Biofilm thickness controls the relative importance of stochastic and deterministic processes in microbial community assembly in moving bed biofilm reactors.

Deterministic and stochastic processes are believed to play a combined role in microbial community assembly, though little is known about the factors determining their relative importance. We investigated the effect of biofilm thickness on community assembly in nitrifying moving bed biofilm reactors using biofilm carriers where maximum biofilm thickness is controlled. We examined the contribution of stochastic and deterministic processes to biofilm assembly in a steady state system using neutral community modelling and community diversity analysis with a null-modelling approach. Our results indicate that the formation of biofilms results in habitat filtration, causing selection for phylogenetically closely related community members, resulting in a substantial enrichment of Nitrospira spp. in the biofilm communities. Stochastic assembly processes were more prevalent in biofilms of 200 µm and thicker, while stronger selection in thinner (50 µm) biofilms could be driven by hydrodynamic and shear forces at the biofilm surface. Thicker biofilms exhibited greater phylogenetic beta-diversity, which may be driven by a variable selection regime caused by variation in environmental conditions between replicate carrier communities, or by drift combined with low migration rates resulting in stochastic historical contingency during community establishment. Our results indicate that assembly processes vary with biofilm thickness, contributing to our understanding of biofilm ecology and potentially paving the way towards strategies for microbial community management in biofilm systems.

DNA- and RNA-SIP Reveal Nitrospira spp. as Key Drivers of Nitrification in Groundwater-Fed Biofilters.

Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier investigations based on 16S rRNA and amoA amplicon analysis, amoA quantitative PCR and metagenomics of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira Here, we hypothesized that these nonclassical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subjected to a continuous supply of NH4+ or NO2- in the presence of 13C-HCO3- (labeled) or 12C-HCO3- (unlabeled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonia oxidation in the biofilter community. A total of 78 (8 by RNA-SIP and 70 by DNA-SIP) and 96 (25 by RNA-SIP and 71 by DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3- uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, and Acidovorax under conditions that favored ammonia oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia-oxidizing taxa was not detected in the SIP fractions. This study provides the first in situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome.IMPORTANCE With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3- uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments.

Copper-Induced Stimulation of Nitrification in Biological Rapid Sand Filters for Drinking Water Production by Proliferation of Nitrosomonas spp.

Copper is a cofactor of the ammonia monooxygenase, an essential enzyme for the activity of ammonia oxidizing prokaryotes (AOP). Copper dosing at less than 1 µg/L stimulated ammonium removal in the poorly nitrifying biological filters of three full-scale drinking water treatment plants. Upon copper dosing, the ammonium concentration in the effluent decreased from up to 0.18 to less than 0.01 mg NH4+-N/L. To investigate how copper dosing affected the filter microbial community, we applied amplicon sequencing and qPCR targeting key nitrifying groups, including complete ammonia oxidizing (comammox) Nitrospira. Copper dosing increased the abundance of different nitrifiers. Multiple Nitrosomonas variants (betaproteobacterial ammonia oxidizers), which initially collectively represented 1% or less of the total community, increased almost 10-fold. Comammox Nitrospira were abundant and increased too, but their relative abundance within the AOP decreased because of Nitrosomonas proliferation. No other consistent change in the filter communities was detected, as well as no adverse effect of copper on the filters functionality. Our results show that copper dosing in three independent treatment plants was associated with consistent growth of AOP and that efficient nitrification was achieved through the joint contribution of comammox Nitrospira and an increasing fraction of betaproteobacterial ammonia oxidizers.

Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira.

The description of comammox Nitrospira spp., performing complete ammonia-to-nitrate oxidation, and their co-occurrence with canonical ß-proteobacterial ammonia oxidizing bacteria (ß-AOB) in the environment, calls into question the metabolic potential of comammox Nitrospira and the evolutionary history of their ammonia oxidation pathway. We report four new comammox Nitrospira genomes, constituting two novel species, and the first comparative genomic analysis on comammox Nitrospira. Unlike canonical Nitrospira, comammox Nitrospira genomes lack genes for assimilatory nitrite reduction, suggesting that they have lost the potential to use external nitrite nitrogen sources. By contrast, compared to canonical Nitrospira, comammox Nitrospira harbor a higher diversity of urea transporters and copper homeostasis genes and lack cyanate hydratase genes. Additionally, the two comammox clades differ in their ammonium uptake systems. Contrary to ß-AOB, comammox Nitrospira genomes have single copies of the two central ammonia oxidation pathway operons. Similar to ammonia oxidizing archaea and some oligotrophic AOB strains, they lack genes involved in nitric oxide reduction. Furthermore, comammox Nitrospira genomes encode genes that might allow efficient growth at low oxygen concentrations. Regarding the evolutionary history of comammox Nitrospira, our analyses indicate that several genes belonging to the ammonia oxidation pathway could have been laterally transferred from ß-AOB to comammox Nitrospira. We postulate that the absence of comammox genes in other sublineage II Nitrospira genomes is the result of subsequent loss.

