Synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under methanethiol stress.

Methanotrophs are able to metabolize volatile organic sulfur compounds (VOSCs), excrete organic carbon during CH4 oxidation, and influence microbial community structure and function of the ecosystem. In return, microbial community structure and environmental factors can affect the growth metabolism of methanotrophs. In this study, Methylomonas koyamae and Hyphomicrobium methylovorum were used for model organisms, and methanethiol (MT) was chosen for a typical VOSC to investigate the synergy effects under VOSC stress. The results showed that when Hyphomicrobium methylovorum was co-cultured with Methylomonas koyamae in the medium with CH4 used as the carbon source, the co-culture had better MT tolerance relative to Methylomonas koyamae and oxidized all CH4 within 120 h, even at the initial MT concentration of 2000 mg m-3. The optimal co-culture ratios of Methylomonas koyamae to Hyphomicrobium methylovorum were 4:1-12:1. Although MT could be converted spontaneously to dimethyl disulfide (DMDS), H2S, and CS2 in air, faster losses of MT, DMDS, H2S, and CS2 were observed in each strain mono-culture and the co-culture. Compared with Hyphomicrobium methylovorum, MT was degraded more quickly in the Methylomonas koyamae culture. During the co-culture, the CH4 oxidation process of Methylomonas koyamae could provide carbon and energy sources for the growth of Hyphomicrobium methylovorum, while Hyphomicrobium methylovorum oxidized MT to help Methylomonas koyamae detoxify. These findings are helpful to understand the synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under MT stress and enrich the role of methanotrophs in the sulfur biogeochemical cycle. KEY POINTS: • The co-culture of Methylomonas and Hyphomicrobium has better tolerance to CH3SH. • Methylomonas can provide carbon sources for the growth of Hyphomicrobium. • The co-culture of Methylomonas and Hyphomicrobium enhances the removal of CH4 and CH3SH.

Detoxification Esterase StrH Initiates Strobilurin Fungicide Degradation in Hyphomicrobium sp. Strain DY-1.

Strobilurin fungicides are widely used in agricultural production due to their broad-spectrum and fungal mitochondrial inhibitory activities. However, their massive application has restrained the growth of eukaryotic algae and increased collateral damage in freshwater systems, notably harmful cyanobacterial blooms (HCBs). In this study, a strobilurin fungicide-degrading strain, Hyphomicrobium sp. strain DY-1, was isolated and characterized successfully. Moreover, a novel esterase gene, strH, responsible for the de-esterification of strobilurin fungicides, was cloned, and the enzymatic properties of StrH were studied. For trifloxystrobin, StrH displayed maximum activity at 50°C and pH 7.0. The catalytic efficiencies (kcat/Km ) of StrH for different strobilurin fungicides were 196.32 ± 2.30 µM-1 · s-1 (trifloxystrobin), 4.64 ± 0.05 µM-1 · s-1 (picoxystrobin), 2.94 ± 0.02 µM-1 · s-1 (pyraclostrobin), and (2.41 ± 0.19)×10-2 µM-1 · s-1 (azoxystrobin). StrH catalyzed the de-esterification of a variety of strobilurin fungicides, generating the corresponding parent acid to achieve the detoxification of strobilurin fungicides and relieve strobilurin fungicide growth inhibition of Chlorella This research will provide insight into the microbial remediation of strobilurin fungicide-contaminated environments.IMPORTANCE Strobilurin fungicides have been widely acknowledged as an essential group of pesticides worldwide. So far, their residues and toxic effects on aquatic organisms have been reported in different parts of the world. Microbial degradation can eliminate xenobiotics from the environment. Therefore, the degradation of strobilurin fungicides by microorganisms has also been reported. However, little is known about the involvement of enzymes or genes in strobilurin fungicide degradation. In this study, a novel esterase gene responsible for the detoxification of strobilurin fungicides, strH, was cloned in the newly isolated strain Hyphomicrobium sp. DY-1. This degradation process detoxifies the strobilurin fungicides and relieves their growth inhibition of Chlorella.

