Elucidating the microbial community associated with the protein preference of sludge-degrading worms.

Sludge predation by aquatic worms results in an increased sludge reduction rate, which is mainly due to the specific removal of a protein fraction from the sludge. As microorganisms play an essential role in sludge hydrolysis a better understanding of the microbial community involved in the worm predation process will provide more insight into the relations between the aquatic worms, their associated microbiome and the efficient sludge reduction. In this study, the microbial community associated with predation by the Tubifex tubifex was investigated. The microbial diversity in the samples of the worm faeces (WF), predated activated sludge and protein-rich substrates were compared. The results indicated that predation on sludge resulted in a microbial change from Actinobacteria (44%) in the sludge, to Proteobacteria (64%) and Bacteriodites (36%) in the WF. Interestingly, the faecal microbial community was more related to the community in (predated) protein-rich substrates than to the community in predated or endogenously respirated activated sludge samples. This similar microbial community could be due to microbial utilisation of protein hydrolysis products. Alternatively, conditions in the worm gut could facilitate a protein hydrolysing community which assists in protein hydrolysis. The genera Burkholderiales, Chryseobacterium and Flavobacterium were found to be associated with predation by T. tubifex.

Unravelling the protein preference of aquatic worms during waste activated sludge degradation.

Worm predation (WP) by Tubifex tubifex was investigated using waste activated sludge (WAS) as the substrate. In order to better understand the sludge degradation mechanisms during WP, the activity of five common hydrolytic enzymes was determined and compared among the initial feed activated sludge, endogenous respirated sludge and worm predated sludge. The results showed that the enzymatic activity decreased upon aerobic (worm) treatment of WAS and that this activity was predominantly associated with the removed solids fraction of the sludge. Interestingly, the protease activity showed a smaller decrease in activity when the worms were present. Flow cell cytometry revealed the release of intestinal bacteria from the worms, which are presumed to be largely responsible for the observed protease activity. Additionally, experiments in which T. tubifex were treated with antibiotics showed that the worms are responsible for a maximum of 73% of the observed proteolytic activity. The remaining 27% is attributed to the intestinal bacteria that exhibit a synergistic relationship with T. tubifex towards protein hydrolysis.

Effect of humic acid on anaerobic digestion of cellulose and xylan in completely stirred tank reactors: inhibitory effect, mitigation of the inhibition and the dynamics of the microbial communities.

Inhibition effect of humic acid (HA) on anaerobic digestion of cellulose and xylan and the mitigation potential of the inhibition were evaluated in controlled fed batch reactors at 30 °C and a hydraulic retention time (HRT) of 20 days. Reactor performances were evaluated by biogas production and metabolite measurements for 220 days. Microbial population dynamics of the reactors were monitored with next-generation 16S rRNA gene sequencing at nine different sampling times. Our results showed that increasing levels of HA inhibited the hydrolysis efficiency of the digestion by 40% and concomitantly reduced the methane yield. Addition of hydrolytic enzymes helped to reverse the negative effects of HA, whereas calcium addition did not reverse HA inhibition. Microbiological analyses showed that the relative abundance of hydrolytic/fermentative bacterial groups such as Clostridiales, Bacteroidales and Anaerolineales was significantly lowered by the presence of HA. HA also affected the archaeal populations. Mostly hydrogenotrophic methanogens were negatively affected by HA. The relative abundance of Methanobacteriaceae, Methanomicrobiales-WCHA208 and Unassigned Thermoplasmata WCHA1-57 were negatively affected by the presence of HA, whereas Methanosaetacea was not affected.

Physical and biochemical changes in sludge upon Tubifex tubifex predation.

Worm predation (WP) on activated sludge leads to increased sludge degradation rates, irrespective of the type of worm used or reactor conditions employed. However, the cause of the increased sludge degradation rates remains unknown. This paper presents a comparative analysis of the physical and biochemical aspects of predated sludge, providing insight into the hydrolytic mechanisms underlying WP. To this end, the sessile worm Tubifex tubifex was used as a model oligochaete and was batch cultivated in an 18-L airlift reactor. Predation on activated sludge showed an average reduction rate of 12 ± 3.8%/d versus 2 ± 1.3%/d for endogenous respirated sludge. Sludge predation resulted in an increased release of inorganic nitrogen, phosphate and soluble chemical oxygen demand (sCOD). The sCOD consisted mainly of polysaccharides; however, fluorescence excitation emission matrix spectroscopy analysis also revealed the presence of Tryptophan-protein-like substances. Results suggest that the released polysaccharides contain a protein-like element. Additionally, soluble iron increased slightly in concentration after WP. The extent of hydrolysis seemed to reach an average plateau of about 40% volatile solids (VS) reduction after 4 days, which is substantially higher than the 29% VS reduction for endogenous decay of activated sludge after 30 days. Furthermore, T. tubifex predominantly consumed the protein fraction of the extracellular polymeric substances. Results suggest that that the worms specifically target a fraction of the sludge that is predominantly biodegradable under aerobic conditions, albeit at significantly higher degradation rates when compared to the endogenous decay of waste activated sludge.

