Metagenomics and metatranscriptomics suggest pathways of 3-chloroaniline degradation in wastewater reactors.

Biological wastewater treatment systems are often affected by shifts in influent quality, including the input of toxic chemicals. Yet the mechanisms underlying the adaptation of activated sludge process performance are rarely studied in a controlled and replicated experimental setting, particularly when challenged with a sustained toxin input. Three replicate bench-scale bioreactors were subjected to a chemical disturbance in the form of 3-chloroaniline (3-CA) over 132 days, after an acclimation period of 58 days, while three control reactors received no 3-CA input. Ammonia oxidation was initially affected by 3-CA. Within three weeks of the experiment, microbial communities in all three treatment reactors adapted to biologically degrade 3-CA resulting in partial ammonia oxidation recovery. Combining process and microbial community data from amplicon sequencing with potential functions gleaned from assembled metagenomics and metatranscriptomics data, two putative degradation pathways for 3-CA were identified. The first pathway, determined from metagenomics data, involves a benzoate dioxygenase and subsequent meta-cleavage of the aromatic ring. The second, determined from intensive short-term sampling for gene expression data in tandem with 3-CA degradation, involves a phenol monooxygenase followed by ortho-cleavage of the aromatic ring. The relative abundances of amplicon sequence variants associated with the genera Gemmatimonas, OLB8, and Taibaiella correlated significantly with 3-CA degradation. Metagenome-assembled genome data also showed the genus OLB8 to be differentially enriched in treatment reactors, making it a strong candidate as 3-CA degrader. Using replicated reactors, this study has demonstrated the impact of a sustained stress on the activated sludge process. The unique and novel features of this study include the identification of putative pathways and potential degraders of 3-CA using long-term and short-term sampling in tandem with multiple methods in a controlled and replicated experiment.

Machine learning-based detection of adventitious microbes in T-cell therapy cultures using long-read sequencing.

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP <71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated, and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimized experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 colony-forming units (CFU)/mL using metagenomic classification. Machine learning can be coupled with long-read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP <71> organisms to 10 CFU/mL. IMPORTANCE This research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long-read nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

Recovery and Analysis of Long-Read Metagenome-Assembled Genomes.

The development of long-read nucleic acid sequencing is beginning to make very substantive impact on the conduct of metagenome analysis, particularly in relation to the problem of recovering the genomes of member species of complex microbial communities. Here we outline bioinformatics workflows for the recovery and characterization of complete genomes from long-read metagenome data and some complementary procedures for comparison of cognate draft genomes and gene quality obtained from short-read sequencing and long-read sequencing.

Controlling anammox speciation and biofilm attachment strategy using N-biotransformation intermediates and organic carbon levels.

Conventional nitrogen removal in wastewater treatment requires a high oxygen and energy input. Anaerobic ammonium oxidation (anammox), the single-step conversion of ammonium and nitrite to nitrogen gas, is a more energy and cost effective alternative applied extensively to sidestream wastewater treatment. It would also be a mainstream treatment option if species diversity and physiology were better understood. Anammox bacteria were enriched up to 80%, 90% and 50% relative abundance, from a single inoculum, under standard enrichment conditions with either stepwise-nitrite and ammonia concentration increases (R1), nitric oxide supplementation (R2), or complex organic carbon from mainstream wastewater (R3), respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica occurred at ammonium and nitrite concentrations > 270 mg NH4-N L-1 and 340 mg NO2-N L-1 respectively. With NO present, heterotrophic growth was inhibited, and Ca. Jettenia coexisted with Ca. B. caroliniensis before diminishing as nitrite increased to 160 mg NO2-N L-1. Organic carbon supplementation led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially formed biofilms on surfaces, whereas Ca. Brocadia sinica formed granules in suspension. Our results indicate that multiple anammox bacteria species co-exist and occupy sub-niches in anammox reactors, and that the dominant population can be reversibly shifted by, for example, changing nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis). Speciation has implications for wastewater process design, where the optimum cell immobilization strategy (i.e. carriers vs granules) depends on which species dominates.