Two-stage oxygen supply strategy for enhancing fed-batch production of pyrroloquinoline quinone in Hyphomicrobium denitrificans FJNU-6.

Oxygen is a vital parameter for pyrroloquinoline quinone (PQQ) biosynthesis. In this study, the effects of oxygen supply on the biosynthesis of PQQ were first investigated systematically with Hyphomicrobium denitrificans FJNU-6. Following a kinetic analysis of the specific cell growth rate (µx) and specific PQQ formation rate (µp) in 5 L benchtop fermentation systems at various oxygen supply levels ranging from 0 to 60%, a novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity. Moreover, the transcription of genes involved in methanol oxidation and PQQ biosynthesis was analyzed throughout the process to outline the effect of oxygen supply on cell metabolism. Furthermore, with constant feeding of methanol at 0-1 g/L after the initial methanol was consumed completely, the PQQ concentration and productivity reached 1070 mg/L and 7.64 mg/L/h, respectively, after 140 h in a 5-L fermenter. The two-stage oxygen supply strategy developed in this study provides an effective and economical strategy for the industrial production of PQQ.Key Points• A novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity.•The transcription of genes involved in methanol oxidation and PQQ biosynthesis was regulated by changes in oxygen supply.• This study offers an effective and economical strategy for industrial or large-scale production of PQQ.

Development of a novel methanotrophic process with the helper micro-organism Hyphomicrobium sp. NM3.

AIMS: Microbial consortia can be more efficient at biological processes than single isolates. The purposes of this study were to design and evaluate a synthetic microbial consortium containing the methanotroph Methylocystis sp. M6 and the helper Hyphomicrobium sp. NM3, and develop a novel methanotrophic process for this consortium utilizing a dialysis membrane. METHODS AND RESULTS: Hyphomicrobium increased the methane-oxidation rate (MOR), biomass and stability at a dilution rate of 0·067 day-1 in fed-batch co-culture. qRT-PCR showed that Methylocystis population increased gradually with time, whereas Hyphomicrobium population remained stable despite cell washing, confirming synergistic population interaction. At 0·1 day-1 , spiking of Hyphomicrobium effectively increased the methanotrophic activity, after which Hyphomicrobium population decreased with time, indicating that the consortium is optimal at <0·1 day-1 . When Hyphomicrobium was grown in dialysis membrane within the bioreactor, MOR increased linearly up to 155·1 ± 1·0 mmol l-1  day-1 at 0·067, 0·1, 0·2 and 0·4 day-1 , which is the highest observed value for a methanotrophic reactor. CONCLUSIONS: Hyphomicrobium sp. NM3 is a promising helper micro-organism for methanotrophs. Hyphomicrobium-methanotroph consortia used concurrently with existing methods can produce an efficient and stable methane oxidation system. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel methanotrophic process is superior to those previously reported in the literature, and can provide efficient and stable methane oxidation.

Catabolic task division between two near-isogenic subpopulations co-existing in a herbicide-degrading bacterial consortium: consequences for the interspecies consortium metabolic model.

Variovorax sp. WDL1 mediates hydrolysis of the herbicide linuron into 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine in a tripartite bacterial consortium with Comamonas testosteroni WDL7 and Hyphomicrobium sulfonivorans WDL6. Although strain WDL1 contains the dcaQTA1A2B operon for DCA oxidation, this conversion is mainly performed by WDL7. Phenotypic diversification observed in WDL1 cultures and scrutiny of the WDL1 genome suggest that WDL1 cultures consist of two dedicated subpopulations, i.e., a linuron-hydrolysing subpopulation (Lin + DCA-) and a DCA-oxidizing subpopulation (Lin-DCA+). Whole genome analysis of strains representing the respective subpopulations revealed that they are identical, aside from the presence of hylA (in Lin + DCA- cells) and the dcaQTA1A2B gene cluster (in Lin-DCA+ cells), and that these catabolic gene modules replace each other at exactly the same locus on a 1380 kb extra-chromosomal element that shows plasmid gene functions including genes for transferability by conjugation. Both subpopulations proliferate in consortium biofilms fed with linuron, but Lin + DCA- cells compose the main WDL1 subpopulation. Our observations instigated revisiting the interactions within the consortium and suggest that the physical separation of two essential linuron catabolic gene clusters in WDL1 by mutually exclusive integration in the same mobile genetic element is key to the existence of WDL1 in a consortium mode.

