Industrial Composting of Sewage Sludge: Study of the Bacteriome, Sanitation, and Antibiotic-Resistant Strains

Wastewater treatment generates a huge amount of sewage sludge, which is a source of environmental pollution. Among the alternatives for the management of this waste, industrial composting stands out as one of the most relevant. The objective of this study was to analyze the bacterial population linked to this process and to determine its effectiveness for the reduction, and even elimination, of microorganisms and pathogens present in these organic wastes. For this purpose, the bacteriome and the fecal bacteria contamination of samples from different sewage sludge industrial composting facilities were evaluated. In addition, fecal bacteria indicators and pathogens, such as Salmonella, were isolated from samples collected at key stages of the process and characterized for antibiotic resistance to macrolide, β-lactam, quinolone, and aminoglycoside families. 16S rRNA phylogeny data revealed that the process clearly evolved toward a prevalence of Firmicutes and Actinobacteria phyla, removing the fecal load. Moreover, antibiotic-resistant microorganisms present in the raw materials were reduced, since these were isolated only in the bio-oxidative phase. Therefore, industrial composting of sewage sludge results in a bio-safe final product suitable for use in a variety of applications.

Aerofilum fasciculatum gen. nov., sp. nov. (Oculatellaceae) and Euryhalinema pallustris sp. nov. (Prochlorotrichaceae) isolated from an Indian mangrove forest

Two novel cyanobacteria (AP3 and AP3b) with thin cells and simple morphology were isolated from two islands of the Indian Sundarbans. The 16S rRNA phylogeny data revealed the distinct lineage of AP3b which was nearest to the clade incorporating the genus Oculatella and Tildeniella. Strain AP3 shared a common ancestor with the species Euryhalinema mangrovii. Additionally, the novel 16S rRNA gene sequences of strains AP3 and AP3b showed similarities about 98% and 93% respectively compared to those of established genera or species to which they were phylogenetically related. Furthermore, the folding patterns of semi-conservative structures like D1-D1’, Box-B and V2 helices of 16S-23S ITS region for both strains AP3 and AP3b displayed significant variations and uniqueness when compared with their respective reference strains (Euryhalinema mangrovii for AP3 and all the genera of Oculatellaceae for AP3b). Strain AP3 shared similar morphological features with its reference strain which confirmed its inter-species relationship. The diagnostic features of AP3b including the presence of necridic cells, aerotopes and a cluster-like growth pattern were found to be very contrasting. Altogether, these results substantiated the establishment of strain AP3b as a novel mono-specific genus named Aerofilum fasciculatum and strain AP3 as the second novel species under the genus Euryhalinema, referred to as Euryhalinema pallustris.

Final Destination? Pinpointing Hyella disjuncta sp. nov. PCC 6712 (Cyanobacteria) Based on Taxonomic Aspects, Multicellularity, Nitrogen Fixation and Biosynthetic Gene Clusters

Unicellular cyanobacteria inhabit a wide range of ecosytems and can be found throughout the phylum offering space for taxonomic confusion. One example is strain PCC 6712 that was described as Chlorogloea sp. (Nostocales) and later assigned to the genus Chroococcidiopsis (Chroococcidiopsidales). We now show that this strain belongs to the order Pleurocapsales and term it Hyella disjuncta based on morphology, genome analyses and 16S-23S ITS rRNA phylogeny. Genomic analysis indicated that H. disjuncta PCC 6712 shared about 44.7% orthologue genes with its closest relative H. patelloides. Furthermore, 12 cryptic biosynthetic gene clusters (BGCs) with potential bioactivity, such as a mycosporine-like amino acid BGC, were detected. Interestingly, the full set of nitrogen fixation genes was found in H. disjuncta PCC 6712 despite its inability to grow on nitrogen-free medium. A comparison of genes responsible for multicellularity was performed, indicating that most of these genes were present and related to those found in other cyanobacterial orders. This is in contrast to the formation of pseudofilaments—a main feature of the genus Hyella—which is weakly expressed in H. disjuncta PCC 6712 but prominent in Hyella patelloides LEGE 07179. Thus, our study pinpoints crucial but hidden aspects of polyphasic cyanobacterial taxonomy.

