Journal of Applied Microbiology

Gerber, E.; Bernard, R.; Castang, S.; Chabot, N.; Coze, F.; Dreux ‐ Zigha, A.; Hauser, E.; Hivin, P.; Joseph, P.; Lazarelli, C.; Letellier, G.; Olive, J.; Leonetti, J.‐P.

doi: 10.1111/jam.12808pmid: 25809882

Summary Deinococcus spp are among the most radiation‐resistant micro‐organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro‐organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.

Dexter, J.; Armshaw, P.; Sheahan, C.; Pembroke, J.T.

doi: 10.1111/jam.12821pmid: 25865951

Summary Ethanol production directly from CO2, utilizing genetically engineered photosynthetic cyanobacteria as a biocatalyst, offers significant potential as a renewable and sustainable source of biofuel. Despite the current absence of a commercially successful production system, significant resources have been deployed to realize this goal. Utilizing the pyruvate decarboxylase from Zymomonas species, metabolically derived pyruvate can be converted to ethanol. This review of both peer‐reviewed and patent literature focuses on the genetic modifications utilized for metabolic engineering and the resultant effect on ethanol yield. Gene dosage, induced expression and cassette optimizat‐ion have been analyzed to optimize production, with production rates of 0·1–0·5 g L−1 day−1 being achieved. The current ‘toolbox’ of molecular manipulations and future directions focusing on applicability, addressing the primary challenges facing commercialization of cyanobacterial technologies are discussed.

Li, X.; Defoirdt, T.; Bossier, P.

doi: 10.1111/jam.12813pmid: 25807847

Aims In this study, we investigated the responsiveness of 15 Vibrio anguillarum strains to three host factors (mucin, bile salts and cholesterol). Methods and Results Three virulence‐related phenotypes were investigated in this respect, i.e. motility, biofilm formation and exopolysaccharide production. Almost all V. anguillarum strains showed a significantly increased motility in the presence of either of the three host factors. Only five of the strains showed increased biofilm formation in the presence of host factors and only three strains showed increased exopolysaccharide production in the presence of the host factors. Conclusions There were no significant correlations between the three putatively virulence‐linked phenotypes (in the absence of host factors) and virulence to sea bass larvae. There was no correlation between responsiveness to the host factors (percentage increase in motility, biofilm formation or exopolysaccharide production in the presence of the three host factors) and virulence to sea bass larvae. However, the responses of these virulence‐related phenotypes upon the addition of either of the three host factors were significantly correlated with each other. This result suggests that the mechanisms by which V. anguillarum responds to these three host factors is linked. Significance and Impact of the Study Although the mechanism by which V. anguillarum responds to the host factors mucin, bile salts and cholesterol seems to be linked, there is no correlation between host factor responsiveness and virulence towards sea bass larvae. This emphasizes that one should be careful when extrapolating results obtained for one particular strain to reach general conclusions on a species of pathogenic bacteria.

Witzig, M.; Boguhn, J.; Zeder, M.; Seifert, J.; Rodehutscord, M.

doi: 10.1111/jam.12829pmid: 25879255

Aims To investigate the effect of donor animal species and their feeding on the composition of the active fraction of rumen microbiota established during in vitro experiments with different forages. Methods and Results Rumen simulation experiments were conducted with maize silage (MS) and grass silage (GS) as substrates. Four experimental runs were performed with ruminal contents of sheep and cows fed either with hay and concentrate or with MS or GS, respectively. Liquid‐ and solid‐associated microbes (LAM and SAM) were fixed for fluorescence in situ hybridization after 14 days of incubation. The LAM were characterized by higher proportions of Lachnospiraceae, Bacteroidetes, Deltaproteobacteria and Methanobacteriaceae at hay‐concentrate‐based feeding of the donor animals whereas counts of Gammaproteobacteria were lower. Similar results were found for SAM. Hay‐concentrate‐based feeding caused a higher ratio of Bacteria:Archaea in SAM. Incubations with GS seemed to enhance Bacteroidetes, Deltaproteobacteria and Archaea whereas numbers of Gammaproteobacteria were enhanced in MS experiments. The effect of the donor animal species was limited to the number of Archaea establishing in vitro being higher for sheep than for cows. Conclusions The feeding of donor animals, more than the animal species itself, affects the composition of the ruminal microbial community that establishes in vitro. Significance and Impact of the Study Data suggest the need of a standardized approach for studying the rumen microbiota in a rumen simulation. Moreover, this study provides fundamental data on the composition of the ruminal microbial community when different diets are fed to donor animals.

