Cultivating the uncultured: Harnessing the "sandwich agar plate" approach to isolate heme-dependent bacteria from marine sediment.

In the classical microbial isolation technique, the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival. In this study, we designed a simple coculture technique named the "sandwich agar plate method," which maintains microbial interactions throughout the isolation and pure culture processes. The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group. Many uncultured species displayed commensal lifestyles. Further study found that heme was the growth-promoting factor of some marine commensal bacteria. Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa. Moreover, our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably. These findings highlight that cocultivation based on the "sandwich agar plate method" could be developed and used to isolate more uncultured bacteria.

Cerina litoralis gen. nov., sp. nov., a novel potential polysaccharide degrading bacterium of the family Flavobacteriaceae, isolated from marine sediment.

The Gram-strain-negative, facultative anaerobic, chemoheterotrophic, short-rod-shaped, non-motile, forming yellow colonies strain, designated F89T, was isolated from marine sediment of Xiaoshi Island, Weihai. Strain F89T grew at 15-37 °C (optimally at 28 °C), at pH 6.0-8.5 (optimally at pH 7.0) and in the presence of 1-5% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain F89T was related to the family Flavobacteriaceae. F89T had highest 16S rRNA gene sequence similarity to Maribacter cobaltidurans MCCC 1K03318T (93.3%). The predominant cellular fatty acids of F89T were iso-C15:0, iso-C15:0 G and Summed Feature 3. The main respiratory quinone of F89T was menaquinone 6 (MK-6), consistent with that observed for all related strains. The polar lipid profile of strain F89T contained phosphatidylethanolamine, two aminolipids and three unidentified polar lipids. The genomic DNA G + C content of strain F89T was 42.7%. Strain F89T encoded 121 glycoside hydrolases and was a potential polysaccharide degrading bacterium. Differential phenotypic and genotypic characteristics of the strain showed that F89T should be classified as a novel genus in Flavobacteriaceae, for which the name Cerina litoralis is proposed. The type strain is F89T (= MCCC 1H00510T = KCTC 92203T).

Global diversity and biogeography of DNA viral communities in activated sludge systems.

BACKGROUND: Activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses that regulate microbial metabolism and nutrient cycling, significantly influencing the stability of AS systems. However, our knowledge about the diversity of viral taxonomic groups and functional traits in global AS systems is still limited. To address this gap, we investigated the global diversity and biogeography of DNA viral communities in AS systems using 85,114 viral operational taxonomic units (vOTUs) recovered from 144 AS samples collected across 54 WWTPs from 13 different countries. RESULTS: AS viral communities and their functional traits exhibited distance-decay relationship (DDR) at the global scale and latitudinal diversity gradient (LDG) from equator to mid-latitude. Furthermore, it was observed that AS viral community and functional gene structures were largely driven by the geographic factors and wastewater types, of which the geographic factors were more important. Carrying and disseminating auxiliary metabolic genes (AMGs) associated with the degradation of polysaccharides, sulfate reduction, denitrification, and organic phosphoester hydrolysis, as well as the lysis of crucial functional microbes that govern biogeochemical cycles were two major ways by which viruses could regulate AS functions. It was worth noting that our study revealed a high abundance of antibiotic resistance genes (ARGs) in viral genomes, suggesting that viruses were key reservoirs of ARGs in AS systems. CONCLUSIONS: Our results demonstrated the highly diverse taxonomic groups and functional traits of viruses in AS systems. Viral lysis of host microbes and virus-mediated HGT can regulate the biogeochemical and nutrient cycles, thus affecting the performance of AS systems. These findings provide important insights into the viral diversity, function, and ecology in AS systems on a global scale. Video Abstract.

Polyethylene Degradation by a Rhodococcous Strain Isolated from Naturally Weathered Plastic Waste Enrichment.