Biofilm Thickness Influences Biodiversity in Nitrifying MBBRs-Implications on Micropollutant Removal.

In biofilm systems for wastewater treatment (e.g., moving bed biofilms reactors-MBBRs) biofilm thickness is typically not under direct control. Nevertheless, biofilm thickness is likely to have a profound effect on the microbial diversity and activity, as a result of diffusion limitation and thus substrate penetration in the biofilm. In this study, we investigated the impact of biofilm thickness on nitrification and on the removal of more than 20 organic micropollutants in laboratory-scale nitrifying MBBRs. We used novel carriers (Z-carriers, AnoxKaldnes) that allowed controlling biofilm thickness at 50, 200, 300, 400, and 500 µm. The impact of biofilm thickness on microbial community was assessed via 16S rRNA gene amplicon sequencing and ammonia monooxygenase (amoA) abundance quantification through quantitative PCR (qPCR). Results from batch experiments and microbial analysis showed that (i) the thickest biofilm (500 µm) presented the highest specific biotransformation rate constants (kbio, L g(-1) d(-1)) for 14 out of 22 micropollutants; (ii) biofilm thickness positively associated with biodiversity, which was suggested as the main factor for the observed enhancement of kbio; (iii) the thinnest biofilm (50 µm) exhibited the highest nitrification rate (gN d(-1) g(-1)), amoA gene abundance and kbio values for some of the most recalcitrant micropollutants (i.e., diclofenac and targeted sulfonamides). Although thin biofilms favored nitrification activity and the removal of some micropollutants, treatment systems based on thicker biofilms should be considered to enhance the elimination of a broad spectrum of micropollutants.

Community Structure in Methanogenic Enrichments Provides Insight into Syntrophic Interactions in Hydrocarbon-Impacted Environments.

The methanogenic biodegradation of crude oil involves the conversion of hydrocarbons to methanogenic substrates by syntrophic bacteria and subsequent methane production by methanogens. Assessing the metabolic roles played by various microbial species in syntrophic communities remains a challenge, but such information has important implications for bioremediation and microbial enhanced energy recovery technologies. Many factors such as changing environmental conditions or substrate variations can influence the composition and biodegradation capabilities of syntrophic microbial communities in hydrocarbon-impacted environments. In this study, a methanogenic crude oil-degrading enrichment culture was successively transferred onto the single long chain fatty acids palmitate or stearate followed by their parent alkanes, hexadecane or octadecane, respectively, in order to assess the impact of different substrates on microbial community composition and retention of hydrocarbon biodegradation genes. 16S rRNA gene sequencing showed that a reduction in substrate diversity resulted in a corresponding loss of microbial diversity, but that hydrocarbon biodegradation genes (such as assA/masD encoding alkylsuccinate synthase) could be retained within a community even in the absence of hydrocarbon substrates. Despite substrate-related diversity changes, all communities were dominated by hydrogenotrophic and acetotrophic methanogens along with bacteria including Clostridium sp., members of the Deltaproteobacteria, and a number of other phyla. Microbial co-occurrence network analysis revealed a dense network of interactions amongst syntrophic bacteria and methanogens that were maintained despite changes in the substrates for methanogenesis. Our results reveal the effect of substrate diversity loss on microbial community diversity, indicate that many syntrophic interactions are stable over time despite changes in substrate pressure, and show that syntrophic interactions amongst bacteria themselves are as important as interactions between bacteria and methanogens in complex methanogenic communities.

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples.

Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H2-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities.

Identification of toluene degraders in a methanogenic enrichment culture.

