Disinfection in a salmon processing plant: Impact on bacterial communities and efficacy towards foodborne bacteria and biofilms.

Salmon aquaculture is the fastest growing food production system in the world. Deficiencies in the quality or safety of salmon can have global repercussions. Controlling food safety aspects during production is therefore essential. Here, we investigate the state of hygiene in a salmon processing plant using next generation sequencing and classical culture-dependent methods to characterize the surface microbiota before and after cleaning and disinfection (C&D) at ten surface sampling points. Total aerobic counts revealed an average reduction in the bacterial loads of 1.1 log CFU/cm2 by C&D. The highest relative abundance in the core microbiota before C&D was assigned to Acinetobacter, Mycoplasmataceae, Pseudomonas and Enterobacteriaceae in descending order. After C&D, we observed a significant increase in the relative abundance of Pseudomonas (p < 0.05). However, variations were found between conveyors, processing machines and drains. To assess the efficacy of commercial disinfectants, we performed susceptibility assays using advanced robotic high-throughput technologies and included foodborne bacteria which may affect food safety and spoilage. These included 128 Pseudomonas isolates, 46 Aeromonas isolates and 59 Enterobacterales isolates sampled from the salmon processing plant. Generally, minimum inhibitory concentrations (MICs) of the disinfectants were below the user concentration recommended by the producer for most isolates. BacTiter-Glo biofilm assays revealed that 30 min exposure to six out of eight commercial disinfectants resulted in an average reduction of relative luminescence >95 % in 59 single-species biofilms selected for screening. However, disinfection alone may not always be sufficient to eradicate biofilms completely. C&D routines must therefore be continuously assessed to maintain food safety and quality. The results from this study can contribute to understand and improve the state of hygiene in salmon processing environments.

Effect of model methanogens on the electrochemical activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes.

Combining anaerobic digestion (AD) and microbial electrochemical technologies (MET) in AD-MET holds great potential. Methanogens have been identified as one cause of decreased electrochemical activity and deterioration of Geobacter spp. biofilm anodes. A better understanding of the different interactions between methanogenic genera/species and Geobacter spp. biofilms is needed to shed light on the observed reduction in electrochemical activity and stability of Geobacter spp. dominated biofilms as well as observed changes in microbial communities of AD-MET. Here, we have analyzed electrochemical parameters and changes in the microbial community of Geobacter spp. biofilm anodes when exposed to three representative methanogens with different metabolic pathways, i.e., Methanosarcina barkeri, Methanobacterium formicicum, and Methanothrix soehngenii. M. barkeri negatively affected the performance and stability of Geobacter spp. biofilm anodes only in the initial batches. In contrast, M. formicicum did not affect the stability of Geobacter spp. biofilm anodes but caused a decrease in maximum current density of ~37%. M. soehngenii induced a coloration change of Geobacter spp. biofilm anodes and a decrease in the total transferred charge by ~40%. Characterization of biofilm samples after each experiment by 16S rRNA metabarcoding, whole metagenome nanopore sequencing, and shotgun sequencing showed a higher relative abundance of Geobacter spp. after exposure to M. barkeri as opposed to M. formicicum or M. soehngenii, despite the massive biofilm dispersal observed during initial exposure to M. barkeri.

Characterization of Microbial Diversity in Decayed Wood from a Spanish Forest: An Environmental Source of Industrially Relevant Microorganisms.

Rotting wood is inhabited by a large diversity of bacteria, fungi, and insects with complex environmental relationships. The aim of this work was to study the composition of the microbiota (bacteria and fungi) in decaying wood from a northwest Spanish forest as a source of industrially relevant microorganisms. The analyzed forest is situated in a well-defined biogeographic area combining Mediterranean and temperate macrobioclimates. Bacterial diversity, determined by metagenome analyses, was higher than fungal heterogeneity. However, a total of 194 different cultivable bacterial isolates (mainly Bacillaceae, Streptomycetaceae, Paenibacillaceae, and Microbacteriaceae) were obtained, in contrast to 343 fungal strains (mainly Aspergillaceae, Hypocreaceae, and Coniochaetaceae). Isolates traditionally known as secondary metabolite producers, such as Actinobacteria and members of the Penicillium genus, were screened for their antimicrobial activity by the detection of antibiotic biosynthetic clusters and competitive bioassays against fungi involved in wood decay. In addition, the ability of Penicillium isolates to degrade cellulose and release ferulic acid from wood was also examined. These results present decaying wood as an ecologically rich niche and a promising source of biotechnologically interesting microorganisms.

Syngas fermentation and microbial electrosynthesis integration as a single process unit.

Industrially relevant syngas (15 % CO, 15% H2, 20% N2 in 50% CO2) fermentation and microbial electrosynthesis were integrated as a single process unit in open and closed-circuit modes. This study examined the impact of electrochemical reducing power from -50 to -400 mV on the acetic acid synthesis and CO inhibition on fermentation. -150 mV vs. Ag/AgCl (3.0 NaCl) was identified as the lowest benchmark potential for improved acetic acid synthesis rate (0.263 mmol L-1h-1), which is 15-fold higher than the open circuit mode's rate. No significant inhibition by CO in the fermentation was observed, while 60% of the gas was consumed. Anodic potential above 2.0 V substantially lowered the product formation. Superseding the fermentation medium with fresh inoculum through a fed-batch operation helped lower the anodic potential.