Polyethylene (PE) is the most widely produced synthetic polymer and the most abundant plastic waste worldwide due to its recalcitrance to biodegradation and low recycle rate. Microbial degradation of PE has been reported, but the underlying mechanisms are poorly understood. Here, we isolated a Rhodococcus strain A34 from 609 day enriched cultures derived from naturally weathered plastic waste and identified the potential key PE degradation enzymes. After 30 days incubation with A34, 1% weight loss was achieved. Decreased PE molecular weight, appearance of C-O and C═O on PE, palmitic acid in the culture supernatant, and pits on the PE surface were observed. Proteomics analysis identified multiple key PE oxidation and depolymerization enzymes including one multicopper oxidase, one lipase, six esterase, and a few lipid transporters. Network analysis of proteomics data demonstrated the close relationships between PE degradation and metabolisms of phenylacetate, amino acids, secondary metabolites, and tricarboxylic acid cycles. The metabolic roadmap generated here provides critical insights for optimization of plastic degradation condition and assembly of artificial microbial communities for efficient plastic degradation.

Short-chain fatty acids (SCFAs) as potential resuscitation factors that promote the isolation and culture of uncultured bacteria in marine sediments.

Many marine bacteria are difficult to culture because they are dormant, rare or found in low-abundances. Enrichment culturing has been widely tested as an important strategy to isolate rare or dormant microbes. However, many more mechanisms remain uncertain. Here, based on 16S rRNA gene high-throughput sequencing and metabolomics technology, it was found that the short-chain fatty acids (SCFAs) in metabolites were significantly correlated with uncultured bacterial groups during enrichment cultures. A pure culture analysis showed that the addition of SCFAs to media also resulted in high efficiency for the isolation of uncultured strains from marine sediments. As a result, 238 strains belonging to 10 phyla, 26 families and 82 species were successfully isolated. Some uncultured rare taxa within Chlorobi and Kiritimatiellaeota were successfully cultured. Amongst the newly isolated uncultured microbes, most genomes, e.g. bacteria, possess SCFA oxidative degradation genes, and these features might aid these microbes in better adapting to the culture media. A further resuscitation analysis of a viable but non-culturable (VBNC) Marinilabiliales strain verified that the addition of SCFAs could break the dormancy of Marinilabiliales in 5 days, and the growth curve test showed that the SCFAs could shorten the lag phase and increase the growth rate. Overall, this study provides new insights into SCFAs, which were first studied as resuscitation factors in uncultured marine bacteria. Thus, this study can help improve the utilisation and excavation of marine microbial resources, especially for the most-wanted or key players. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00187-w.

Leader peptide removal in lasso peptide biosynthesis based on penultimate isoleucine residue.

Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster (bsf) from Bradymonas sediminis FA350, a Gram-negative and facultatively prey-dependent bacterium that belongs to a novel bacterial order Bradymonadales in the class Deltaproteobacteria. The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.

Gaoshiqia sediminis gen. nov., sp. nov., isolated from coastal sediment.

A Gram-stain-negative, facultative anaerobic, motile, rod-shaped and orange bacterium, designated A06T, was obtained off the coast of Weihai, PR China. Cells were 0.4-0.5×0.6-1.0 µm in size. Strain A06T grew at 20-40 °C (optimum, 33 °C), at pH 6.0-8.0 (optimum, pH 6.5-7.0) and in the presence of 0-8 % NaCl (w/v) (optimum, 2 %). Cells were oxidase and catalase positive. Menaquinone-7 was detected as the major respiratory quinone. The dominant cellular fatty acids were identified as C15:0 2-OH, iso-C15:0, anteiso-C15:0 and iso-C15:1 ω6c. The DNA G+C content of strain A06T was 46.1 mol%. The polar lipids were phosphatidylethanolamine, one aminolipid, one glycolipid and three unidentified lipids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain A06T is a member of the family Prolixibacteraceae and exhibited the highest sequence similarity to Mangrovibacterium diazotrophicum DSM 27148T (94.3 %). Based on its phylogenetic and phenotypic characteristics, strain A06T is considered to represent a novel genus in the family Prolixibacteraceae, for which the name Gaoshiqia gen. nov. is proposed. The type species is Gaoshiqia sediminis sp. nov., with type strain A06T (=KCTC 92029T=MCCC 1H00491T). The identification and acquisition of microbial species and genes in sediments will help broaden the understanding of microbial resources and lay a foundation for its application in biotechnology. Strain A06T uses an enrichment method, so the isolation of strain A06T is of great significance to the enrichment of marine microbial resources.