Metaproteomics Applied to Activated Sludge for Industrial Wastewater Treatment Revealed a Dominant Methylotrophic Metabolism of Hyphomicrobium zavarzinii.

In biological wastewater treatments, microbial populations of the so-called activated sludge work together in the abatement of pollutants. In this work, the metabolic behavior of the biomass of a lab-scale plant treating industrial pharmaceutical wastewater was investigated through a metaproteomic approach. The complete treatment process included a membrane biological reactor (MBR) coupled with an advanced oxidation process (AOP) for partial breakdown of non-biodegradable molecules. Proteins from biomass samples collected pre- and post-AOP application were investigated by two-dimensional gel electrophoresis (2DE), mass spectrometry (MS), and finally identified by database search. Results showed that most proteins remained constant between pre- and post-AOP. Methanol dehydrogenase (MDH) belonging to Hyphomicrobium zavarzinii appeared as the most constantly expressed protein in the studied consortium. Other identified proteins belonging to Hyphomicrobium spp. revealed a predominant methylotrophic metabolism, and H. zavarzinii appeared as a key actor in the studied microbial community.

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans, Hyphomicrobium denitrificans, and Hyphomicrobium zavarzinii.

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.

Potentialities of coupling biological processes (biotrickler/biofilter) for the degradation of a mixture of sulphur compounds.

This study deals with the potential of biological processes combining a biotrickler and a biofilter to treat a mixture of sulphur-reduced compounds including dimethyl sulphide (DMS), dimethyl disulphide (DMDS) and hydrogen sulphide (H2S). As a reference, duplicated biofilters were implemented, and operating conditions were similar for all bioprocesses. The first step of this work was to determine the efficiency removal level achieved for each compound of the mixture and in a second step, to assess the longitudinal distribution of biodegradation activities and evaluate the total bacteria, Hyphomicrobium sp. and Thiobacillus thioparus densities along the bed height. A complete removal of hydrogen sulphide is reached at the start of the experiment within the first stage (biotrickler) of the coupling. This study highlighted that the coupling of a biotrickling filter and a biofilter is an interesting way to improve both removal efficiency levels (15-20% more) and kinetics of recalcitrant sulphur compounds such as DMS and DMDS. The total cell densities remained similar (around 1 × 10(10) 16S recombinant DNA (rDNA) copies g dry packing material) for duplicated biofilters and the biofilter below the biotrickling filter. The relative abundances of Hyphomicrobium sp. and T. thioparus have been estimated to an average of 10 ± 7.0 and 0.23 ± 0.07%, respectively, for all biofilters. Further investigation should allow achieving complete removal of DMS by starting the organic sulphur compound degradation within the first stage and surveying microbial community structure colonizing this complex system.

Hyphomicrobium nitrativorans sp. nov., isolated from the biofilm of a methanol-fed denitrification system treating seawater at the Montreal Biodome.

