Epilithic Microbial Community Functionality in Deep Oligotrophic Continental Bedrock.

The deep terrestrial biosphere hosts vast sessile rock surface communities and biofilms, but thus far, mostly planktic communities have been studied. We enriched deep subsurface microbial communities on mica schist in microcosms containing bedrock groundwater from the depth of 500 m from Outokumpu, Finland. The biofilms were visualized using scanning electron microscopy, revealing numerous different microbial cell morphologies and attachment strategies on the mica schist surface, e.g., bacteria with outer membrane vesicle-like structures, hair-like extracellular extensions, and long tubular cell structures expanding over hundreds of micrometers over mica schist surfaces. Bacterial communities were analyzed with amplicon sequencing showing that Pseudomonas, Desulfosporosinus, Hydrogenophaga, and Brevundimonas genera dominated communities after 8-40 months of incubation. A total of 21 metagenome assembled genomes from sessile rock surface metagenomes identified genes involved in biofilm formation, as well as a wide variety of metabolic traits indicating a high degree of environmental adaptivity to oligotrophic environment and potential for shifting between multiple energy or carbon sources. In addition, we detected ubiquitous organic carbon oxidation and capacity for arsenate and selenate reduction within our rocky MAGs. Our results agree with the previously suggested interaction between the deep subsurface microbial communities and the rock surfaces, and that this interaction could be crucial for sustaining life in the harsh anoxic and oligotrophic deep subsurface of crystalline bedrock environment.

CO2 and carbonate as substrate for the activation of the microbial community in 180 m deep bedrock fracture fluid of Outokumpu Deep Drill Hole, Finland.

Microbial communities in deep subsurface environments comprise a large portion of Earth's biomass, but the metabolic activities in these habitats are largely unknown. Here the effect of CO2 and carbonate on the microbial community of an isolated groundwater fracture zone at 180 m depth of the Outokumpu Deep Scientific Drill Hole (Finland) was tested. Outokumpu groundwater at 180 m depth contains approximately 0.45 L L-1 dissolved gas of which methane contributes 76%. CO2, on the other hand, is scarce. The number of microbial cells with intracellular activity in the groundwater was low when examined with redox staining. Fluorescence Assisted Cell Sorting (FACS) analyses indicated that only 1% of the microbial community stained active with the redox sensing dye in the untreated groundwater after 4 weeks of starvation. However, carbon substrate and sulfate addition increased the abundance of fluorescent cells up to 7%. CO2 and CO2 + sulfate activated the greatest number of microbes, especially increasing the abundance of Pseudomonas sp., which otherwise was present at only low abundance in Outokumpu. Over longer exposure time (2 months) up to 50% of the bacterial cells in the groundwater were shown to incorporate inorganic carbon from carbonate into biomass. Carbon recapture is an important feature in this ecosystem since it may decrease the rate of carbon loss in form of CO2 released from cellular processes.

Bacterial populations on brewery filling hall surfaces as revealed by next-generation sequencing.

Due to the presence of moisture and nutrients, brewery filling line surfaces are susceptible to unwanted microbial attachment. Knowledge of the attaching microbes will aid in designing hygienic control of the process. In this study the bacterial diversity present on brewery filling line surfaces was revealed by next generation sequencing. The two filling lines studied maintained their characteristic bacterial community throughout three sampling times (13-163 days). On the glass bottle line, γ-proteobacteria dominated (35-82% of all OTUs), whereas on the canning line α-, ß- and γ-proteobacteria and actinobacteria were most common. The most frequently detected genera were Acinetobacter, Propinobacterium and Pseudomonas. The halophilic genus Halomonas was commonly detected, which might be due to its tolerance to alkaline foam cleaners. This study has revealed a detailed overall picture of the bacterial groups present on filling line surfaces. Further effort should be given to determine the efficacy of washing procedures on different bacterial groups.

Microbially induced corrosion of carbon steel in deep groundwater environment.