Niche Modification by Sulfate-Reducing Bacteria Drives Microbial Community Assembly in Anoxic Marine Sediments.

Sulfate-reducing bacteria (SRB) are essential functional microbial taxa for degrading organic matter (OM) in anoxic marine environments. However, there are little experimental data regarding how SRB regulates microbial communities. Here, we applied a top-down microbial community management approach by inhibiting SRB to elucidate their contributions to the microbial community during OM degradation. Based on the highly replicated microcosms (n = 20) of five different incubation stages, we found that many microbial community properties were influenced after inhibiting SRB, including the composition, structure, network, and community assembly processes. We also found a strong coexistence pattern between SRB and other abundant phylogenetic lineages via positive frequency-dependent selection. The relative abundances of the families Synergistaceae, Peptostreptococcaceae, Dethiosulfatibacteraceae, Prolixibacteraceae, Marinilabiliaceae, and Marinifilaceae were simultaneously suppressed after inhibiting SRB during OM degradation. A close association between SRB and the order Marinilabiliales among coexisting taxa was most prominent. They contributed to preserved modules during network successions, were keystone nodes mediating the networked community, and contributed to homogeneous ecological selection. The molybdate tolerance test of the isolated strains of Marinilabiliales showed that inhibited SRB (not the inhibitor of SRB itself) triggered a decrease in the relative abundance of Marinilabiliales. We also found that inhibiting SRB resulted in reduced pH, which is unsuitable for the growth of most Marinilabiliales strains, while the addition of pH buffer (HEPES) in SRB-inhibited treatment microcosms restored the pH and the relative abundances of these bacteria. These data supported that SRB could modify niches to affect species coexistence. IMPORTANCE Our model offers insight into the ecological properties of SRB and identifies a previously undocumented dimension of OM degradation. This targeted inhibition approach could provide a novel framework for illustrating how functional microbial taxa associate the composition and structure of the microbial community, molecular ecological network, and community assembly processes. These findings emphasize the importance of SRB during OM degradation. Our results proved the feasibility of the proposed study framework, inhibiting functional taxa at the community level, for illustrating when and to what extent functional taxa can contribute to ecosystem services.

Aequorivita vitellina sp. nov. and Aequorivita xiaoshiensis sp. nov., isolated from marine sediment.

Two Gram-stain-negative, strictly aerobic, chemoheterotrophic, short-rod-shaped and non-motile strains, forming yellow colonies and designated F47161T and F64183T, were isolated from marine sediment of Xiaoshi Island, Wei Hai, PR China. Strain F47161T grew at 15-37 °C (optimally at 30 °C) and pH 6.0-9.0 (optimally at pH 7.5) and in the presence of 1-9 % (w/v) NaCl (optimally at 3 %). Strain F64183T grew at 10-37 °C (optimally at 30 °C) and pH 6.0-8.5 (optimally at pH 7.0) and in the presence of 1-8 % (w/v) NaCl (optimally at 3 %). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that F47161T and F64183T were related to members of the genus Aequorivita. The strains shared 97.4 % 16S rRNA gene sequence similarity to each other. F47161T and F64183T shared highest 16S rRNA gene sequence similarity to Aequorivita sinensis JCM 19789T, and the values were 97.5 % and 98.4 %, respectively. The predominant cellular fatty acids of both F64183T and F47161T were iso-C15 : 0 and iso-C17 : 0 3-OH, but the predominant fatty acids of F47161T also included anteiso-C15 : 0. The sole respiratory quinone of F47161T and F64183T was menaquinone 6 (MK-6), consistent with that observed for all related strains. Between F47161T and F64183T, the average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values were 75.8 % and 20.5 %, respectively, and between the novel isolates (F47161T and F64183T) and A. sinensis JCM 19789T they were 76.0 % and 94.2 % and 20.6 % and 57.1 %, respectively. The genomic DNA G+C contents of F47161T and F64183T was 37.3 % and 34.5 %, respectively. The polar lipid profiles of F47161T and F64183T contained phosphatidylethanolamine, two aminolipids, one glycolipid, one phosphoglycolipid and two unidentified polar lipids. Differential phenotypic and genotypic characteristics of the two strains indicated that the two strains should be classified as representing two novel species of the genus Aequorivita, for which the names Aequorivita vitellina sp. nov. and Aequorivita xiaoshiensis sp. nov. are proposed. The type strains are F47161T (=MCCC 1H00509T=KCTC 92017T) and F64183T (=MCCC 1H00507T=KCTC 92016T), respectively.