A budding prosthecate bacterial strain, designated NL23(T), was isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. Phylogenetic analysis based on 16S rRNA (rRNA) gene sequences showed that the strain was affiliated with the genus Hyphomicrobium of the Alphaproteobacteria and was most closely related to Hyphomicrobium zavarzinii with 99.4 % sequence similarity. Despite this high level of 16S rRNA gene sequence similarity, DNA-DNA hybridization assays showed that strain NL23(T) was only distantly related to H. zavarzinii ZV-622(T) (12 %). Strain NL23(T) grew aerobically, but also had the capacity to grow under denitrifying conditions in the presence of nitrate without nitrite accumulation. Growth occurred at pH 7.0-9.5, with 0-1 % NaCl and at temperatures of 15-35 °C. Major fatty acids were C18 : 1ω7c or ω6c (84.6 %) and C18 : 0 (8.5 %), and major quinones were Q8 (5 %) and Q9 (95 %). The complete genome of the strain was sequenced and showed a DNA G+C content of 63.8 mol%. Genome analysis predicted open reading frames (ORF) encoding the key enzymes of the serine pathway as well as enzymes involved in methylotrophy. Also, ORF encoding a periplasmic nitrate reductase (Nap), a nitrite reductase (Nir), a nitric oxide reductase (Nor) and a nitrous oxide reductase (Nos) were identified. Our results support that strain NL23(T) represents a novel species within the genus Hyphomicrobium, for which the name Hyphomicrobium nitrativorans sp. nov. is proposed. The type strain is NL23(T) ( = ATCC BAA-2476(T) = LMG 27277(T)).

Dimethyl sulfide biofiltration using immobilized Hyphomicrobium VS and Thiobacillus thioparus TK-m in sugarcane bagasse.

Sugarcane bagasse was used as a carrier material of microorganisms in two different biofilters used to remove dimethyl sulfide (DMS) from a gas stream. The first biofilter was inoculated with Hyphomicrobium VS and the second with Thiobacillus thioparus Tk-m. During the operation of the biofilters the empty bed residence time (EBRT) was varied from 90 to 180 seconds and the inlet concentration of DMS from 12 to 50 ppmv. The inlet load was varied in the range of 0.62 to 5.2 (g DMS/m3 h). The maximum elimination capacity (EC) of the biofilter inoculated with Hyphomicrobium VS was 5 g DMS/m3 h; however, for the biofilter inoculated with T. thioparus Tk-m the maximum EC was 3.9 g DMS/m3 h. For T. thioparus TK-m the maximum removal efficiency (RE) obtained was 85.1 +/- 5.2% at 12 ppmv DMS inlet concentration, inlet load of 0.62 g DMS/m3 h and 180 s EBRT. The highest removal efficiency for Hyphomicrobium VS was 97.6 + 4.8% at 12 ppmv DMS inlet concentration, load of 0.62 g DMS/m3 h and 180 s EBRT.

Complete genome sequence of the chloromethane-degrading Hyphomicrobium sp. strain MC1.

Hyphomicrobium sp. strain MC1 is an aerobic methylotroph originally isolated from industrial sewage. This prosthecate bacterium was the first strain reported to grow with chloromethane as the sole carbon and energy source. Its genome, consisting of a single 4.76-Mb chromosome, is the first for a chloromethane-degrading bacterium to be formally reported.

Purification and characterization of dimethylsulfide monooxygenase from Hyphomicrobium sulfonivorans.

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH2-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K(m) of 17.2 (± 0.48) µM for DMS (k(cat) = 5.45 s⁻¹) and a V(max) of 1.25 (± 0.01) µmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²(+), Cd²(+), and Pb²(+) ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH2-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.

Growth kinetics of Hyphomicrobium and Thiobacillus spp. in mixed cultures degrading dimethyl sulfide and methanol.

The growth kinetics of Hyphomicrobium spp. and Thiobacillus spp. on dimethyl sulfide (DMS) and methanol (in the case of Hyphomicrobium spp.) in an enrichment culture created from a biofilter cotreating DMS and methanol were studied. Specific growth rates of 0.099 h(-1) and 0.11 h(-1) were determined for Hyphomicrobium spp. and Thiobacillus spp., respectively, growing on DMS at pH 7. These specific growth rates are double the highest maximum specific growth rate for bacterial growth on DMS reported to date in the literature. When the pH of the medium was decreased from pH 7 to pH 5, the specific growth rate of Hyphomicrobium spp. decreased by 85%, with a near 100-fold decline in the yield of Hyphomicrobium 16S rRNA gene copies in the mixed culture. Through the same pH shift, the specific growth rate and 16S rRNA gene yield of Thiobacillus spp. remained similar. When methanol was used as a substrate, the specific growth rate of Hyphomicrobium spp. declined much less over the same pH range (up to 30%) while the yield of 16S rRNA gene copies declined by only 50%. Switching from an NH(4)(+)-N-based source to a NO(3)(-)-N-based source resulted in the same trends for the specific growth rate of these microorganisms with respect to pH. This suggests that pH has far more impact on the growth kinetics of these microorganisms than the nitrogen source. The results of these mixed-culture batch experiments indicate that the increased DMS removal rates observed in previous studies of biofilters cotreating DMS and methanol are due to the proliferation of DMS-degrading Hyphomicrobium spp. on methanol at pH levels not conducive to high growth rates on DMS alone.