Sutili, F.J.; Lima Silva, L.; Gressler, L.T.; Gressler, L.T.; Battisti, E.K.; Heinzmann, B.M.; Vargas, A.C.; Baldisserotto, B.

doi: 10.1111/jam.12812pmid: 25810355

Aims The aims of this study were to investigate the in vitro antibacterial activity of the essential oils (EOs) of Hesperozygis ringens (HREO), popularly known as ‘espanta‐pulga’ and two different species of basil, Ocimum gratissimum (OGEO) and Ocimum americanum (OAEO), as well as, the potential of these products to be used in silver catfish (Rhamdia quelen) infected with Aeromonas hydrophila. Methods and Results OGEO and HREO showed better antibacterial activity in vitro. Subinhibitory concentrations of all EOs inhibited haemolysis caused by Aer. hydrophila in fish erythrocytes (100% reduction for OAEO at 100 μg ml−1 and more than 90% for HREO and OGEO at 150 μg ml−1). However, OAEO and HREO showed the best survival results (75 and 70% respectively) after their use as treatment (therapeutic baths—1 h daily/5 days) in silver catfish experimentally infected with Aer. hydrophila. A second in vivo assay using healthy fish was conducted to verify the potential of the EOs (preventive baths—1 h daily/5 days) to promote fish survival. Fish exposed to HREO and OAEO and their diluent (ethanol) showed significant lower haematocrit values and higher complement system activity compared to control. Plasma cortisol level was significantly higher in the groups exposed to both EOs. There was no significant difference in survival of silver catfish challenged with Aer. hydrophila after preventive baths with HREO, OAEO and control group. Conclusions All tested EOs showed in vitro antibacterial properties against Aer. hydrophila and HREO and OAEO showed potential to be used in the treatment of infected fish. Significance and Impact of the Study These products can be used in aquaculture as therapeutic and prophylactic agents against fish pathogens, with antimicrobial and/or immunostimulant properties.

Guo, L.; Long, M.; Huang, Y.; Wu, G.; Deng, W.; Yang, X.; Li, B.; Meng, Y.; Cheng, L.; Fan, L.; Zhang, H.; Zou, L.

doi: 10.1111/jam.12820pmid: 25846200

Aims The study aims to demonstrate the antimicrobial and disinfectant resistance phenotypes and genotypes of Escherichia coli isolates obtained from giant pandas (Ailuropoda melanoleuca). Methods and Results Antimicrobial testing was performed according to the standard disk diffusion method. The minimal inhibitory concentrations (MICs) of disinfectants were determined using the agar dilution method. All isolates were screened for the presence of antimicrobial and disinfectant resistance genes and further analysed for genetic relatedness by pulse‐field gel electrophoresis (PFGE). Results showed that 46·6% of the isolates were resistant to at least one antimicrobial. Escherichia coli isolates showed resistance to fewer antimicrobials as panda age increased. Among antimicrobial‐resistant E. coli isolates, the antimicrobial resistance genes blaCTX‐M (88·2%) and sul1 (92·3%) were most prevalent. The disinfectant resistance genes emrE, ydgE/ydgF, mdfA and sugE(c) were commonly present (68·2–98·9%), whereas qac and sugE(p) were relatively less prevalent (0–21·3%). The frequencies of resistance genes tended to be higher in E. coli isolated in December than in July, and PFGE profiles were also more diverse in isolates in December. The qacEΔ1 and sugE(p) genes were higher in adolescent pandas than in any other age groups. PFGE revealed that antimicrobial resistance correlated well with sampling time and habitat. Conclusions This study demonstrated that antimicrobial and disinfectant resistance was common in giant panda‐derived E. coli, and the antimicrobial resistance was associated with sampling time and habitat. Escherichia coli could serve as a critical vector in spreading disinfectant and antimicrobial resistance. Significance and Impact of the Study This is the first study that demonstrated the phenotypic and genetic characterizations of antimicrobial and disinfectant resistance in E. coli isolates from more than 60 giant pandas. Frequent transfer of pandas to other cages may lead to the dissemination of antimicrobial resistance. The study highlights the need for regularly monitoring the antimicrobial and disinfectant resistance in bacteria from giant pandas.

Kelsey, D.J.; Nieto‐Delgado, C.; Cannon, F.S.; Brennan, R.A.

doi: 10.1111/jam.12823pmid: 25857444

Aims To examine organic neem compounds for their effective growth inhibition of saprotrophic soft‐rot fungi on anthracite bricks bound with collagen and lignin for use in iron foundry cupolas as an alternative fuel source. Methods and Results Azadirachtin, crude neem oil (NO), and clarified neem oil extract (CNO) were combined with copper to inhibit the growth of the soft‐rot fungus, Chaetomium globosum. A synergistic interaction was observed between CNO and a low dose of copper on nutrient media (two‐factor anova with triplicate replication: P < 0·05). Interaction was confirmed on lab‐scale collagen‐lignin‐anthracite briquettes by measuring their unconfined compressive (UC) strength. The effective collagen strength of the briquettes was enhanced by applying CNO to their surface prior to inoculation: the room temperature UC strength of the briquettes was 28 ± 4·6% greater when CNO (0·4 mg cm−2) was surface‐applied, and was 43 ± 3·0% greater when CNO plus copper (0·14 μg cm−2) were surface‐applied. Conclusion Surface application of CNO and copper synergistically prevents fungal growth on bindered anthracite briquettes and increases their room temperature strength. Significance and Impact of the Study This novel organic fungicidal treatment may increase the storage and performance of anthracite bricks in iron foundries, thereby saving 15–20% of the energy used in conventional coke production.