Shotgun metagenomic sequencing analysis of ocular surface microbiome in Singapore residents with mild dry eye.

The ocular surface microbiome has implications for ocular surface inflammation and immunology. Previous shotgun metagenomics analyses were performed in China, showing results that differed according to environment and age. Patients with Sjogren's syndrome were reported to have altered conjunctival microbiome, but such studies have not been done in milder dry eye. The aim of this study is to describe the conjunctival microbiome in people with mild dry eye in Singapore. Samples were collected from 14 participants with mild dry eye and 10 age-matched comparison participants recruited from Singapore National Eye Centre (SNEC) clinics. Shotgun metagenomic sequencing analysis was employed to evaluate the conjunctival microbiome composition. Proteobacteria formed the predominant phylum in the conjunctiva. As in a study from a coastal city in China, Achromobacter spp. was numerically most abundant. Compared to age-matched controls, the conjunctival microbial composition in mild dry eye was similar. Several microorganisms, including Streptococcus spp. increased in representation with age, and the abundance of Staphylococcus correlated with Schirmer readings. In addition, when cultured corneal epithelial cells were exposed to three strains of Achromobacter xylosoxidans, cytokines such as TNF-α and IL-6 were upregulated in the cell lysates and supernatants. Ourresults suggest that age is an important factor that affects composition of the conjunctival microbiome, and relative abundance of specific microorganism may vary according to the environment of the human host.

Adapt or perish.

Microbial communities in wastewater treatment plants provide insights into the development and mechanisms of antimicrobial resistance.

Recovery of High Quality Metagenome-Assembled Genomes From Full-Scale Activated Sludge Microbial Communities in a Tropical Climate Using Longitudinal Metagenome Sampling.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.

The phylogeny, ecology and ecophysiology of the glycogen accumulating organism (GAO) Defluviicoccus in wastewater treatment plants.

This comprehensive review looks critically what is known about members of the genus Defluviicoccus, an example of a glycogen accumulating organism (GAO), in wastewater treatment plants, but found also in other habitats. It considers the operating conditions thought to affect its performance in activated sludge plants designed to remove phosphorus microbiologically, including the still controversial view that it competes with the polyphosphate accumulating bacterium Ca. Accumulibacter for readily biodegradable substrates in the anaerobic zone receiving the influent raw sewage. It looks at its present phylogeny and what is known about it's physiology and biochemistry under the highly selective conditions of these plants, where the biomass is recycled continuously through alternative anaerobic (feed); aerobic (famine) conditions encountered there. The impact of whole genome sequence data, which have revealed considerable intra- and interclade genotypic diversity, on our understanding of its in situ behaviour is also addressed. Particular attention is paid to the problems in much of the literature data based on clone library and next generation DNA sequencing data, where Defluviicoccus identification is restricted to genus level only. Equally problematic, in many publications no attempt has been made to distinguish between Defluviicoccus and the other known GAO, especially Ca. Competibacter, which, as shown here, has a very different ecophysiology. The impact this has had and continues to have on our understanding of members of this genus is discussed, as is the present controversy over its taxonomy. It also suggests where research should be directed to answer some of the important research questions raised in this review.

Comparative Genomics of Members of the Genus Defluviicoccus With Insights Into Their Ecophysiological Importance.

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca. Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published (Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca. Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanusT , the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus.

Global warming readiness: Feasibility of enhanced biological phosphorus removal at 35 °C.

Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30 °C to be achievable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors for > 300 days at 30 °C and 35 °C, respectively, and followed the dynamics of the communities of polyphosphate accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescence in situ hybridization analyses. Stable and nearly complete phosphorus (P) removal was achieved at 30 °C; similarly, long term P removal was stable at 35 °C with effluent PO43-_P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Candidatus Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. A slow-feeding strategy effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. Comparative cycle studies revealed higher carbon and P cycling activity of Ca. Accumulibacter when the temperature was increased from 30 °C to 35 °C, implying that the lowered P removal performance at 35 °C was not a direct effect of temperature, but a result of higher metabolic rates of carbon (and/or P) utilization of PAOs and GAOs, the resultant carbon deficiency, and escalated community competition. An increase in the TOC-to-PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited PAOs. In general, a slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at 35 °C, representing basic conditions suitable for full-scale treatment plants experiencing higher water temperatures.

Prospects for multi-omics in the microbial ecology of water engineering.

Advances in high-throughput sequencing technologies and bioinformatics approaches over almost the last three decades have substantially increased our ability to explore microorganisms and their functions - including those that have yet to be cultivated in pure isolation. Genome-resolved metagenomic approaches have enabled linking powerful functional predictions to specific taxonomical groups with increasing fidelity. Additionally, related developments in both whole community gene expression surveys and metabolite profiling have permitted for direct surveys of community-scale functions in specific environmental settings. These advances have allowed for a shift in microbiome science away from descriptive studies and towards mechanistic and predictive frameworks for designing and harnessing microbial communities for desired beneficial outcomes. Water engineers, microbiologists, and microbial ecologists studying activated sludge, anaerobic digestion, and drinking water distribution systems have applied various (meta)omics techniques for connecting microbial community dynamics and physiologies to overall process parameters and system performance. However, the rapid pace at which new omics-based approaches are developed can appear daunting to those looking to apply these state-of-the-art practices for the first time. Here, we review how modern genome-resolved metagenomic approaches have been applied to a variety of water engineering applications from lab-scale bioreactors to full-scale systems. We describe integrated omics analysis across engineered water systems and the foundations for pairing these insights with modeling approaches. Lastly, we summarize emerging omics-based technologies that we believe will be powerful tools for water engineering applications. Overall, we provide a framework for microbial ecologists specializing in water engineering to apply cutting-edge omics approaches to their research questions to achieve novel functional insights. Successful adoption of predictive frameworks in engineered water systems could enable more economically and environmentally sustainable bioprocesses as demand for water and energy resources increases.

Dynamics of the fecal microbiome and antimicrobial resistome in commercial piglets during the weaning period.

This study aimed to characterize the alteration of the fecal microbiome and antimicrobial resistance (AMR) determinants in 24 piglets at day 3 pre-weaning (D. - 3), weaning day (D.0), days 3 (D.3) and 8 post-weaning (D.8), using whole-genome shotgun sequencing. Distinct clusters of microbiomes and AMR determinants were observed at D.8 when Prevotella (20.9%) was the major genus, whereas at D. - 3-D.3, Alistipes (6.9-12.7%) and Bacteroides (5.2-8.5%) were the major genera. Lactobacillus and Escherichia were notably observed at D. - 3 (1.2%) and D. - 3-D.3 (0.2-0.4%), respectively. For AMR, a distinct cluster of AMR determinants was observed at D.8, mainly conferring resistance to macrolide-lincosamide-streptogramin (mefA), ß-lactam (cfxA6 and aci1) and phenicol (rlmN). In contrast, at D. - 3-D.3, a high abundance of determinants with aminoglycoside (AMG) (sat, aac(6')-aph(2''), aadA and acrF), ß-lactam (fus-1, cepA and mrdA), multidrug resistance (MDR) (gadW, mdtE, emrA, evgS, tolC and mdtB), phenicol (catB4 and cmlA4), and sulfonamide patterns (sul3) was observed. Canonical correlation analysis (CCA) plot associated Escherichia coli with aac(6')-aph(2''), emrA, mdtB, catB4 and cmlA4 at D. - 3, D.0 and/or D.3 whereas at D.8 associations between Prevotella and mefA, cfxA6 and aci1 were identified. The weaning age and diet factor played an important role in the microbial community composition.

Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation.

BACKGROUND: Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. RESULTS: Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. CONCLUSIONS: The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.

Influence of Extraction Solvent on Nontargeted Metabolomics Analysis of Enrichment Reactor Cultures Performing Enhanced Biological Phosphorus Removal (EBPR).

Metabolome profiling is becoming more commonly used in the study of complex microbial communities and microbiomes; however, to date, little information is available concerning appropriate extraction procedures. We studied the influence of different extraction solvent mixtures on untargeted metabolomics analysis of two continuous culture enrichment communities performing enhanced biological phosphate removal (EBPR), with each enrichment targeting distinct populations of polyphosphate-accumulating organisms (PAOs). We employed one non-polar solvent and up to four polar solvents for extracting metabolites from biomass. In one of the reactor microbial communities, we surveyed both intracellular and extracellular metabolites using the same set of solvents. All samples were analysed using ultra-performance liquid chromatography mass spectrometry (UPLC-MS). UPLC-MS data obtained from polar and non-polar solvents were analysed separately and evaluated using extent of repeatability, overall extraction capacity and the extent of differential abundance between physiological states. Despite both reactors demonstrating the same bioprocess phenotype, the most appropriate extraction method was biomass specific, with methanol: water (50:50 v/v) and methanol: chloroform: water (40:40:20 v/v) being chosen as the most appropriate for each of the two different bioreactors, respectively. Our approach provides new data on the influence of solvent choice on the untargeted surveys of the metabolome of PAO enriched EBPR communities and suggests that metabolome extraction methods need to be carefully tailored to the specific complex microbial community under study.

Recovery of complete genomes and non-chromosomal replicons from activated sludge enrichment microbial communities with long read metagenome sequencing.

New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.

Microbial abundance and community composition in biofilms on in-pipe sensors in a drinking water distribution system.

Collecting biofilm samples from drinking water distribution systems (DWDSs) is challenging due to limited access to the pipes during regular operations. We report here the analysis of microbial communities in biofilm and water samples collected from sensors installed in a DWDS where monochloramine is used as a residual disinfectant. A total of 52 biofilm samples and 14 bulk water samples were collected from 17 pipe sections representing different water ages. Prokaryotic genome copies (bacterial and archaeal 16S rRNA genes, Mycobacterium spp., ammonia-oxidizing bacteria (AOB), and cyanobacteria) were quantified with droplet digital PCR, which revealed the abundance of these genes in both biofilm and water samples. Prokaryotic 16S rRNA gene sequencing analysis was carried out for a subset of the samples (12 samples from four sites). Mycobacterium and AOB species were dominant in the DWDS sections with low water age and sufficient residual monochloramine, whereas Nitrospira species (nitrite-oxidizing bacteria) dominated in the sections with higher water age and depleted monochloramine level, suggesting the occurrence of nitrification in the studied DWDS. The present study provides novel information on the abundance and identity of prokaryotes in biofilms and water in a full-scale operational DWDS.

Integration of time-series meta-omics data reveals how microbial ecosystems respond to disturbance.

The development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive phenotypic plasticity and niche complementarity in oleaginous microbial populations from a biological wastewater treatment plant. We perform meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover four distinct fundamental niche types. Throughout the time-series, we observe a major, transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst community members. Ex situ bioreactor experiments confirm that responses occur within five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our results show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity, and set the foundation for future ecological engineering efforts.

Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation.

