Revealing the dissimilar structure of microbial communities in different WWTPs that treat fish-canning wastewater with different NaCl content

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research was supported by the Spanish Government (Spanish Ministry of Science and Innovation) through TREASURE-TECHNOSALT (CTQ2017-83225-C2-1-R) and TREASURE-MICROSALT (CTQ2017-83225-C2-2-R) projects. The authors Alba Roibas-Rozas and Anuska Mosquera-Corral belong to the Galician Competitive Research Group GRC-ED431C 2017-29 and to the CRETUS Strategic Partnership (ED431E 2018/01) . All these programmes are co-funded by FEDER (UE) . Funding for open access charge: Universidad de Granada/CBUA.Studies that characterize the microbial communities in wastewater treatment plants (WWTPs) are numerous, yet similar studies in industrial WWTPs treating fish-canning effluents are limited. The microbial communities in samples of 4 fish-canning WWTPs that operated under different NaCl concentrations were investigated by qPCR and partial 16S rRNA gene Illumina sequencing. The absolute abundances of key microbial populations (Total Bacteria, Archaea and Fungi, ammonium oxidizing bacteria (AOB), Mycolata, Candidatus Microthrix, Ca. Accumulibacter and Ca. Competibacter) presented statistical differences among the WWTPs. The NaCl concentration negatively affected the absolute abundance of Bacteria and Fungi, filamentous, and phosphate (PAO) and glycogen (GAO) accumulating bacteria, while AOB and Ca. Microthrix populations were statistically higher in the WWTP with higher NaCl contents. On the other hand, the main bacterial operational taxonomic units (OTUs) were classified as members of Kouleothrix (Chloroflexia, Chloroflexi) and Tetrasphaera (Actinomycetia, Actinobacteria), family Beijerinckiaceae (Alphaproteobacteria, Proteobacteria), order Betaproteobacteriales (Gammaproteobacteria, Proteobacteria), Sphingobacteriales (Sphingobacteriia, Bacteroidetes) and Frankiales (Actinobacteria, Actinobacteria), class Anaerolineae (Chloroflexi), phylum Chloroflexi and Bacteria_unclassified. The structure of the bacterial community was highly dissimilar among the 4 WWTPs, as the identities of the dominant OTUs differed significantly among them. Therefore, the individual characteristics of the different WWTPs, mainly NaCl concentration, were responsible for the narrow assemblage of the bacterial communities. Different OTUs belonging to the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes and Proteobacteria were revealed as salttolerant. Taking into account these results, NaCl content was an important driver of the abundance of microbial populations and the bacterial community structure in the analysed industrial facilities.Spanish Government (Spanish Ministry of Science and Innovation) CTQ2017-83225-C2-1-R CTQ2017-83225-C2-2-REuropean CommissionGalician Competitive Research Group GRC-ED431C 2017-29CRETUS Strategic Partnership ED431E 2018/0

Diversity of culturable nocardioform actinomycetes from wastewater treatment plants in Spain and their role in the biodegradability of aromatic compounds

[EN] Currently, municipal and industrial wastewater treatment plants (WWTPs) are mainly focusing on reduction of biological oxygen demand and on the removal of nutrients. However, there are microorganisms that interfere with the process. In this environment, there is a large diversity of microorganisms that have not been studied in detail and that could provide real and practical solutions to the foaming problems. Among such microorganisms, Gram-positive actinomycete bacteria are of special interest because they are known for producing secondary metabolites as well as chemically diverse compounds and for their capacity to degrade recalcitrant pollutants. Three different media were chosen to isolate actinomycetes from 28 WWTPs in Spain. A total of 189 activated sludge samples were collected; 126 strains were isolated and identified to belong to 1 suborder, i.e. Corynebacterineae, and 7 genera, i.e. Corynebacterium, Dietzia, Gordonia, Mycobacterium, Rhodococcus, Tsukamurella and Williamsia. Furthermore, 71 strains were capable of biodegrading at least 1 aromatic product, and that 27 of them amplified for catA gene. The results of this research help us understand the complexity of the foam-forming microbial populations in Spain and it shows that WWTPs can be a good source of microorganisms that can degrade phenol or naphthalene.This work was supported by grants from Entidad Publica de Saneamiento de Aguas Residuales (EPSAR) de la Comunitat Valenciana.Soler Hernández, A.; García Hernández, J.; Zornoza-Zornoza, AM.; Alonso Molina, JL. (2017). Diversity of culturable nocardioform actinomycetes from wastewater treatment plants in Spain and their role in the biodegradability of aromatic compounds. Environmental Technology. 39(2):172-181. https://doi.org/10.1080/09593330.2017.1296897S17218139