Park, K.C.; Whitney, C.G.E.; Kozera, C.; O'Leary, S.J.B.; McGinn, P.J.

doi: 10.1111/jam.12818pmid: 25845886

Aims The objective of the study was to isolate the microalgae strains from treated municipal wastewater in both summer and winter seasons in order to identify strains better suited for nutrient remediation and biofuel production under either cooler or warmer temperatures. Methods and Results Fifty‐six strains in total were isolated and identified by DNA sequencing from effluent samples collected from a local wastewater treatment plant during the summer and winter of 2011. Screening of 41 isolates based on the fatty acid productivity at either 22 or 10°C resulted in the selection of 12 strains organized into two groups of 6—the M (mild) and C (cool) groups, respectively. Four of the C‐group strains were isolated from the winter sample, while four of the M‐group isolates were isolated from the summer sample. Fatty acid pools in M‐group strains were heavily regulated in response to growth temperature while C‐group strains were more insensitive. In three of the six C‐group strains, the rates of biomass and fatty acid productivity at 10°C exceeded the corresponding rates at 22°C. Conversely, M group were always more productive at 22 compared to 10°C. Mixotrophic strategies to enhance productivity were generally unsuccessful in M‐group strains at 22°C but proved to be more effective in C‐group cultures at 10°C. Conclusions In general, C‐group strains appeared better suited for growth in municipal wastewater at 10°C, while M‐group strains were better suited at 22°C. On balance, C‐group isolates were more likely to come from winter wastewater samples while M‐group strains were more likely to come from the summer sample. Significance and Impact of the Study Our results demonstrate that the effects of temperature on microalgal growth for wastewater remediation can be mitigated somewhat by isolation and careful selection of strains adapted to seasonal wastewater conditions.

Goyari, S.; Devi, S.H.; Bengyella, L.; Khan, M.; Sharma, C.K.; Kalita, M.C.; Talukdar, N.C.

doi: 10.1111/jam.12816pmid: 25833715

Aims Elucidation of different physico‐chemical parameters and the secretory enzymes released by Talaromyces verruculosus SGMNPf3 during cellulosic biomass degradation. Methods and Results We determined the optimal pH, temperature and time course parameters for the efficient degradation of different natural and commercial cellulosic substrates by T. verruculosus SGMNPf3, previously isolated from a forest soil. The optimal growth of the fungus and production of its cellulases were obtained when the culture condition was maintained at pH 3·3 and temperature 30°C. Activity of the crude cellulases was maximum at 60°C. Activity of cellulase enzymes produced on natural cellulose substrates was higher than that on commercial cellulose substrates. A continuous increase in cellulase activity at different time points indicated no apparent end product inhibition. This might be attributed to the high individual cellulases, notably β‐glucosidase (316·1 μmol g−1) production. Zymogram of extracellular crude proteins showed two dominant extracellular protein bands of molecular weight 72·3 and 61·4 kDa, indicating their cellulolytic nature. MALDI‐TOF and LC‐MS/MS analysis of the 2DE spots also identified several enzymes including β‐glucosidase involved in the process of cellulose degradation. Conclusions Based on its optimal parameters for cellulolytic activities, we suggest that the fungus is acido‐mesophilic. There was apparently no end‐product inhibition of the cellulase activity and this is attributed to the ability of the fungus to produce sufficient β‐glucosidase. The dominant proteins secreted by the fungus were confirmed to be cellulases. Significance and Impact of the Study The high individual cellulase activities, better cellulase production on natural substrates and apparent absence of end‐product inhibition are characteristics of T. verruculosus SGMNPf3 for use in harvesting naturally endowed energy in cellulosic biomass.

Reznicek, O.; Facey, S.J.; Waal, P.P.; Teunissen, A.W.R.H.; Bont, J.A.M.; Nijland, J.G.; Driessen, A.J.M.; Hauer, B.

doi: 10.1111/jam.12825pmid: 25882005

Aims Saccharomyces cerevisiae does not express any xylose‐specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. Methods and Results Three rounds of error‐prone PCR were used to generate mutants with improved xylose‐transport characteristics. After developing a fast and reliable high‐throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild‐type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. Conclusions Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co‐consumption. Significance and Impact of the Study Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co‐consumption of new substrates. Using a high‐throughput screening system based on flow‐through cytometry, various mutants were identified with improved xylose‐transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.