantly changed when the electron acceptor was changed from nitrate to selenite after long-term selenite reduction. High-throughput 16S rRNA gene sequencing indicated that Methylococcus (26%) became the predominant microbe performing selenite reduction and methane oxidation and the possible pathways of AOM accompanied with selenite reduction were proposed. This study revealed more potential relation during the biogeochemical cycle of carbon, nitrogen, and selenium.

Mass transfer affects reactor performance, microbial morphology, and community succession in the methane-dependent denitrification and anaerobic ammonium oxidation co-culture.

Denitrifying anaerobic methane oxidation (DAMO) combining anaerobic ammonium oxidation (Anammox) process is a novel nitrogen removal technology. However, the roles of methane transfer (gas phase) and nitrogen transfer (liquid phase) in the heterogeneous process remain unclear. In this study, granular DAMO and Anammox co-cultures were inoculated from a hollow-fiber membrane bioreactor into a sequence batch reactor (SBR). Since the methane transfer became limited in SBR, the nitrate removal rate first decreased and then increased to 10 mg/(L∙day), while the ammonium removal rate did not recover and was around 2 mg/(L∙day). The activity of DAMO archaea and Anammox bacteria decreased noticeably. Furthermore, granular aggregates dispersed into small granules and ultimately became flocs with poor settleability in SBR. The content of extracellular polymeric substances decreased, especially that of proteins and humics. DAMO archaea decreased by 94.6% and Anammox bacteria decreased by 72%. In summary, the limitation of methane transfer affected DAMO and Anammox processes more notably than nitrogen transfer, resulting in lower nitrogen removal, granule disruption, and microbial community succession.

Degradation of organic pollutants by anaerobic methane-oxidizing microorganisms using methyl orange as example.

Anaerobic oxidation of methane (AOM) microorganisms widespread in nature and they are able to utilize methane as electron donor to reduce sulfate, nitrate, nitrite, and high valence metals. However, whether persistent organic contaminants can also be degraded remains unknown. In this study, the organic pollutant methyl orange (MO) was used to address this open question. The initial concentration of MO affected its degradation efficiency and higher concentration (>100 mg/L) caused considerable inhibition. A 13CH4 isotope experiment indicated that methane oxidation was involved in MO degradation, which produced N, N-dimethyl-p-phenylenediamine, and 4-aminobenzenesulfonic acid corresponded stoichiometrically. During the long-term experiment, the maximum degradation rate was 47.91 mg/(L·d). The percentage of Candidatus Methanoperedens and Pseudoxanthomonas significantly increased after 30-d of MO degradation under CH4 conditions; moreover, Candidatus Methanoperedens dominated (46.83%) the microbial community. Candidatus Methanoperedens, either alone or in combination with Pseudoxanthomonas, utilized methane as the sole carbon source to degrade MO via direct interspecies electron transfer or the syntrophy pathway. This study will add to our understanding of the functions and applications of AOM microorganisms.

Humic substances as electron acceptors for anaerobic oxidation of methane driven by ANME-2d.

Humic substances (humics) are ubiquitous in terrestrial and aquatic environments where they can serve as electron acceptors for anaerobic oxidation of organic compounds. Methane is a powerful greenhouse gas, as well as the least reactive organic molecule. Anaerobic oxidation of methane (AOM) coupled to microbial reduction of various electron acceptors plays a crucial role in mitigating methane emissions. Here, we reported that humics could serve as terminal electron acceptors for AOM using enriched nitrate-reducing AOM microorganisms. AOM coupled to the reduction of humics was demonstrated based on the production of 13C-labelled carbon dioxide, and AOM activity was evaluated with different methane partial pressures and electron acceptor concentrations. After three-cycle reduction, both AOM activity and copy numbers of the archaea 16S rRNA and mcrA genes were the highest when anthraquinone-2,6-disulfonic acid and anthraquinone-2-sulfonic acid were electron acceptors. The high-throughput sequencing results suggested that ANME-2d were the dominant methane oxidation archaea after humics reduction, although the partner bacteria NC10 trended downward, other reported humics reduction bacteria (Geobactor and Anammox) appeared. The potential electron transfer models from ANME-2d to humics were proposed. These results enable a better understanding of available electron acceptors for AOM in natural environments and broaden our insight into the significant role of ANME-2d.