The metallic low and intermediate level radioactive waste generally consists of carbon steel and stainless steels. The corrosion rate of carbon steel in deep groundwater is typically low, unless the water is very acidic or microbial activity in the environment is high. Therefore, the assessment of microbially induced corrosion of carbon steel in deep bedrock environment has become important for evaluating the safety of disposal of radioactive waste. Here we studied the corrosion inducing ability of indigenous microbial community from a deep bedrock aquifer. Carbon steel coupons were exposed to anoxic groundwater from repository site 100 m depth (Olkiluoto, Finland) for periods of 3 and 8 months. The experiments were conducted at both in situ temperature and room temperature to investigate the response of microbial population to elevated temperature. Our results demonstrate that microorganisms from the deep bedrock aquifer benefit from carbon steel introduced to the nutrient poor anoxic deep groundwater environment. In the groundwater incubated with carbon steel the planktonic microbial community was more diverse and 100-fold more abundant compared to the environment without carbon steel. The betaproteobacteria were the most dominant bacterial class in all samples where carbon steel was present, whereas in groundwater incubated without carbon steel the microbial community had clearly less diversity. Microorganisms induced pitting corrosion and were found to cluster inside the corrosion pits. Temperature had an effect on the species composition of microbial community and also affected the corrosion deposits layer formed on the surface of carbon steel.

Cereulide produced by Bacillus cereus increases the fitness of the producer organism in low-potassium environments.

Cereulide, produced by certain Bacillus cereus strains, is a lipophilic cyclic peptide of 1152 Da that binds K(+) ions with high specificity and affinity. It is toxic to humans, but its role for the producer organism is not known. We report here that cereulide operates for B. cereus to scavenge potassium when the environment is growth limiting for this ion. Cereulide-producing B. cereus showed higher maximal growth rates (µ(max)) than cereulide non-producing B. cereus in K(+)-deficient medium (K(+) concentration ~1 mM). The cereulide-producing strains grew faster in K(+)-deficient than in K(+)-rich medium with or without added cereulide. Cereulide non-producing B. cereus neither increased µ(max) in K(+)-deficient medium compared with K(+)-rich medium, nor benefited from added cereulide. Cereulide-producing strains outcompeted GFP-labelled Bacillus thuringiensis in potassium-deficient (K(+) concentration ~1 mM) but not in potassium-rich (K(+) concentration ~30 mM) medium. Exposure to 2 µM cereulide in potassium-free medium lacking an energy source caused, within seconds, a major efflux of cellular K(+) from B. cereus not producing cereulide as well as from Bacillus subtilis. Cereulide depleted the cereulide non-producing B. cereus and B. subtilis cells of a major part of their K(+) stores, but did not affect cereulide-producing B. cereus strains. Externally added 6-10 µM cereulide triggered the generation of biofilms and pellicles by B. cereus. The results indicate that both endogenous and externally accessible cereulide supports the fitness of cereulide-producing B. cereus in environments where the potassium concentration is low.

Pseudoxanthomonas bacteria that drive deposit formation of wood extractives can be flocculated by cationic polyelectrolytes.

Runnability problems caused by suspended bacteria in water using industries, have, in contrast to biofilms, received little attention. We describe here that Pseudoxanthomonas taiwanensis, a wide-spread and abundant bacterium in paper machine water circuits, aggregated dispersions of wood extractives ("pitch") and resin acid, under conditions prevailing in machine water circuits (10(9) cfu ml(-1), pH 8, 45°C). The aggregates were large enough (up to 50 µm) so that they could be expected to clog wires and felts and to reduce dewatering of the fiber web. The Pseudoxanthomonas bacteria were negatively charged over a pH range of 3.2-10. Cationic polyelectrolytes of the types used as retention aids or fixatives to flocculate "anionic trash" in paper machines were effective in flocculating the Pseudoxanthomonas bacteria. The polyelectrolyte most effective for this purpose was of high molecular weight (7-8 × 10(6) g mol(-1)) and low charge density (1 meq g(-1)), whereas polyelectrolytes that effectively zeroed the electrophoretic mobility (i.e., neutralized the negative charge) of the bacterium were less effective in flocculating the bacteria. Based on the results, we concluded that the polyelectrolytes functioning by bridging mechanism, rather than by neutralization of the negative charge, may be useful as tools for reducing harmful deposits resulting from interaction of bacteria with wood extractives in warm water industry.

Adsorption of bacteria and polycations on model surfaces of cellulose, hemicellulose and wood extractives studied by QCM-D.

Quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM) were used as the tools to study the adsorption of bacteria onto surfaces of silica and polystyrene coated with materials related to papermaking. Cationic polyelectrolytes used as fixatives and retention aids in paper industry were found to promote irreversible adsorption of the ubiquitous white water bacterium, Pseudoxanthomonas taiwanensis, onto model surfaces of cellulose (pH 8). The high charged low molecular weight polyelectrolyte, poly(diallyldimethyl) ammonium chloride (pDADMAC) adsorbed to silica surface as a flat and rigid layer, whereas the low charged cationic polyacryl amide (C-PAM) of high molecular weight adsorbed as a thick and loose layer. AFM images showed that the polyelectrolytes accumulated as layers around each bacterial cell. In the presence of wood hemicellulose (O-acetyl-galactoglucomannan) the bacteria adsorbed massively, as large, tightly packed rafts (up to 0.05mm in size) onto the polystyrene crystal surface coated with wood extractives (pH 4.7). AFM and FESEM micrographs also showed large naked areas (with no bacteria) in between the bacterial rafts on the crystal surface. In this case, QCM-D only incompletely responded to the massiveness of the bacterial adsorption. The results indicate that cationic polymers can be used to increase the retention of bacteria from the process water onto the fibre web and that, depending on the balance between hemicelluloses and wood extractives and pH of the process waters, bacteria can be drawn from process waters onto surfaces.

Yeasts in malting, with special emphasis on Wickerhamomyces anomalus (synonym Pichia anomala).

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. The production of malt (malting) exploits the biochemical reactions of a natural process, grain germination. In addition to germinating grain, the malting process includes another metabolically active component: a diverse microbial community that includes various types of bacteria and fungi. Therefore, malting can be considered as a complex ecosystem involving two metabolically active groups. Yeasts and yeast-like fungi are an important part of this ecosystem, but previously the significance of yeasts in malting has been largely underestimated. Characterization and identification of yeasts in industrial processes revealed 25 ascomycetous yeasts belonging to 10 genera, and 18 basidiomycetous yeasts belonging to 7 genera. In addition, two ascomycetous yeast-like fungi belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Several ascomycetous yeast strains showed strong antagonistic activity against field and storage moulds, Wickerhamomyces anomalus (synonym Pichia anomala) being the most effective species. Malting studies revealed that W. anomalus VTT C-04565 effectively restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. In order to broaden the antimicrobial spectrum and to improve malt brewhouse performance, W. anomalus could be combined with other starter cultures such as Lactobacillus plantarum. Well-characterized microbial mixtures consisting of barley and malt-derived microbes open up several possibilities to improve malt properties and to ensure the safety of the malting process.

Deinobacterium chartae gen. nov., sp. nov., an extremely radiation-resistant, biofilm-forming bacterium isolated from a Finnish paper mill.

A rod-shaped, non-spore-forming, non-motile, aerobic, oxidase and catalase-positive and radiation-resistant bacterium (designated strain K4.1(T)) was isolated from biofilm collected from a Finnish paper mill. The bacterium grew as pale pink colonies on oligotrophic medium at 12 to 50 °C (optimum 37 to 45 °C) and at pH 6 to 10.3. The DNA G+C content of the strain was 66.8 l%. According to 16S rRNA gene sequence analysis, strain K4.1(T) was distantly related to the genus Deinococcus, sharing highest similarity with Deinococcus pimensis (90.0  %). In the phylogenetic tree, strain K4.1(T) formed a separate branch in the vicinity of the genus Deinococcus. The peptidoglycan type was A3ß with L-Orn-Gly-Gly and the quinone system was determined to be MK-8. The polar lipid profile of strain K4.1(T) differed markedly from that of the genus Deinococcus. The predominant lipid of strain K4.1(T) was an unknown aminophospholipid and it did not contain the unknown phosphoglycolipid predominant in the polar lipid profiles of deinococci analysed to date. Two of the predominant fatty acids of the strain, 15 : 0 anteiso and 17 : 0 anteiso, were lacking or present in small amounts in species of the genus Deinococcus. Phylogenetic distinctness and significant differences in the polar lipid and fatty acid profiles suggest classification of strain K4.1(T) as a novel genus and species in the family Deinococcaceae, for which we propose the name Deinobacterium chartae gen. nov., sp. nov. The type strain is K4.1(T) (=DSM 21458(T) =HAMBI 2721(T)).

Ultrastructure of biofilms formed on barley kernels during malting with and without starter culture.

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. In the malting process, dry barley kernels are steeped in water which initiates germination and invigorates microbial growth on the kernels. In the present study, field emission scanning electron microscopy (FESEM) was used to visualize the microbial community within the tissues of barley kernels before and after the steeping, with and without Lactobacillus plantarum E76 added as a starter culture. The results show that the community of 10(8)cfu g(-1) on dry, stored barley kernels increased 5-10 fold during the steeping forming a dense biofilm of bacteria and fungi with slimy exopolymeric matrix. FESEM revealed that crevices between the outer epidermis and the testa of sound barley kernels were heavily colonized with microbes, whereas there were only few microbes on the outer surface of the husks, in the aleurone layer or in the endosperm underneath an intact testa layer. The microbes frequently possessed appendages forming bridging them to the kernel and the individual microbial cells to each other. The L. plantarum added to the steeping water reduced the amount of exopolymeric matrix in the biofilm and improved the wort filterability.

