Proteogenomic analysis of Georgfuchsia toluolica revealed unexpected concurrent aerobic and anaerobic toluene degradation.

Denitrifying Betaproteobacteria play a key role in the anaerobic degradation of monoaromatic hydrocarbons. We performed a multi-omics study to better understand the metabolism of the representative organism Georgfuchsia toluolica strain G5G6 known as a strict anaerobe coupling toluene oxidation with dissimilatory nitrate and Fe(III) reduction. Despite the genomic potential for degradation of different carbon sources, we did not find sugar or organic acid transporters, in line with the inability of strain G5G6 to use these substrates. Using a proteomics analysis, we detected proteins of fumarate-dependent toluene activation, membrane-bound nitrate reductase, and key components of the metal-reducing (Mtr) pathway under both nitrate- and Fe(III)-reducing conditions. High abundance of the multiheme cytochrome MtrC implied that a porin-cytochrome complex was used for respiratory Fe(III) reduction. Remarkably, strain G5G6 contains a full set of genes for aerobic toluene degradation, and we detected enzymes of aerobic toluene degradation under both nitrate- and Fe(III)-reducing conditions. We further detected an ATP-dependent benzoyl-CoA reductase, reactive oxygen species detoxification proteins, and cytochrome c oxidase indicating a facultative anaerobic lifestyle of strain G5G6. Correspondingly, we found diffusion through the septa a substantial source of oxygen in the cultures enabling concurrent aerobic and anaerobic toluene degradation by strain G5G6.

Reconsidering hydrolysis kinetics for anaerobic digestion of waste activated sludge applying cascade reactors with ultra-short residence times.

Hydrolysis is considered to be the rate-limiting step in anaerobic digestion of waste activated sludge (WAS). In this study, an innovative 4 stages cascade anaerobic digestion system was researched to (1) comprehensively clarify whether cascading configuration enhances WAS hydrolysis, and to (2) better understand the governing hydrolysis kinetics in this system. The cascade system consisted of three 2.2 L ultra-short solids retention times (SRT) continuous stirred tank reactors (CSTRs) and one 15.4 L CSTR. The cascade system was compared with a reference conventional CSTR digester (22 L) in terms of process performance, hydrolytic enzyme activities and microbial community dynamics under mesophilic conditions (35 °C). The results showed that the cascade system achieved a high and stable total chemical oxygen demand (tCOD) reduction efficiency of 40-42%, even at 12 days total SRT that corresponded to only 1.2 days SRT each in the first three reactors of the cascade. The reference-CSTR converted only 31% tCOD into biogas and suffered process deterioration at the applied low SRTs. Calculated specific hydrolysis rates in the first reactors of the cascade system were significantly higher compared to the reference-CSTR, especially at the lowest applied SRTs. The activities of several hydrolytic enzymes produced in the different stages revealed that protease, cellulase, amino peptidases, and most of the tested glycosyl-hydrolases had significantly higher activities in the first three small digesters of the cascade system, compared to the reference-CSTR. This increase in hydrolytic enzyme production by far exceeded the increase in specific hydrolysis rate, indicating that hydrolysis was limited by solids-surface availability for enzymatic attack. Correspondingly, high relative abundances of hydrolytic-fermentative bacteria and hydrogenotrophic methanogens as well as the presence of syntrophic bacteria were found in the first three digesters of the cascade system. However, in the fourth reactor, acetoclastic methanogens dominated, similarly as in the reference-CSTR. Overall, the results concluded that using multiple CSTRs that are operated at low SRTs in a cascade mode of operation significantly improved the enzymatic hydrolysis rate and extend in anaerobic WAS digestion. Moreover, the governing hydrolysis kinetics in the cascading reactors were far more complex than the generally assumed simplified first-order kinetics.

Temperature susceptibility of a mesophilic anaerobic membrane bioreactor treating saline phenol-containing wastewater.