Flow-based method for biofilm microbiota enrichment and exploration of metagenomes.

Most bacteria live in biofilms in their natural habitat rather than the planktonic cell stage that dominates during traditional laboratory cultivation and enrichment schemes. The present study describes the establishment of a flow-based enrichment method based on multispecies biofilm communities for directing biofilm functionality using an environmental inoculum. By controlling flow conditions and physio-chemical properties, the set-up aims to simulate natural conditions ex situ for biofilm formation. The functionality of the method was demonstrated by enrichment of biofilm microbiomes using consortia from a warm compost pile and industrial waste materials as growth substrate, and further exploring the metagenomes by biotechnological tools. The 16S rRNA gene sequencing results revealed a difference in consortium composition and especially in genus abundance, in flow experiments compared to traditional liquid-shake experiments after enrichment, indicating good biofilm development and increased abundance of biofilm-forming taxa. The shotgun sequence mining demonstrated that different enzymes classes can be targeted by enriching biofilms on different substrates such as oat husk, pine saw dust, and lignin. The flow-based biofilm method is effective in reducing bacterial consortia complexity and in selecting biofilm-forming bacteria, and it is possible to enrich the biofilm community in various directions based on the choice of sample material, environmental conditions, and nutritional preferences, targeting enzymes or enzyme classes of industrial interest.

Impact of electrochemical reducing power on homoacetogenesis.

Homoacetogenesis was performed in a microbial electrosynthesis single-chamber reactor at open and closed circuits modes. The aim is to investigate how an applied reducing power affects acetic acid synthesis and H2 gas-liquid mass transfer. At a cathode voltage of -175 mV vs. Ag/AgCl (3.0 NaCl), the acetic acid synthesis rate ramped up to 0.225 mmol L-1h-1 due to additional electrons and protons liberation from carbon-free sources such as water and ammonium via anodic oxidation. The study sets a new lowest benchmark that acetic acid can be bioelectrochemical synthesized at - 175 mV. The applied reducing power did not increase the H2 gas-liquid mass transfer because the direct electron transfer from cathode to microorganisms reduced the demand for H2 in the fermentation medium. Microbial analysis shows a high presence of Veillonellaceae spore-forming clostridia, which are identified as homoacetogens.

Multiplex droplet digital PCR assay for detection of Flavobacterium psychrophilum and Yersinia ruckeri in Norwegian aquaculture.

We report the development of ddPCR assays for single and simultaneous detection of the bacterial pathogens Flavobacterium psychrophilum and Yersinia ruckeri in water from land-based recirculation aquaculture systems (RAS), producing Atlantic salmon (Salmo salar) smolt. The method was tested and verified for use in water analyses from RAS production sites, and proved to be specific and with sensitivity 0.0011 ng DNA for F. psychrophilum and 1.24 ng for Y. ruckeri. These bacteria are important fish pathogens that have caused reoccurring salmonid infection disease in RAS. Monitoring pathogen levels in water samples could be a useful alternative surveillance strategy to evaluate operational risk assessment connected to stress factors. Water quality is essential for fish health and growth in RAS production in general, and high or increasing levels of these pathogens in the RAS water may generate an early indication of unfavourable conditions in the RAS environment, and give directions to operational actions. This approach may reduce fish mortality, reduce production loss, and offer more effective and targeted preventive measures within RAS production.

Corrigendum: Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

[This corrects the article DOI: 10.3389/fmicb.2016.01481.].

Biodegradation of n-alkanes on oil-seawater interfaces at different temperatures and microbial communities associated with the degradation.

Oil biodegradation studies have mainly focused on microbial processes in dispersions, not specifically on the interfaces between the oil and the seawater in the dispersions. In this study, a hydrophobic adsorbent system, consisting of Fluortex fabrics, was used to investigate biodegradation of n-alkanes and microbial communities on oil-seawater interfaces in natural non-amended seawater. The study was performed over a temperature range from 0 to 20 °C, to determine how temperature affected biodegradation at the oil-seawater interfaces. Biodegradation of n-alkanes were influenced both by seawater temperature and chain-length. Biotransformation rates of n-alkanes decreased by reduced seawater temperature. Low rate coefficients at a seawater temperature of 0 °C were probably associated with changes in physical-chemical properties of alkanes. The primary bacterial colonization of the interfaces was predominated by the family Oceanospirillaceae at all temperatures, demonstrating the wide temperature range of these hydrocarbonoclastic bacteria. The mesophilic genus Oleibacter was predominant at the seawater temperature of 20 °C, and the psychrophilic genus Oleispira at 5 and 0 °C. Upon completion of n-alkane biotransformation, other oil-degrading and heterotrophic bacteria became abundant, including Piscirickettsiaceae (Cycloclasticus), Colwelliaceae (Colwellia), Altermonadaceae (Altermonas), and Rhodobacteraceae. This is one of a few studies that describe the biodegradation of oil, and the microbial communities associated with the degradation, directly at the oil-seawater interfaces over a large temperature interval.

Novel archaeal thermostable cellulases from an oil reservoir metagenome.

Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.

6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.

D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg2+ and Mn2+ and displayed similar K' values (56-75 µM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower V max (49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg2+ and Mn2+, and displayed similar thermostabilities. The PFKs have similar K m values for fructose 6-phosphate (0.61-0.94 µM) and for ATP (0.38-0.82 µM), while Pfk1-MGA3 had a 2-fold lower V max (6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.

Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.