This study describes the first direct functional assignment of a highly abundant extracellular protein from a key environmental and biotechnological biofilm performing an anaerobic ammonium oxidation (anammox) process. Expression levels of Brosi_A1236, belonging to a class of proteins previously suggested to be cell surface associated, were in the top one percentile of all genes in the "Candidatus Brocadia sinica"-enriched biofilm. The Brosi_A1236 structure was computationally predicted to consist of immunoglobulin-like anti-parallel ß-strands, and circular dichroism conducted on the isolated surface protein indicated that ß-strands are the dominant higher-order structure. The isolated protein was stained positively by the ß-sheet-specific stain thioflavin T, along with cell surface- and matrix-associated regions of the biofilm. The surface protein has a large unstructured content, including two highly disordered domains at its C terminus. The disordered domains bound to the substratum and thereby facilitated the adhesion of negatively charged latex microspheres, which were used as a proxy for cells. The disordered domains and isolated whole surface protein also underwent liquid-liquid phase separation to form liquid droplets in suspension. Liquid droplets of disordered protein wet the surfaces of microspheres and bacterial cells and facilitated their coalescence. Furthermore, the surface layer protein formed gels as well as ordered crystalline structures. These observations suggest that biophysical remodeling through phase transitions promotes aggregation and biofilm formation.IMPORTANCE By employing biophysical and liquid-liquid phase separation concepts, this study revealed how a highly abundant extracellular protein enhances the key environmental and industrial bioprocess of anaerobic ammonium oxidation (anammox). Extracellular proteins of environmental biofilms are understudied and poorly annotated in public databases. Understanding the function of extracellular proteins is also increasingly important for improving bioprocesses and resource recovery. Here, protein functions were assessed based on theoretical predictions of intrinsically disordered domains, known to promote adhesion and liquid-liquid phase separation, and available surface layer protein properties. A model is thus proposed to explain how the protein promotes aggregation and biofilm formation by extracellular matrix remodeling and phase transitions. This work provides a strong foundation for functional investigations of extracellular proteins involved in biofilm development.

The Isolate Caproiciproducens sp. 7D4C2 Produces n-Caproate at Mildly Acidic Conditions From Hexoses: Genome and rBOX Comparison With Related Strains and Chain-Elongating Bacteria.

Bulk production of medium-chain carboxylates (MCCs) with 6-12 carbon atoms is of great interest to biotechnology. Open cultures (e.g., reactor microbiomes) have been utilized to generate MCCs in bioreactors. When in-line MCC extraction and prevention of product inhibition is required, the bioreactors have been operated at mildly acidic pH (5.0-5.5). However, model chain-elongating bacteria grow optimally at neutral pH values. Here, we isolated a chain-elongating bacterium (strain 7D4C2) that grows at mildly acidic pH. We studied its metabolism and compared its whole genome and the reverse ß-oxidation (rBOX) genes to other bacteria. Strain 7D4C2 produces lactate, acetate, n-butyrate, n-caproate, biomass, and H2/CO2 from hexoses. With only fructose as substrate (pH 5.5), the maximum n-caproate specificity (i.e., products per other carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose and n-butyrate (electron acceptor) were combined as a substrate. A comparison of 7D4C2 cultures with fructose and n-butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasing n-caproate specificity from ∼92% at mildly acidic pH (pH 4.5-5.0) to ∼24% at alkaline pH (pH 9.0). Moreover, when carboxylates were extracted from the broth (undissociated n-caproic acid was ∼0.3 mM), the n-caproate selectivity (i.e., product per substrate fed) was 42.6 ± 19.0% higher compared to 7D4C2 cultures without extraction. Based on the 16S rRNA gene sequence, strain 7D4C2 is most closely related to the isolates Caproicibacter fermentans (99.5%) and Caproiciproducens galactitolivorans (94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2, C. fermentans, and C. galactitolivorans belong to the same genus of Caproiciproducens. Their rBOX genes are conserved and located next to each other, forming a gene cluster, which is different than for other chain-elongating bacteria such as Megasphaera spp. In conclusion, Caproiciproducens spp., comprising strain 7D4C2, C. fermentans, C. galactitolivorans, and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster. Caproiciproducens sp. 7D4C2 (DSM 110548) was studied here to understand n-caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars into n-caproate.