This study examined the temperature susceptibility of a continuous-flow lab-scale anaerobic membrane bioreactor (AnMBR) to temperature shifts from 35 °C to 55 °C and its bioconversion robustness treating synthetic phenolic wastewater at 16 gNa+.L-1. During the experiment, the mesophilic reactor was subjected to stepwise temperature increases by 5 °C. The phenol conversion rates of the AnMBR decreased from 3.16 at 35 °C to 2.10 mgPh.gVSS-1.d-1 at 45 °C, and further decreased to 1.63 mgPh.gVSS-1.d-1 at 50 °C. At 55 °C, phenol conversion rate stabilized at 1.53 mgPh.gVSS-1.d-1 whereas COD removal efficiency was 38% compared to 95.5% at 45 °C and 99.8% at 35 °C. Interestingly, it was found that the phenol degradation process was less susceptible for the upward temperature shifts than the methanogenic process. The temperature increase implied twenty-one operational taxonomic units from the reactor's microbial community with significant differential abundance between mesophilic and thermophilic operation, and eleven of them are known to be involved in aromatic compounds degradation. Reaching the upper-temperature limits for mesophilic operation was associated with the decrease in microbial abundance of the phyla Firmicutes and Proteobacteria, which are linked to syntrophic phenol degradation. It was also found that the particle size decreased from 89.4 µm at 35 °C to 21.0 µm at 55 °C. The accumulation of small particles and higher content of soluble microbial protein-like substances led to increased transmembrane pressure which negatively affected the filtration performance. Our findings indicated that at high salinity a mesophilic AnMBR can tolerate a temperature up to 45 °C without being limited in the phenol conversion capacity.

Impact of long-term salinity exposure in anaerobic membrane bioreactors treating phenolic wastewater: Performance robustness and endured microbial community.

Industrial wastewaters are becoming increasingly associated with extreme conditions such as the presence of refractory compounds and high salinity that adversely affect biomass retention or reduce biological activity. Hence, this study evaluated the impact of long-term salinity increase to 20 gNa+.L-1 on the bioconversion performance and microbial community composition in anaerobic membrane bioreactors treating phenolic wastewater. Phenol removal efficiency of up to 99.9% was achieved at 14 gNa+.L-1. Phenol conversion rates of 5.1 mgPh.gVSS-1.d-1, 4.7 mgPh.gVSS-1.d-1, and 11.7 mgPh.gVSS-1.d-1 were obtained at 16 gNa+.L-1,18 gNa+.L-1 and 20 gNa+.L-1, respectively. The AnMBR's performance was not affected by short-term step-wise salinity fluctuations of 2 gNa+.L-1 in the last phase of the experiment. It was also demonstrated in batch tests that the COD removal and methane production rate were higher at a K+:Na+ ratio of 0.05, indicating the importance of potassium to maintain the methanogenic activity. The salinity increase adversely affected the transmembrane pressure likely due to a particle size decrease from 185 µm at 14 gNa+.L-1 to 16 µm at 20 gNa+.L-1. Microbial community was dominated by bacteria belonging to the Clostridium genus and archaea by Methanobacterium and Methanosaeta genus. Syntrophic phenol degraders, such as Pelotomaculum genus were found to be increased when the maximum phenol conversion rate was attained at 20 gNa+.L-1. Overall, the observed robustness of the AnMBR performance indicated an endured microbial community to salinity changes in the range of the sodium concentrations applied.

Lignocellulose-derived thin stillage composition and efficient biological treatment with a high-rate hybrid anaerobic bioreactor system.