Predation capacity of Bradymonabacteria, a recently discovered group in the order Bradymonadales, isolated from marine sediments.

Bacterial predation is a vital feeding behavior that affects community structure and maintains biodiversity. However, predatory bacterial species in coastal sediments are comparatively poorly described. In this study, the predation capacity of all nine culturable Bradymonabacteria strains belonging to the recently discovered order Bradymonadales was determined against different types of prey. The predatory efficiency of Bradymonabacteria increased as the initial prey proportion in a mixed culture decreased. When the initial prey proportion was 0.5, the number of surviving prey bacterial cells significantly decreased after 4 h of predation with the Bradymonabacteria strains TMQ1, SEH01, B210 and FA350. However, growth of the prey strain occurred in the presence of the Bradymonabacteria strains TMQ4, TMQ2, TMQ3, V1718 and YN101. When the initial prey proportion decreased to 0.1 or 0.01, most of the Bradymonabacteria strains preyed efficiently. Furthermore, established neighboring colonies of prey were destroyed by Bradymonabacteria. This invading predation capacity was determined by the predation ability of the strain and its motility on the agar surface. Our findings provide new insights into the potential ecological significance of predatory Bradymonabacteria, which may serve as a potential probiotic for use in the aquaculture.

The Widespread Use of Nanomaterials: The Effects on the Function and Diversity of Environmental Microbial Communities.

In recent years, as an emerging material, nanomaterials have rapidly expanded from laboratories to large-scale industrial productions. Along with people's productive activities, these nanomaterials can enter the natural environment of soil, water and atmosphere through various ways. At present, a large number of reports have proved that nanomaterials have certain toxic effects on bacteria, algae, plants, invertebrates, mammalian cell lines and mammals in these environments, but people still know little about the ecotoxicology of nanomaterials. Most relevant studies focus on the responses of model strains to nanomaterials in pure culture conditions, but these results do not fully represent the response of microbial communities to nanomaterials in natural environments. Over the years, the effect of nanomaterials infiltrated into the natural environment on the microbial communities has become a popular topic in the field of nano-ecological environment research. It was found that under different environmental conditions, nanomaterials have various effects on the microbial communities. The medium; the coexisting pollutants in the environment and the structure, particle size and surface modification of nanomaterials may cause changes in the structure and function of microbial communities. This paper systematically summarizes the impacts of different nanomaterials on microbial communities in various environments, which can provide a reference for us to evaluate the impacts of nanomaterials released into the environment on the microecology and has certain guiding significance for strengthening the emission control of nanomaterials pollutants.

Robiginitalea marina sp. nov., isolated from coastal sediment.