Degradation of methamidophos by Hyphomicrobium species MAP-1 and the biochemical degradation pathway.

Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of methamidophos. Further, this strain could utilize methamidophos as its sole carbon, nitrogen and phosphorus source for growth and could completely degrade 3,000 mg l(-1) methamidophos in 84 h under optimal conditions (pH 7.0, 30 degrees C). The enzyme responsible for methamidophos degradation was mainly located on the cell inner membrane (90.4%). During methamidophos degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC-MS) analysis. Using this information, a biochemical degradation pathway of methamidophos by Hyphomicrobium sp. MAP-1 was proposed for the first time. Methamidophos is first cleaved at the P-N bond to form O,S-dimethyl hydrogen thiophosphate and NH(3). Subsequently, O,S-dimethyl hydrogen thiophosphate is hydrolyzed at the P-O bond to release -OCH(3) and form S-methyl dihydrogen thiophosphate. O,S-dimethyl hydrogen thiophosphate can also be hydrolyzed at the P-S bond to release -SCH(3) and form methyl dihydrogen phosphate. Finally, S-methyl dihydrogen thiophosphate and methyl dihydrogen phosphate are likely transformed into phosphoric acid.

Atomic resolution structure of pseudoazurin from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans: structural insights into its spectroscopic properties.

The crystal structure of native pseudoazurin (HdPAz) from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans has been determined at a resolution of 1.18 A. After refinement with SHELX employing anisotropic displacement parameters and riding H atoms, R(work) and R(free) were 0.135 and 0.169, respectively. Visualization of the anisotropic displacement parameters as thermal ellipsoids provided insight into the atomic motion within the perturbed type 1 Cu site. The asymmetric unit includes three HdPAz molecules which are tightly packed by head-to-head cupredoxin dimer formation. The shape of the Cu-atom ellipsoid implies significant vibrational motion diagonal to the equatorial xy plane defined by the three ligands (two His and one Cys). The geometric parameters of the type 1 Cu site in the HdPAz structure differ unambiguously from those of other pseudoazurins. It is demonstrated that their structural aspects are consistent with the unique visible absorption spectrum.

Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria.

The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of (13)C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived (13)C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real-time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors.

Architecture and spatial organization in a triple-species bacterial biofilm synergistically degrading the phenylurea herbicide linuron.

Members of a triple-species 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea (linuron)-mineralizing consortium, i.e. the linuron- and 3,4-dichloroaniline-degrading Variovorax sp. WDL1, the 3,4-dichloroaniline-degrading Comamonas testosteroni WDL7 and the N,O-dimethylhydroxylamine-degrading Hyphomicrobium sulfonivorans WDL6, were cultivated as mono- or multi-species biofilms in flow cells irrigated with selective or nonselective media, and examined with confocal laser scanning microscopy. In contrast to mono-species biofilms of Variovorax sp. WDL1, the triple-species consortium biofilm degraded linuron completely through apparent synergistic interactions. The triple-species linuron-fed consortium biofilm displayed a heterogeneous structure with an irregular surface topography that most resembled the topography of linuron-fed mono-species WDL1 biofilms, indicating that WDL1 had a dominating influence on the triple-species biofilm architecture. This architecture was dependent on the carbon source supplied, as the biofilm architecture of WDL1 growing on alternative carbon sources was different from that observed under linuron-fed conditions. Linuron-fed triple-species consortium biofilms consisted of mounds composed of closely associated WDL1, WDL7 and WDL6 cells, while this association was lost when the consortium was grown on a nonselective carbon source. In addition, under linuron-fed conditions, microcolonies displaying associated growth developed rapidly after inoculation. These observations indicate that the spatial organization in the linuron-fed consortium biofilm reflected the metabolic interactions within the consortium.