BACKGROUND: This study aims to chemically characterize thin stillage derived from lignocellulosic biomass distillation residues in terms of organic strength, nutrient, and mineral content. The feasibility of performing anaerobic digestion on these stillages at mesophilic (40 °C) and thermophilic (55 °C) temperatures to produce methane was demonstrated. The microbial communities involved were further characterized. RESULTS: Energy and sugar cane stillage have a high chemical oxygen demand (COD of 43 and 30 g/L, respectively) and low pH (pH 4.3). Furthermore, the acetate concentration in sugar cane stillage was high (45 mM) but was not detected in energy cane stillage. There was also a high amount of lactate in both types of stillage (35-37 mM). The amount of sugars was 200 times higher in energy cane stillage compared to sugar cane stillage. Although there was a high concentration of sulfate (18 and 23 mM in sugar and energy cane stillage, respectively), both thin stillages were efficiently digested anaerobically with high COD removal under mesophilic and thermophilic temperature conditions and with an organic loading rate of 15-21 g COD/L/d. The methane production rate was 0.2 L/g COD, with a methane percentage of 60 and 64, and 92 and 94 % soluble COD removed, respectively, by the mesophilic and thermophilic reactors. Although both treatment processes were equally efficient, there were different microbial communities involved possibly arising from the differences in the composition of energy cane and sugar cane stillage. There was more acetic acid in sugar cane stillage which may have promoted the occurrence of aceticlastic methanogens to perform a direct conversion of acetate to methane in reactors treating sugar cane stillage. CONCLUSIONS: Results showed that thin stillage contains easily degradable compounds suitable for anaerobic digestion and that hybrid reactors can efficiently convert thin stillage to methane under mesophilic and thermophilic conditions. Furthermore, we found that optimal conditions for biological treatment of thin stillage were similar for both mesophilic and thermophilic reactors. Bar-coded pyrosequencing of the 16S rRNA gene identified different microbial communities in mesophilic and thermophilic reactors and these differences in the microbial communities could be linked to the composition of the thin stillage.

Genome and proteome analysis of Pseudomonas chloritidismutans†AW-1T that grows on n-decane with chlorate or oxygen as electron acceptor.

Growth of Pseudomonas chloritidismutans AW-1T on C7 to C12 n-alkanes with oxygen or chlorate as electron acceptor was studied by genome and proteome analysis. Whole genome shotgun sequencing resulted in a 5 Mbp assembled sequence with a G + C content of 62.5%. The automatic annotation identified 4767 protein-encoding genes and a putative function could be assigned to almost 80% of the predicted proteins. The distinct phylogenetic position of P. chloritidismutans AW-1T within the Pseudomonas stutzeri cluster became clear by comparison of average nucleotide identity values of sequenced genomes. Analysis of the proteome of P. chloritidismutans AW-1T showed the versatility of this bacterium to adapt to aerobic and anaerobic growth conditions with acetate or n-decane as substrates. All enzymes involved in the alkane oxidation pathway were identified. An alkane monooxygenase was detected in n-decane-grown cells, but not in acetate-grown cells. The enzyme was found when grown in the presence of oxygen or chlorate, indicating that under both conditions an oxygenase-mediated pathway is employed for alkane degradation. Proteomic and biochemical data also showed that both chlorate reductase and chlorite dismutase are constitutively present, but most abundant under chlorate-reducing conditions.

Microbial respiration with chlorine oxyanions: diversity and physiological and biochemical properties of chlorate- and perchlorate-reducing microorganisms.

Chlorine oxyanions are valuable electron acceptors for microorganisms. Recent findings have shed light on the natural formation of chlorine oxyanions in the environment. These suggest a permanent introduction of respective compounds on Earth, long before their anthropogenic manufacture. Microorganisms that are able to grow by the reduction of chlorate and perchlorate are affiliated with phylogenetically diverse lineages, spanning from the Proteobacteria to the Firmicutes and archaeal microorganisms. Microbial reduction of chlorine oxyanions can be found in diverse environments and different environmental conditions (temperature, salinities, pH). It commonly involves the enzymes perchlorate reductase (Pcr) or chlorate reductase (Clr) and chlorite dismutase (Cld). Horizontal gene transfer seems to play an important role for the acquisition of functional genes. Novel and efficient Clds were isolated from microorganisms incapable of growing on chlorine oxyanions. Archaea seem to use a periplasmic Nar-type reductase (pNar) for perchlorate reduction and lack a functional Cld. Chlorite is possibly eliminated by alternative (abiotic) reactions. This was already demonstrated for Archaeoglobus fulgidus, which uses reduced sulfur compounds to detoxify chlorite. A broad biochemical diversity of the trait, its environmental dispersal, and the occurrence of relevant enzymes in diverse lineages may indicate early adaptations of life toward chlorine oxyanions on Earth.