A Gram-stain-negative, orange, aerobic, non-motile, rod-shaped marine bacterium, designated as 2V75T, was isolated from the coastal sediment of Xiaoshi Island, Weihai, China. The strain 2V75T grew at 20-45 °C (optimum, 37 °C), from pH 7.0 to 9.0 (optimum, pH 7.0) and in the presence of 0.5-5% (w/v) NaCl (optimum, 3%). Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain 2V75T was affiliated to the genus Robiginitalea and had the highest sequence similarity with R. biformata KCTC 12146T (93.7%). The ANI values between strain 2V75T and R. biformata KCTC 12146T were 72.6%, respectively. The DNA G + C content was 54.8 mol%. MK-6 was the only respiratory quinone. Based on the phenotypic, phylogenetic and chemotaxonomic data, strain 2V75T should be classified as a novel species in the genus Robiginitalea, for which the name Robiginitalea marina is proposed. The type strain is 2V75T (= KCTC 92035T = MCCC 1H00484T).

Psychroserpens luteolus sp. nov., isolated from Gelidium, reclassification of Ichthyenterobacterium magnum as Psychroserpens magnus comb. nov., Flavihalobacter algicola as Psychroserpens algicola comb. nov., Arcticiflavibacter luteus as Psychroserpens luteus comb. nov.

A Gram-stain-negative, motile by gliding, catalase positive, facultative anaerobic strain, designated strain XSD401T, was isolated from an unidentified Gelidium species of Xiaoshi Island, Shandong Province, China. The 16S rRNA gene sequence comparisons indicated strain XSD401T had a sequence similarity of 96.9% with Psychroserpens damuponensis KCTC 23539T and 96.3% with Psychroserpens burtonensis DSM 12212T. The G + C content of the genomic DNA was 33.9%. The ANI values between strain XSD401T and P. damuponensis KCTC 23539T, P. burtonensis DSM 12212T, were 76.9% and 76.9%, respectively. The dDDH values between strain XSD401T and P. damuponensis KCTC 23539T, P. burtonensis DSM 12212T, were 20.4% and 20.3%, respectively. The AAI values and POCP values of these 8 species were all over 72% and 50%. Combined with the results of comparative genomic analysis, Ichthyenterobacterium magnum, Flavihalobacter algicola and Arcticiflavibacter luteus were reclassified into Psychroserpens. Furthermore, the differences in morphology, physiology and genotype from the previously described taxa support the classification of strain XSD401T as a representative of the genus Psychroserpens, for which the name Psychroserpens luteolus sp. nov. is proposed. The type strain is XSD401T (= MCCC 1H00396T = KCTC 72684T = JCM 33931T).

Salinity Gradient Controls Microbial Community Structure and Assembly in Coastal Solar Salterns.

Salinity acts as a critical environmental filter on microbial communities in natural systems, negatively affecting microbial diversity. However, how salinity affects microbial community assembly remains unclear. This study used Wendeng multi-pond saltern as a model to evaluate the prokaryotic community composition and diversity and quantify the relative importance of ecological processes across salinity gradients. The results showed that low-saline salterns (45-80 g/L) exhibited higher bacterial diversity than high-saline salterns (175-265 g/L). The relative abundance of taxa assigned to Halomicrobiaceae, Rhodobacteraceae, Saprospiraceae, and Thiotrichaceae exhibited a hump-shaped dependence on increasing salinity. Salinity and pH were the primary environmental factors that directly or indirectly determined the composition and diversity of prokaryotic communities. Microbial co-occurrence network dynamics were more complex in the sediment than in the water of salterns. An infer Community Assembly Mechanisms by Phylogenetic-bin-based null model analysis (iCAMP) showed that microbial community assembly in sediment and water differed. Our findings provide more information about microbial community structure and the importance of various ecological processes in controlling microbial community diversity and succession along salinity gradients in water and sediment.

Description of Lujinxingia vulgaris sp. nov., isolated from coastal sediment via prey-traps.