A fast method for the quantification of methylamine in fermentation broths by gas chromatography.

The objective of this study was to develop a method for the quantitative analysis of the methylamine concentration in fermentation broths of Hyphomicrobium zavarzinii ZV 580 cultures. For this purpose an established method for the quantification of free amino acids in such matrices was adapted and validated. The detection limit was 10 microM, the calibration curve showed good linearity (R2=0.9998) in the concentration range between 0.1 and 8 mM. The standard deviation of the injection-to-injection reproducibility (n=10) of the retention coefficient was <1%, that of the peak area<5%. In case of the sample-to-sample reproducibility (n=8), the standard deviation was <5% for the retention coefficient and <10% for the peak area. The validated method was successfully applied for monitoring a fed-batch bioprocess (starting volume: 8L, initial methylamine hydrochloride concentration: 10 mM) producing a dye-linked formaldehyde dehydrogenase in H. zavarzinii ZV 580.

Denitrification with carbon addition--kinetic considerations.

The Blue Plains Advanced Wastewater Treatment Plant (Washington, D.C.) uses methanol as an external carbon source in a postdenitrification process, to achieve low effluent total nitrogen concentrations. This becomes more difficult in winter, at lower mixed liquor temperatures and higher flows, as a consequence of the kinetic behavior of the methanol-utilizing heterotrophs. The paper reports on an experimental batch test study conducted on Blue Plains postdenitrification sludge to investigate (1) the maximum specific growth rate of methanol-utilizing heterotrophs (Mu(METH)); (2) the temperature dependency of the growth rate; and (3) the efficacy of alternate substrates (ethanol, acetate, and sugar). A limited number of tests were conducted on sludge from two other treatment plants with methanol addition.

A novel bacterial sulfur oxidation pathway provides a new link between the cycles of organic and inorganic sulfur compounds.

Dimethylsulfide (DMS) plays a globally significant role in carbon and sulfur cycling and impacts Earth's climate because its oxidation products serve as nuclei for cloud formation. While the initial steps of aerobic DMS degradation and the fate of its carbon atoms are reasonably well documented, oxidation of the contained sulfur is largely unexplored. Here, we identified a novel pathway of sulfur compound oxidation in the ubiquitously occurring DMS-degrader Hyphomicrobium denitrificans XT that links the oxidation of the volatile organosulfur compound with that of the inorganic sulfur compound thiosulfate. DMS is first transformed to methanethiol from which sulfide is released and fully oxidized to sulfate. Comparative proteomics indicated thiosulfate as an intermediate of this pathway and pointed at a heterodisulfide reductase (Hdr)-like system acting as a sulfur-oxidizing entity. Indeed, marker exchange mutagenesis of hdr-like genes disrupted the ability of H. denitrificans to metabolize DMS and also prevented formation of sulfate from thiosulfate provided as an additional electron source during chemoorganoheterotrophic growth. Complementation with the hdr-like genes under a constitutive promoter rescued the phenotype on thiosulfate as well as on DMS. The production of sulfate from an organosulfur precursor via the Hdr-like system is previously undocumented and provides a new shunt in the biogeochemical sulfur cycle. Furthermore, our findings fill a long-standing knowledge gap in microbial dissimilatory sulfur metabolism because the Hdr-like pathway is abundant not only in chemoheterotrophs, but also in a wide range of chemo- and photolithoautotrophic sulfur oxidizers acting as key players in global sulfur cycling.