Bradyrhizobium agreste sp. nov., Bradyrhizobium glycinis sp. nov. and Bradyrhizobium diversitatis sp. nov., isolated from a biodiversity hotspot of the genus Glycine in Western Australia

Strains of the genus Bradyrhizobium associated with agronomically important crops such as soybean (Glycine max) are increasingly studied; however, information about symbionts of wild Glycine species is scarce. Australia is a genetic centre of wild Glycine species and we performed a polyphasic analysis of three Bradyrhizobium strains—CNPSo 4010T, CNPSo 4016T, and CNPSo 4019T—trapped from Western Australian soils with Glycine clandestina, Glycine tabacina and Glycine max, respectively. The phylogenetic tree of the 16S rRNA gene clustered all strains into the Bradyrhizobium japonicum superclade; strains CNPSo 4010T and CNPSo 4016T had Bradyrhizobium yuanmingense CCBAU 10071T as the closest species, whereas strain CNPSo 4019T was closer to Bradyrhizobium liaoningense LMG 18230T. The multilocus sequence analysis (MLSA) with five housekeeping genes—dnaK, glnII, gyrB, recA and rpoB—confirmed the same clusters as the 16S rRNA phylogeny, but indicated low similarity to described species, with nucleotide identities ranging from 93.6 to 97.6% of similarity. Considering the genomes of the three strains, the average nucleotide identity and digital DNA–DNA hybridization values were lower than 94.97 and 59.80 %, respectively, with the closest species. In the nodC phylogeny, strains CNPSo 4010T and CNPSo 4019T grouped with Bradyrhizobium zhanjiangense and Bradyrhizobium ganzhouense , respectively, while strain CNPSo 4016T was positioned separately from the all symbiotic Bradyrhizobium species. Other genomic (BOX-PCR), phenotypic and symbiotic properties were evaluated and corroborated with the description of three new lineages of Bradyrhizobium . We propose the names of Bradyrhizobium agreste sp. nov. for CNPSo 4010T (=WSM 4802T=LMG 31645T) isolated from Glycine clandestina, Bradyrhizobium glycinis sp. nov. for CNPSo 4016T (=WSM 4801T=LMG 31649T) isolated from Glycine tabacina and Bradyrhizobium diversitatis sp. nov. for CNPSo 4019T (=WSM 4799T=LMG 31650T) isolated from G. max.

A new Cnemaspis Strauch, 1887 (Squamata: Gekkonidae) from the northern Western Ghats, Maharashtra, India

A new species belonging to the genus Cnemaspis Strauch, 1887 is described from the Rangana Fort mountain range of the northern Western Ghats, Maharashtra, India. A preliminary 16S rRNA phylogeny of Indian Cnemaspis is provided and the phylogenetic position of the new species is established within the goaensis clade. The new species, Cnemaspis ranganaensis sp. nov. can easily be distinguished from all Indian congeners by having 3 or 4 spine-like tubercles on the flanks, conical tubercles absent on flank, dorsal pholidosis heterogeneous, presence of precloacal and femoral pores in males, 8 or 9 poreless scales between femoral and precloacal pores; gular scales flat, smooth; 93–101 paravertebral scales, 59–63 mid-dorsal scales; 93–101 longitudinal scales from mental to cloaca, 30–31 scales across belly; ventral scales smooth, imbricate; tail with small, granular, keeled, pointed, subimbricate scales intermixed with enlarged, strongly keeled, conical tubercles forming whorls; scales on ventral side of tail imbricate, smooth, with three rows of large, slightly elongated median subcaudals. The new species is closely related to C. goaensis, from which it differs by a genetic divergence of 1.9-3.0%. Molecular phylogenetic reconstruction supports the distinctiveness of the new species.

Description of a new member of the family Erysipelotrichaceae: Dakotella fusiforme gen. nov., sp. nov., isolated from healthy human feces