Mitigation of Humic Acid Inhibition in Anaerobic Digestion of Cellulose by Addition of Various Salts.

Humic compounds are inhibitory to the anaerobic hydrolysis of cellulosic biomass. In this study, the impact of salt addition to mitigate the inhibitory effects of humic compounds was investigated. The experiment was conducted using batch tests to monitor the anaerobic hydrolysis of cellulose in the presence of humic acid. Sodium, potassium, calcium, magnesium and iron salts were tested separately for their efficiency to mitigate humic acid inhibition. All experiments were done under mesophilic conditions (30 °C) and at pH 7. Methane production was monitored online, using the Automatic Methane Potential Test System. Methane production, soluble chemical oxygen demand and volatile fatty acid content of the samples were measured to calculate the hydrolysis efficiencies. Addition of magnesium, calcium and iron salts clearly mitigated the inhibitory effects of humic acid and hydrolysis efficiencies reached up to 75%, 65% and 72%, respectively, which were similar to control experiments. Conversely, potassium and sodium salts addition did not mitigate the inhibition and hydrolysis efficiencies were found to be less than 40%. Mitigation of humic acid inhibition via salt addition was also validated by inductively coupled plasma atomic emission spectroscopy analyses, which showed the binding capacity of different cations to humic acid.

Rare earth metals are essential for methanotrophic life in volcanic mudpots.

Growth of Methylacidiphilum fumariolicum SolV, an extremely acidophilic methanotrophic microbe isolated from an Italian volcanic mudpot, is shown to be strictly dependent on the presence of lanthanides, a group of rare earth elements (REEs) such as lanthanum (Ln), cerium (Ce), praseodymium (Pr) and neodymium (Nd). After fractionation of the bacterial cells and crystallization of the methanol dehydrogenase (MDH), it was shown that lanthanides were essential as cofactor in a homodimeric MDH comparable with one of the MDHs of Methylobacterium extorquens AM1. We hypothesize that the lanthanides provide superior catalytic properties to pyrroloquinoline quinone (PQQ)-dependent MDH, which is a key enzyme for both methanotrophs and methylotrophs. Thus far, all isolated MxaF-type MDHs contain calcium as a catalytic cofactor. The gene encoding the MDH of strain SolV was identified to be a xoxF-ortholog, phylogenetically closely related to mxaF. Analysis of the protein structure and alignment of amino acids showed potential REE-binding motifs in XoxF enzymes of many methylotrophs, suggesting that these may also be lanthanide-dependent MDHs. Our findings will have major environmental implications as metagenome studies showed (lanthanide-containing) XoxF-type MDH is much more prominent in nature than MxaF-type enzymes.

Genomic and Physiological Analysis of Carbon Storage in the Verrucomicrobial Methanotroph "Ca. Methylacidiphilum Fumariolicum" SolV.

"Candidatus Methylacidiphilum fumariolicum" SolV is a verrucomicrobial methanotroph that can grow in extremely acidic environments at high temperature. Strain SolV fixes carbon dioxide (CO(2)) via the Calvin-Benson-Bassham cycle with methane as energy source, a trait so far very unusual in methanotrophs. In this study, the ability of "Ca. M. fumariolicum" to store carbon was explored by genome analysis, physiological studies, and electron microscopy. When cell cultures were depleted for nitrogen, glycogen storage was clearly observed in cytoplasmic storage vesicles by electron microscopy. After cessation of growth, the dry weight kept increasing and the bacteria were filled up almost entirely by glycogen. This was confirmed by biochemical analysis, which showed that glycogen accumulated to 36% of the total dry weight of the cells. When methane was removed from the culture, this glycogen was consumed within 47 days. During the period of glycogen consumption, the bacteria kept their viability high when compared to bacteria without glycogen (from cultures growing exponentially). The latter bacteria lost viability already after a few days when starved for methane. Analysis of the draft genome of "Ca. M. fumariolicum" SolV demonstrated that all known genes for glycogen storage and degradation were present and also transcribed. Phylogenetic analysis of these genes showed that they form a separate cluster with "Ca. M. infernorum" V4, and the most closely related other sequences only have an identity of 40%. This study presents the first physiological evidence of glycogen storage in the phylum Verrucomicrobia and indicates that carbon storage is important for survival at times of methane starvation.

Metabolic Regulation of "Ca. Methylacidiphilum Fumariolicum" SolV Cells Grown Under Different Nitrogen and Oxygen Limitations.