Two Gram-stain negative, facultative anaerobic, oxidase-negative, catalase-positive bacilli, designated as strains TMQ4T and TMQ2, were isolated from Xiaoshi Island, China, using prey-traps. Growth was observed within the ranges 25-45 °C (optimally at 37 °C), pH 6.5-9.0 (optimally at pH 7.5-8.0) and 1-8% NaCl (optimally at 3-4%, w/v). The draft genome sequences of strains TMQ4T and TMQ2 contained 184 contigs of 5,609,735 bp with a G+C content of 64.4% and 148 contigs of 5,589,985 bp with a G+C content of 65.0%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that both strains belonged to the genus Lujinxingia with the similarity of 98.9%. The phylogenetic and phylogenomic topologies and analyses demonstrated that both strains clustered together and differentiated from the closest neighbour, Lujinxingia sediminis SEH01T. Genomic analyses showed that two strains lost the biosynthesis pathway of several chemical compounds. Iso-C15:0 was contained in the predominant cellular fatty acids in both strains. The major polar lipids of both strains consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified lipids; and the respiratory quinone was menaquinone MK-7 for both strains. Both strains predated other bacteria, including Owenweeksia hongkongensis JCM 12287T and Paraliobacillus ryukyuensis DSM 15140T, and were lured with one prey Acinetobacter baumannii ATCC 19606T in prey-trap. Combining genomic analyses, two strains had the predatory indices of 2, similar to representative typical bacterial predators. The physiological, biochemical, and phylogenetic properties suggest that the two strains represent a novel species within the genus Lujinxingia. The name Lujinxingia vulgaris sp. nov. is proposed, with strain TMQ4T (= KCTC 62851T = MCCC 1H00392T) as type strain and strain TMQ2 (= KCTC 72,079 = MCCC 1H00381) as reference strain.

Strategies for culturing active/dormant marine microbes.

Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service. However, many of their functions and phenotypes remain unknown because indigenous marine bacteria are mostly difficult to culture. Although many novel techniques have brought previously uncultured microbes into laboratory culture, there are still many most-wanted or key players that need to be cultured from marine environments. This review discusses possible reasons for 'unculturable microbes' and categorizes uncultured bacteria into three groups: dominant active bacteria, rare active bacteria, and dormant bacteria. This review also summarizes advances in cultivation techniques for culturing each group of unculturable bacteria. Simulating the natural environment is an effective strategy for isolating dominant active bacteria, whereas culturomics and enrichment culture methods are proposed for isolating rare active bacteria. For dormant bacteria, resuscitation culture is an appropriate strategy. Furthermore, the review provides a list of the most-wanted bacteria and proposes potential strategies for culturing these bacteria in marine environments. The review provides new insight into the development of strategies for the cultivation of specific groups of uncultured bacteria and therefore paves the way for the detection of novel microbes and their functions in marine ecosystems.

Inducible expression of agar-degrading genes in a marine bacterium Catenovulum maritimus Q1T and characterization of a ß-agarase.

Agar-degrading bacteria are crucial drivers for the carbon cycle in the marine environments due to their ability that use algae as a carbon source. Although numerous agar-degrading bacteria and agarases have been reported, little is known about expression levels of agar-degrading genes in wild strains. Here, the genome of an agar-hydrolyzing marine bacterium, Catenovulum maritimus Q1T, was sequenced and annotated with 11 agarase and 2 neoagarooligosaccharide hydrolase genes. Quantitative PCR revealed that all the annotated agar-degrading genes were expressed consistently that initially upregulated and then gradually downregulated under agarose induction. Moreover, the presence of glucose inhibited the agar-degrading ability, in terms of both gene expression and enzymatic activity. These facts indicated the agar-degrading ability of wild bacteria was mainly induced by agarose and repressed by the available carbon source. Additionally, a ß-agarase, AgaQ1, belonging to the GH16 family, with high expression in strain Q1T, was cloned and characterized. Biochemical analysis showed that the recombinant AgaQ1 was substrate-specific, yielding neoagarotetraose and neoagarohexaose as the main products. It exhibited optimal activity at 40 °C, pH 8.0, and an agarose concentration of 1.6% (w/v). Besides, AgaQ1 showed a high-specific activity (757.7 U/mg) and stable enzymatic activity under different ion or agent treatments; thus, AgaQ1 has great potential in industrial applications. KEY POINTS: • The genome of C. maritimus Q1T was sequenced and annotated with 11 agarases and 2 Nabh genes. • The expression of agar-degrading genes in the strain C. maritimus Q1T was induced by agarose. • Glucose was the carbon source utilized prior to agarose for bacterial growth. • A ß-agarase, AgaQ1, with high expression and activity was identified.