A Gram-positive, non-motile, rod-shaped facultative anaerobic bacterial strain SG502T was isolated from healthy human fecal samples in Brookings, SD, USA. The comparison of the 16S rRNA gene placed the strain within the family Erysipelotrichaceae. Within this family, Clostridium innocuum ATCC 14501T, Longicatena caecimuris strain PG-426-CC-2, Eubacterium dolichum DSM 3991T and E. tortuosum DSM 3987T(=ATCC 25548T) were its closest taxa with 95.28%, 94.17%, 93.25%, and 92.75% 16S rRNA sequence identities respectively. The strain SG502T placed itself close to C. innocuum in the 16S rRNA phylogeny. The members of genus Clostridium within family Erysipelotrichaceae was proposed to be reassigned to genus Erysipelatoclostridium to resolve the misclassification of genus Clostridium. Therefore, C. innocuum was also classified into this genus temporarily with the need to reclassify it in the future because of its difference in genomic properties. Similarly, genome sequencing of the strain and comparison with its 16S phylogenetic members and proposed members of the genus Erysipelatoclostridium, SG502T warranted a separate genus even though its 16S rRNA similarity was >95% when comapred to C. innocuum. The strain was 71.8% similar at ANI, 19.8% [17.4–22.2%] at dDDH and 69.65% similar at AAI to its closest neighbor C. innocuum. The genome size was nearly 2,683,792 bp with 32.88 mol% G+C content, which is about half the size of C. innocuum genome and the G+C content revealed 10 mol% difference. Phenotypically, the optimal growth temperature and pH for the strain SG502T were 37 °C and 7.0 respectively. Acetate was the major short-chain fatty acid product of the strain when grown in BHI-M medium. The major cellular fatty acids produced were C18:1ω9c, C18:0and C16:0. Thus, based on the polyphasic analysis, for the type strain SG502T (=DSM 107282T= CCOS 1889T), the name Dakotella fusiforme gen. nov., sp. nov., is proposed.

Description of Stenomitos kolaenensis and S. hiloensis sp. nov. (Leptolyngbyaceae, Cyanobacteria) with an emendation of the genus

Stenomitos is a recently established cyanobacterial genus, some species of which appear to be cryptic. Here we describe two new species in this genus, Stenomitos kolaensis sp. nov. isolated from the Al-Fe humic podzols of a boreal forest near Nikel town, Murmansk region, Russia and S. hiloensis sp. nov. isolated from a basaltic seep wall on Akeola Road, Hilo, Hawaii, USA. Phylogenetic analyses were conducted on the 16S and 16S-23S ITS rRNA gene regions using Bayesian Inference, and Maximum Likelihood. Phylogenetic analysis of the 16S-23S ITS rRNA region resulted in both S. kolaensis and S. hiloensis forming separate clades from other Stenomitos lineages. Antarctic strains of Stenomitos frigidus (previously reported as “Leptolyngbya frigida”) show that species to be polyphyletic and in need of revision. The structure of the conserved ITS regions (Box-B, D1-D1ʹ, V2 and V3 helices) provided support for separation of the species, and the p-distances among aligned ITS regions further confirmed that a number of species exist within the genus. S. kolaensis and S. hiloensis can be distinguished from other described Stenomitos species (S. rutilans and S. tremulus) by their geographical distribution, habitat preference, 16S rRNA phylogeny, and differences in the secondary structure of the 16S-23S ITS region.

Description of a new marine planktonic cyanobacterial species Synechococcus moorigangaii (Order Chroococcales) from Sundarbans mangrove ecosystem

The genus Synechococcus is widespread across marine environments globally including in coastal habitats. In this study, culture of a new isolate (CMS01) of Synechococcus has been established and described based on polyphasic taxonomy from the world’s largest mangrove ecosystem, Sundarbans. This planktonic photoautrotroph has been proposed as a new species Synechococcus moorigangaii sp. nov. belonging to the order Chroococcales. The cells representing this proposed new species are solitary and can also form chain comprising of 4–6 cells. The shape of cell is oval to cylindrical and length ranges from 1.2–3 µm while the width ranges from 0.8–2 µm. The distribution pattern of photosynthetic filaments was found to be from the periphery of cell. Based on robust phylogeny of 16S rRNA, in addition to functional genes such as psbA, ureC, rbcL, and cbbA (multi gene phylogeny), the proposed new species differed from closest described species of Synechococcus under order Chroococcales. The fatty acid analysis indicated the presence of C12 and C14 chain fatty acids exclusive to isolate CMS01. The new isolate can grow across a range of salinity and in presence of different nitrogen sources. It has the ability to fix atmospheric di-nitrogen into ammonium ion. This new isolate of Synechococcus spp. is the first marine planktonic cyanobacterium described from a mangrove ecosystem and characterized using polyphasic approaches. Based on 16S rRNA phylogeny, this proposed new species clustered with Synechococcus strains PCC 7117, PCC 73109, PCC 7002, PCC 7003, PCC 7376 and NKNG15041c belonging to the order Chroococcales. The new species Synechococcus moorigangaii sp. nov. can serve as a model organism to understand ecophysiology and adaptation of planktonic cyanobacterial communities in mangrove ecosystems.