Microbe repelling coated stainless steel analysed by field emission scanning electron microscopy and physicochemical methods.

Coating of stainless steel with diamond-like carbon or certain fluoropolymers reduced or almost eliminated adhesion and biofilm growth of Staphylococcus epidermidis, Deinococcus geothermalis, Meiothermus silvanus and Pseudoxanthomonas taiwanensis. These species are known to be pertinent biofilm formers on medical implants or in the wet-end of paper machines. Field emission scanning electron microscopic analysis showed that Staph. epidermidis, D. geothermalis and M. silvanus grew on stainless steel using thread-like organelles for adhesion and biofilm formation. The adhesion threads were fewer in number on fluoropolymer-coated steel than on plain steel and absent when the same strains were grown in liquid culture. Psx. taiwanensis adhered to the same surfaces by a mechanism involving cell ghosts on which the biofilm of live cells grew. Hydrophilic (diamond-like carbon) or hydrophobic (fluoropolymer) coatings reduced the adherence of the four test bacteria on different steels. Selected topographic parameters, including root-mean-square roughness (S (q)), skewness (S (sk)) and surface kurtosis (S (ku)), were analysed by atomic force microscopy. The surfaces that best repelled microbial adhesion of the tested bacteria had higher skewness values than those only slightly repelling. Water contact angle, measured (theta (m)) or roughness corrected (theta (y)), affected the tendency for biofilm growth in a different manner for the four test bacteria.

Architecture of Deinococcus geothermalis biofilms on glass and steel: a lectin study.

Deinococcus geothermalis is resistant to chemical and physical stressors and forms tenuous biofilms in paper industry. The architecture of its biofilms growing on glass and on stainless acid proof steel was studied with confocal laser scanning microscopy and fluorescent lectins and nanobeads as in situ probes. Hydrophobic nanobeads adhered to the biofilms but did not penetrate to biofilm interior. In contrast, the biofilms were readily permeable towards many different lectins. A skeletal network of glycoconjugates, reactive with Dolichos biflorus and Maclura pomifera lectins, was prominent in the space inside the biofilm colony core but absent on the exterior. Cells in the core space of the biofilm were interconnected by a network of adhesion structures, reactive with Amaranthus caudatus lectin but with none of the 65 other tested lectins. The glycoconjugates connecting the individual cells to steel reacted with Phaseolus vulgaris lectin whereas those connecting to glass mainly reacted with A. caudatus lectin. Envelopes of all cells in the D. geothermalis biofilm reacted with several other lectins, with many different specificities. We conclude that numerous different glycoconjugates are involved in the adhesion and biofilm formation of D. geothermalis, possibly contributing its unique survival capacity when exposed to dehydration, biocidal chemicals and other extreme conditions.

Destruction of Deinococcus geothermalis biofilm by photocatalytic ALD and sol-gel TiO2 surfaces.

The aim of the present work was to explore possibilities of photocatalytic TiO2 coating for reducing biofilms on non-living surfaces. The model organism, Deinococcus geothermalis, known to initiate growth of durable, colored biofilms on machine surfaces in the paper industry, was allowed to form biofilms on stainless steel, glass and TiO2 film coated glass or titanium. Field emission electron microscopy revealed that the cells in the biofilm formed at 45 degrees C under vigorous shaking were connected to the surface by means of numerous adhesion threads of 0.1-0.3 microm in length. Adjacent cells were connected to one another by threads of 0.5-1 microm in length. An ultrastructural analysis gave no indication for the involvement of amorphous extracellular materials (e.g., slime) in the biofilm. When biofilms on photocatalytic TiO2 surfaces, submerged in water, were exposed to 20 W h m(-2) of 360 nm light, both kinds of adhesion threads were completely destroyed and the D. geothermalis cells were extensively removed (from >10(7) down to below 10(6) cells cm(-2)). TiO2 films prepared by the sol-gel technique were slightly more effective than those prepared by the ALD technique. Doping of the TiO2 with sulfur did not enhance its biofilm-destroying capacity. The results show that photocatalytic TiO2 surfaces have potential as a self-cleaning technology for warm water using industries.