FISH Handbook for Biological Wastewater Treatment

The FISH Handbook for Biological Wastewater Treatment provides all the required information for the user to be able to identify and quantify important microorganisms in activated sludge and biofilms by using fluorescence in situ hybridization (FISH) and epifluorescence microscopy. It has for some years been clear that most microorganisms in biological wastewater systems cannot be reliably identified and quantified by conventional microscopy or by traditional culture-dependent methods such as plate counts. Therefore, molecular biological methods are vital and must be introduced instead of, or in addition to, conventional methods. At present, FISH is the most widely used and best tested of these methods. This handbook presents all relevant information from the literature and, based on the extensive experience of the authors, advice and recommendations are given for reliable FISH identification and quantification

The Effect of Media Composition on Nitrile Hydratase Activity and Stability, and on Cell Envelope Components of Rhodococcus DAP 96253

Rhodococcus is an important industrial organism that possesses diverse metabolic capabilities, it also has a unique cell envelope, composed of an outer layer of mycolic acids and glycolipids (free or bound lipids generally linked to the sugar trehalose). Rhodococcus is able to transform nitriles to the corresponding amide by the enzyme Nitrile Hydratase (NHase), therefore rhodococcal cells can be utilized as biocatalysts in the detoxification of nitrile waste water or in the production of industrially important amides such as acrylamide. However, the NHase within the native cells must be stable with high activity. This research examined how NHase activity and stability can be increased in native cells by changing growth media composition, the impact on the rhodococcal cell envelope was also studied. Growth media composition was altered by supplementing different sugars such as fructose, maltose or maltodextrin to replace glucose in rich solid media containing cobalt and urea for induction of NHase. The supplementation of maltose or maltodextrin resulted in significantly higher NHase activities and greater NHase stability at 55„aC. The supplementation of these different sugars was shown to alter cellular and lipid bound trehalose levels, a sugar known to stabilize proteins and a component of the rhodococcal cell envelope. Cells that had higher levels of cellular trehalose had significantly greater NHase stability at 55„aC. The effect of the different sugar supplements and inducers of NHase, such as cobalt, on cell envelope components such as mycolic acids and glycolipids were examined by High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). The results showed that changes in mycolic acids and glycolipids occurred when the cells were grown in the presence of different sugar supplements and when the cells were induced for NHase. Susceptibility of Rhodococcus sp DAP 96253 to different antibiotics was examined to indicate if changes were occurring in the cell envelope. Differences in antibiotic susceptibility were observed when the cells were grown on media with different sugar supplements and when the cells were induced for NHase. In the presence of cobalt Rhodococcus sp DAP 96253 showed a significant increase in sensitivity to antibiotics. Changes in growth media composition influences the cell envelope of Rhodococcus sp DAP 96253 and also affects NHase activity and stability. Therefore, achieving increased enzyme activity and stability is not entirely dependent on the actual enzyme, but is related to other aspects of the cell, such as the cell envelope and metabolites of the cell

The Oxidative Reaction of Potassium Permanganate with Mycolic Acids Leads to a Unique Diagnostic Pattern for Mycobacterium Tuberculosis

In an oxidative reaction, potassium permanganate reacts with the mycolic acid component of _M.tuberculosis_ and gives rise to a unique saponified pattern specific for _M.tuberculosis_. This brown orange pattern makes it possible to identify _M.tuberculosis_ in a typical laboratory sample. The method avoids the complexity of other methods with special regard to save time and costs