Bradymonabacteria, a novel bacterial predator group with versatile survival strategies in saline environments.

BACKGROUND: Bacterial predation is an important selective force in microbial community structure and dynamics. However, only a limited number of predatory bacteria have been reported, and their predatory strategies and evolutionary adaptations remain elusive. We recently isolated a novel group of bacterial predators, Bradymonabacteria, representative of the novel order Bradymonadales in δ-Proteobacteria. Compared with those of other bacterial predators (e.g., Myxococcales and Bdellovibrionales), the predatory and living strategies of Bradymonadales are still largely unknown. RESULTS: Based on individual coculture of Bradymonabacteria with 281 prey bacteria, Bradymonabacteria preyed on diverse bacteria but had a high preference for Bacteroidetes. Genomic analysis of 13 recently sequenced Bradymonabacteria indicated that these bacteria had conspicuous metabolic deficiencies, but they could synthesize many polymers, such as polyphosphate and polyhydroxyalkanoates. Dual transcriptome analysis of cocultures of Bradymonabacteria and prey suggested a potential contact-dependent predation mechanism. Comparative genomic analysis with 24 other bacterial predators indicated that Bradymonabacteria had different predatory and living strategies. Furthermore, we identified Bradymonadales from 1552 publicly available 16S rRNA amplicon sequencing samples, indicating that Bradymonadales was widely distributed and highly abundant in saline environments. Phylogenetic analysis showed that there may be six subgroups in this order; each subgroup occupied a different habitat. CONCLUSIONS: Bradymonabacteria have unique living strategies that are transitional between the "obligate" and the so-called facultative predators. Thus, we propose a framework to categorize the current bacterial predators into 3 groups: (i) obligate predators (completely prey-dependent), (ii) facultative predators (facultatively prey-dependent), and (iii) opportunistic predators (prey-independent). Our findings provide an ecological and evolutionary framework for Bradymonadales and highlight their potential ecological roles in saline environments. Video abstract.

Long-Term Investigation into the Membrane Fouling Behavior in Anaerobic Membrane Bioreactors for Municipal Wastewater Treatment Operated at Two Different Temperatures.

In this study, the characteristics of activated sludge flocs were investigated and their effects on the evolution of membrane fouling were considered in the anaerobic membrane bioreactors (AnMBR), which were operated at 25 and 35 °C for municipal wastewater treatment. It was found that the membrane fouling rate of the AnMBR at 25 °C was more severe than that at 35 °C. The membrane fouling trends were not consistent with the change in the concentration of soluble microbial product (SMP). The larger amount of SMP in the AnMBR at 35 °C did not induce more severe membrane fouling than that in the AnMBR at 25 °C. However, the polysaccharide and protein concentration of extracellular polymeric substance (EPS) was higher in the AnMBR at 25 °C in comparison with that in the AnMBR at 35 °C, and the protein/polysaccharide ratio of the EPS in the AnMBR at 25 °C was higher in contrast to that in the AnMBR at 35 °C. Meanwhile, the fouling tendencies measured for the AnMBRs could be related to the characteristics of loosely bound EPS and tightly bound EPS. The analysis of the activated sludge flocs characteristics indicated that a smaller sludge particle size and more fine flocs were observed at the AnMBR with 25 °C. Therefore, the membrane fouling potential in the AnMBR could be explained by the characteristics of activated sludge flocs.