Methanogenic biodegradation involves the cooperative metabolism of syntrophic bacteria that catalyse the initial attack and subsequent degradation of hydrocarbons, and methanogens that convert intermediates such as hydrogen and carbon dioxide, formate, and/or acetate to methane. The identity of syntrophic microbes and the nature of their interactions with other syntrophs and methanogens are not well understood. Furthermore, it is difficult to isolate the organisms responsible for the initial activation and subsequent degradation of hydrocarbon substrates under methanogenic conditions due to the thermodynamic relationships that exist among microbes in methanogenic communities. We used time-resolved RNA stable isotope probing and RT-qPCR to identify the organisms involved in the initial attack on toluene and subsequent degradation reactions in a highly enriched toluene-degrading methanogenic culture. Our results reveal the importance of a Desulfosporosinus sp. in anaerobic toluene activation in the culture. Other organisms that appear to play roles in toluene degradation include Syntrophaceae, Desulfovibrionales and Chloroflexi. The high bacterial diversity observed in this culture and the extensive labelling of different phylogenetic groups over the course of the stable isotope probing experiment highlight the complexity of the relationships that exist in methanogenic ecosystems.

Syntrophic biodegradation of hydrocarbon contaminants.

Anaerobic environments are crucial to global carbon cycling wherein the microbial metabolism of organic matter occurs under a variety of redox conditions. In many anaerobic ecosystems, syntrophy plays a key role wherein microbial species must cooperate, essentially as a single catalytic unit, to metabolize substrates in a mutually beneficial manner. Hydrocarbon-contaminated environments such as groundwater aquifers are typically anaerobic, and often methanogenic. Syntrophic processes are needed to biodegrade hydrocarbons to methane, and recent studies suggest that syntrophic hydrocarbon metabolism can also occur in the presence of electron acceptors. The elucidation of key features of syntrophic processes in defined co-cultures has benefited greatly from advances in 'omics' based tools. Such tools, along with approaches like stable isotope probing, are now being used to monitor carbon flow within an increasing number of hydrocarbon-degrading consortia to pinpoint the key microbial players involved in the degradative pathways. The metagenomic sequencing of hydrocarbon-utilizing consortia should help to further identify key syntrophic features and define microbial interactions in these complex communities.

Methanogenic toluene metabolism: community structure and intermediates.

Toluene is a model compound used to study the anaerobic biotransformation of aromatic hydrocarbons. Reports indicate that toluene is transformed via fumarate addition to form benzylsuccinate or by unknown mechanisms to form hydroxylated intermediates under methanogenic conditions. We investigated the mechanism(s) of syntrophic toluene metabolism by a newly described methanogenic enrichment from a gas condensate-contaminated aquifer. Pyrosequencing of 16S rDNA revealed that the culture was comprised mainly of Clostridiales. The predominant methanogens affiliated with the Methanomicrobiales. Methane production from toluene ranged from 72% to 79% of that stoichiometrically predicted. Isotope studies using (13)C(7) toluene showed that benzylsuccinate and benzoate transiently accumulated revealing that members of this consortium can catalyse fumarate addition and subsequent reactions. Detection of a BssA gene fragment in this culture further supported fumarate addition as a mechanism of toluene activation. Transient formation of cresols, benzylalcohol, hydroquinone and methylhydroquinone suggested alternative mechanism(s) for toluene metabolism. However, incubations of the consortium with (18)O-H(2)O showed that the hydroxyl group in these metabolites did not originate from water and abiotic control experiments revealed abiotic formation of hydroxylated species due to reactions of toluene with sulfide and oxygen. Our results suggest that toluene is activated by fumarate addition, presumably by the dominant Clostridiales.

Metabolic production of a novel polymer feedstock, 3-carboxy muconate, from vanillin.

Vanillin can be produced on a commercial scale by depolymerising renewable lignin. One product of microbial metabolism of vanillin by common soil microbes, such as Acinetobacter baylyi, is a tricarboxylic acid with a butadiene backbone known as 3-carboxy muconate (3CM). Three enzymes, 4-hydroxy benzaldehyde dehydrogenase, vanillate monooxygenase and protocatechuate 3,4-dioxygenase, catalyse the biotransformation of vanillin to 3CM. These three enzymes were metabolically engineered into an Escherichia coli host, giving a biocatalyst that converted vanillin into 3CM. The biocatalyst was found to give 100% yield of 3CM from 1 mM of vanillin after 39 h. The rate-limiting reaction was identified as the conversion of vanillate to 3,4-dihydroxybenzoate catalysed by vanillate monooxygenase. Low expression of the reductase subunit of this enzyme was identified as contributing to the reduced rate of this reaction. Proof of principle of a novel application for 3CM was demonstrated when it was converted into a trimethyl ester derivative and copolymerised with styrene.