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC.

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a ß-hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to ß-hydroxybutyrate that may be converted to the energy and carbon storage compound, poly-ß-hydroxybutyrate. Accordingly, we confirmed the formation of poly-ß-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor.

Metabolic response of Alicycliphilus denitrificans strain BC toward electron acceptor variation.

Alicycliphilus denitrificans is a versatile, ubiquitous, facultative anaerobic bacterium. Alicycliphilus denitrificans strain BC can use chlorate, nitrate, and oxygen as electron acceptor for growth. Cells display a prolonged lag-phase when transferred from nitrate to chlorate and vice versa. Furthermore, cells adapted to aerobic growth do not easily use nitrate or chlorate as electron acceptor. We further investigated these responses of strain BC by differential proteomics, transcript analysis, and enzyme activity assays. In nitrate-adapted cells transferred to chlorate and vice versa, appropriate electron acceptor reduction pathways need to be activated. In oxygen-adapted cells, adaptation to the use of chlorate or nitrate is likely difficult due to the poorly active nitrate reduction pathway and low active chlorate reduction pathway. We deduce that the Nar-type nitrate reductase of strain BC also reduces chlorate, which may result in toxic levels of chlorite if cells are transferred to chlorate. Furthermore, the activities of nitrate reductase and nitrite reductase appear to be not balanced when oxygen-adapted cells are shifted to nitrate as electron acceptor, leading to the production of a toxic amount of nitrite. These data suggest that strain BC encounters metabolic challenges in environments with fluctuations in the availability of electron acceptors. All MS data have been deposited in the ProteomeXchange with identifier PXD000258.

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601(T.).

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601(T) have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601(T) is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601(T) are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601(T) and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601(T). Genes involved in cyclohexanol degradation were only found in strain K601(T). Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.

Genome sequences of Alicycliphilus denitrificans strains BC and K601T.

Alicycliphilus denitrificans strain BC and A. denitrificans strain K601(T) degrade cyclic hydrocarbons. These strains have been isolated from a mixture of wastewater treatment plant material and benzene-polluted soil and from a wastewater treatment plant, respectively, suggesting their role in bioremediation of soil and water. Although the strains are phylogenetically closely related, there are some clear physiological differences. The hydrocarbon cyclohexanol, for example, can be degraded by strain K601(T) but not by strain BC. Furthermore, both strains can use nitrate and oxygen as an electron acceptor, but only strain BC can use chlorate as electron acceptor. To better understand the nitrate and chlorate reduction mechanisms coupled to the oxidation of cyclic compounds, the genomes of A. denitrificans strains BC and K601(T) were sequenced. Here, we report the complete genome sequences of A. denitrificans strains BC and K601(T).

Nitrate and (per)chlorate reduction pathways in (per)chlorate-reducing bacteria.

The reduction of (per)chlorate and nitrate in (per)chlorate-reducing bacteria shows similarities and differences. (Per)chlorate reductase and nitrate reductase both belong to the type II DMSO family of enzymes and have a common bis(molybdopterin guanine dinucleotide)molybdenum cofactor. There are two types of dissimilatory nitrate reductases. With respect to their localization, (per)chlorate reductase is more similar to the dissimilatory periplasmic nitrate reductase. However, the periplasmic, unlike the membrane-bound, respiratory nitrate reductase, is not able to use chlorate. Structurally, (per)chlorate reductase is more similar to respiratory nitrate reductase, since these reductases have analogous subunits encoded by analogous genes. Both periplasmic (per)chlorate reductase and membrane-bound nitrate reductase activities are induced under anoxic conditions in the presence of (per)chlorate and nitrate respectively. During microbial (per)chlorate reduction, molecular oxygen is generated. This is not the case for nitrate reduction, although an atypical reaction in nitrite reduction linked to oxygen formation has been described recently. Microbial oxygen production during reduction of oxyanions may enhance biodegradation of pollutants under anoxic conditions.