Aerobic methanotrophic bacteria can use methane as their sole energy source. The discovery of "Ca. Methylacidiphilum fumariolicum" strain SolV and other verrucomicrobial methanotrophs has revealed that the ability of bacteria to oxidize CH(4) is much more diverse than has previously been assumed in terms of ecology, phylogeny, and physiology. A remarkable characteristic of the methane-oxidizing Verrucomicrobia is their extremely acidophilic phenotype, growing even below pH 1. In this study we used RNA-Seq to analyze the metabolic regulation of "Ca. M. fumariolicum" SolV cells growing at µ(max) in batch culture or under nitrogen fixing or oxygen limited conditions in chemostats, all at pH 2. The analysis showed that two of the three pmoCAB operons each encoding particulate methane monoxygenases were differentially expressed, probably regulated by the available oxygen. The hydrogen produced during N(2) fixation is apparently recycled as demonstrated by the upregulation of the genes encoding a Ni/Fe-dependent hydrogenase. These hydrogenase genes were also upregulated under low oxygen conditions. Handling of nitrosative stress was shown by the expression of the nitric oxide reductase encoding genes norB and norC under all conditions tested, the upregulation of nitrite reductase nirK under oxygen limitation and of hydroxylamine oxidoreductase hao in the presence of ammonium. Unraveling the gene regulation of carbon and nitrogen metabolism helps to understand the underlying physiological adaptations of strain SolV in view of the harsh conditions of its natural ecosystem.

Draft genome sequence of the volcano-inhabiting thermoacidophilic methanotroph Methylacidiphilum fumariolicum strain SolV.

The draft genome of Methylacidiphilum fumariolicum SolV, a thermoacidophilic methanotroph of the phylum Verrucomicrobia, is presented. Annotation revealed pathways for one-carbon, nitrogen, and hydrogen catabolism and respiration together with central metabolic pathways. The genome encodes three orthologues of particulate methane monooxygenases. Sequencing of this genome will help in the understanding of methane cycling in volcanic environments.

Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon.

Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical ß-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical ß-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient ß-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.

Autotrophic methanotrophy in verrucomicrobia: Methylacidiphilum fumariolicum SolV uses the Calvin-Benson-Bassham cycle for carbon dioxide fixation.

Genome data of the extreme acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicumstrain SolV indicated the ability of autotrophic growth. This was further validated by transcriptome analysis, which showed that all genes required for a functional Calvin-Benson-Bassham (CBB) cycle were transcribed. Experiments with (13)CH(4) or (13)CO(2) in batch and chemostat cultures demonstrated that CO(2) is the sole carbon source for growth of strain SolV. In the presence of CH(4), CO(2) concentrations in the headspace below 1% (vol/vol) were growth limiting, and no growth was observed when CO(2)concentrations were below 0.3% (vol/vol). The activity of the key enzyme of the CBB cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), measured with a (13)C stable-isotope method was about 70 nmol CO(2) fixed · min(-1)· mg of protein(-1). An immune reaction with antibody against the large subunit of RuBisCO on Western blots was found only in the supernatant fractions of cell extracts. The apparent native mass of the RuBisCO complex in strain SolV was about 482 kDa, probably consisting of 8 large (53-kDa) and 8 small (16-kDa) subunits. Based on phylogenetic analysis of the corresponding RuBisCO gene, we postulate that RuBisCO of the verrucomicrobial methanotrophs represents a new type of form I RuBisCO.

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.

Nitrogen fixation by the verrucomicrobial methanotroph 'Methylacidiphilum fumariolicum' SolV.

The ability to utilize atmospheric nitrogen (N(2)) as a sole nitrogen source is an important trait for prokaryotes. Knowledge of N(2) fixation by methanotrophs is needed to understand their role in nitrogen cycling in different environments. The verrucomicrobial methanotroph 'Methylacidiphilum fumariolicum' strain SolV was investigated for its ability to fix N(2). Physiological studies were combined with nitrogenase activity measurements and phylogenetic analysis of the nifDHK genes, encoding the subunits of the nitrogenase. 'M. fumariolicum' SolV was able to fix N(2) at low oxygen (O(2)) concentration (0.5 %, v/v) in chemostat cultures. This low oxygen concentration was also required for an optimal nitrogenase activity [47.4 nmol ethylene h(-1) (mg cell dry weight)(-1)]. Based on acetylene reduction assay and growth experiments, the nitrogenase of strain SolV seems to be extremely oxygen sensitive compared to most proteobacterial methanotrophs. The activity of the nitrogenase was not inhibited by ammonium concentrations up to 94 mM. This is believed to be the first report on the physiology of N(2) fixation within the phylum Verrucomicrobia.

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.