Investigation of Cr(VI) reduction potential and mechanism by Caldicellulosiruptor saccharolyticus under glucose fermentation condition.

This study examined the microbial reduction of hexavalent chromium [Cr(VI)] by an extremely thermophilic bacterium, Caldicellulosiruptor saccharolyticus, under glucose fermentation conditions at 70°C. Experimentation with different initial Cr(VI) concentrations confirmed that C. saccharolyticus had the ability to reduce Cr(VI) and immobilize Cr(III). At a concentration of 40mg/L, Cr(VI) was completely reduced within 12h, and 97% of the reduction product Cr(III) precipitated on the cell surface. Cr(VI) reduction was accelerated by the addition of neutral red (NR, an electron mediator), resulting in the reduction time shortened to 1h. The addition of CuCl2, a Ni-Fe hydrogenase inhibitor, also enhanced Cr(VI) reduction. Additionally, analysis of the relationship between Cr(VI) reduction and glucose fermentation suggested that different electron sources acted during CuCl2 and NR conditions. Hydrogen served as an electron donor under normal fermentation and NR conditions with the catalysis of Ni-Fe hydrogenase. However, when the activity of Ni-Fe hydrogenase was inhibited by CuCl2, C. saccharolyticus directly used reduction equivalents during glucose fermentation for intracellular Cr(VI) reduction. Therefore, our findings demonstrated high Cr(VI) reduction ability and different electron transfer pathways during Cr(VI) reduction by C. saccharolyticus.

Chromium isotope fractionation during Cr(VI) reduction in a methane-based hollow-fiber membrane biofilm reactor.

Chromium (Cr) isotope fractionation analysis is a promising tool for monitoring Cr(VI) reduction in natural aqueous systems. In addition, large amounts of CH4 in natural aqueous sediments are oxidized to CO2 through methanotrophs, thereby mitigating emissions to the atmosphere. However, the investigations on the Cr(VI) reduction process with methanotrophs, and the associated Cr isotope fractionation patterns are scarce. In this study, we have shown that Cr(VI) reduction can occur in the presence of CH4 as the sole electron donor in a hollow-fiber membrane reactor (HfMBR) after direct bacteria enrichment from sediment samples. Products of the methane oxidation by the methanotrophs are used by microbes to reduce Cr(VI) as shown by the progressive increase in δ53Cr with time in the CH4 feed reactor. The isotope fractionation factor (ε) of -2.62 ± 0.20‰ was obtained from the application of the Rayleigh distillation model. The results of Cr isotope fractionation analysis also explained the decrease of Cr(VI) concentration in the N2 feed reactor, where the δ53Cr values remained steady in the first two weeks but significantly increased in the last two weeks, indicating that physical adsorption and subsequent Cr(VI) reduction occurred. This study extended the application of Cr isotope fractionation, showing the suitability of this method for clarifying different Cr(VI) removal processes.

The content of trace element iron is a key factor for competition between anaerobic ammonium oxidation and methane-dependent denitrification processes.

Coupling of anaerobic ammonium oxidation (Anammox) with denitrifying anaerobic methane oxidation (DAMO) is a sustainable pathway for nitrogen removal and reducing methane emissions from wastewater treatment processes. However, studies on the competitive relation between Anammox bacteria and DAMO bacteria are limited. Here, we investigated the effects of variations in the contents of trace element iron on Anammox and DAMO microorganisms. The short-term results indicated that optimal concentrations of iron, which obviously stimulated the activity of Amammox bacteria, DAMO bacteria and DAMO archaea, were 80, 20, and 80 µM, respectively. The activity of Amammox bacteria increased more significant than DAMO bacteria with increasing contents of trace element iron. After long-term incubation with high content of trace element iron of 160 µM in the medium, Candidatus Brocadia (Amammox bacteria) outcompeted Candidatus Methylomirabilis oxyfera (DAMO bacteria), and ANME-2d (DAMO archaea) remarkably increased in number and dominated the co-culture systems (64.5%). Meanwhile, with further addition of iron, the removal rate of ammonium and nitrate increased by 13.6 and 9.2 times, respectively, when compared with that noted in the control. As far as we know, this study is the first to explore the important role of trace element iron contents in the competition between Anammox bacteria and DAMO bacteria and further enrichment of DAMO archaea by regulating the contents of trace element iron.