Ultralong C100 Mycolic Acids Support the Assignment of Segniliparus as a New Bacterial Genus

Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated α-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus α-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane

Bacteriophages of wastewater foaming-associated filamentous Gordonia reduce host levels in raw activated sludge

Filamentous bacteria are a normal and necessary component of the activated sludge wastewater treatment process, but the overgrowth of filamentous bacteria results in foaming and bulking associated disruptions. Bacteriophages, or phages, were investigated for their potential to reduce the titer of foaming bacteria in a mixed-microbial activated sludge matrix. Foaming-associated filamentous bacteria were isolated from activated sludge of a commercial wastewater treatment plan and identified as Gordonia species by 16S rDNA sequencing. Four representative phages were isolated that target G. malaquae and two un-named Gordonia species isolates. Electron microscopy revealed the phages to be siphophages with long tails. Three of the phages - GordTnk2, Gmala1, and GordDuk1 - had very similar ~76 kb genomes, with >93% DNA identity. These genomes shared limited synteny with Rhodococcus equi phage ReqiDocB7 and Gordonia phage GTE7. In contrast, the genome of phage Gsput1 was smaller (43 kb) and was not similar enough to any known phage to be placed within an established phage type. Application of these four phages at MOIs of 5–15 significantly reduced Gordonia host levels in a wastewater sludge model by approximately 10-fold as compared to non-phage treated reactors. Phage control was observed for nine days after treatment

Draft genome sequence of Dietzia maris DSM 43672, a gram-positivebacterium of the mycolata group

Here, we report the draft genome sequence of Dietzia maris, known previously as Rhodococcus maris. It is 3,505,372 bp in size with a G+C content of 73%. The draft genome sequence will improve our understanding of Dietzia maris related to other mycolata species and constitutes a basic tool for exploring the cell wall proteins

Dynamics of PHA-Accumulating Bacterial Communities Fed with Lipid-Rich Liquid Effluents from Fish-Canning Industries

This research was supported by the Spanish Government (Spanish Ministry of Science and Innovation) through the TREASURE-MICROSALT (CTQ2017-83225-C2-2-R) and ECOPOLYVER-MACROPOLYVER (PID2020-112550RC22) projects.The biosynthesis of polyhydroxyalkanoates (PHAs) from industrial wastes by mixed microbial cultures (MMCs) enriched in PHA-accumulating bacteria is a promising technology to replace petroleum-based plastics. However, the populations’ dynamics in the PHA-accumulating MMCs are not well known. Therefore, the main objective of this study was to address the shifts in the size and structure of the bacterial communities in two lab-scale sequencing batch reactors (SBRs) fed with fish-canning effluents and operated under non-saline (SBR-N, 0.5 g NaCl/L) or saline (SBR-S, 10 g NaCl/L) conditions, by using a combination of quantitative PCR and Illumina sequencing of bacterial 16S rRNA genes. A double growth limitation (DGL) strategy, in which nitrogen availability was limited and uncoupled to carbon addition, strongly modulated the relative abundances of the PHA-accumulating bacteria, leading to an increase in the accumulation of PHAs, independently of the saline conditions (average 9.04 wt% and 11.69 wt%, maximum yields 22.03 wt% and 26.33% SBR-N and SBR-S, respectively). On the other hand, no correlations were found among the PHAs accumulation yields and the absolute abundances of total Bacteria, which decreased through time in the SBR-N and did not present statistical differences in the SBR-S. Acinetobacter, Calothrix, Dyella, Flavobacterium, Novosphingobium, Qipengyuania, and Tsukamurella were key PHA-accumulating genera in both SBRs under the DGL strategy, which was revealed as a successful tool to obtain a PHA-enriched MMC using fish-canning effluents.Spanish Government (Spanish Ministry of Science and Innovation) CTQ2017-83225-C2-2-R PID2020-112550RC2