Biofilm-Forming Ability and Effect of Sanitation Agents on Biofilm-Control of Thermophile Geobacillus sp. D413 and Geobacillus toebii E134.

Geobacillus sp. D413 and Geobacillus toebii E134 are aerobic, non-pathogenic, endospore-forming, obligately thermophilic bacilli. Gram-positive thermophilic bacilli can produce heat-resistant spores. The bacteria are indicator organisms for assessing the manufacturing process's hygiene and are capable of forming biofilms on surfaces used in industrial sectors. The present study aimed to determine the biofilm-forming properties of Geobacillus isolates and how to eliminate this formation with sanitation agents. According to the results, extracellular DNA (eDNA) was interestingly not affected by the DNase I, RNase A, and proteinase K. However, the genomic DNA (gDNA) was degraded by only DNase I. It seemed that the eDNA had resistance to DNase I when purified. It is considered that the enzymes could not reach the target eDNA. Moreover, the eDNA resistance may result from the conserved folded structure of eDNA after purification. Another assumption is that the eDNA might be protected by other extracellular polymeric substances (EPS) and/or extracellular membrane vesicles (EVs) structures. On the contrary, DNase I reduced unpurified eDNA (mature biofilms). Biofilm formation on surfaces used in industrial areas was investigated in this work: the D413 and E134 isolates adhered to all surfaces. Various sanitation agents could control biofilms of Geobacillus isolates. The best results were provided by nisin for D413 (80%) and α-amylase for E134 (98%). This paper suggests that sanitation agents could be a solution to control biofilm structures of thermophilic bacilli.

Geobacillus strains that have potential value in microbial enhanced oil recovery.

Bacteria from the genus Geobacillus are generally obligately thermophilic, with a unique bioenergy production capacity and unique enzymes. Geobacillus species were isolated primarily from hot springs, oilfields, and associated soils. They often exhibit unique survival patterns in these extreme oligotrophic environments. With the development of the microbial resources found in oilfields, Geobacillus spp. have been proven as valuable bacteria in many reports related to oilfields. After the isolation of Geobacillus by culture methods, more evidence was found that they possess the abilities of hydrocarbon utilization and bioemulsifier production. This paper mainly summarizes some characteristics of the Geobacillus species found in the oilfield environment, focusing on the inference and analysis of hydrocarbon degradation and bioemulsifier synthesis based on existing research, which may reveal their potential value in microbial enhanced oil recovery. It also provides references for understanding microbes in extreme environments.

A design of experiments approach for the rapid formulation of a chemically defined medium for metabolic profiling of industrially important microbes.

Geobacillus thermoglucosidans DSM2542 is an industrially important microbe, however the complex nutritional requirements of Geobacilli confound metabolic engineering efforts. Previous studies have utilised semi-defined media recipes that contain complex, undefined, biologically derived nutrients which have unknown ingredients that cannot be quantified during metabolic profiling. This study used design of experiments to investigate how individual nutrients and interactions between these nutrients contribute to growth. A mathematically derived defined medium has been formulated that has been shown to robustly support growth of G. thermoglucosidans in two different environmental conditions (96-well plate and shake flask) and with a variety of lignocellulose-based carbohydrates. This enabled the catabolism of industrially relevant carbohydrates to be investigated.

Short communication: Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species.

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of ß-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006.

Genetic toolbox for controlled expression of functional proteins in Geobacillus spp.

Species of genus Geobacillus are thermophilic bacteria and play an ever increasing role as hosts for biotechnological applications both in academia and industry. Here we screened a number of Geobacillus strains to determine which industrially relevant carbon sources they can utilize. One of the strains, G. thermoglucosidasius C56-YS93, was then chosen to develop a toolbox for controlled gene expression over a wide range of levels. It includes a library of semi-synthetic constitutive promoters (76-fold difference in expression levels) and an inducible promoter from the xylA gene. A library of synthetic in silico designed ribosome binding sites was also created for further tuning of translation. The PxylA was further used to successfully express native and heterologous xylanases in G. thermoglucosidasius. This toolbox enables fine-tuning of gene expression in Geobacillus species for metabolic engineering approaches in production of biochemicals and heterologous proteins.

An improved agar medium for growth of Geobacillus thermoglucosidarius strains.

Geobacillus species have potential applications in many biotechnological processes. They are fastidious in their vitamin and amino acid requirements. A new semi-defined agar medium (SDM) was developed which gave consistently high viable cell counts of various G. thermoglucosidasius strains (5×108-6×108cfu/ml) under aerobic conditions at 70°C.

Magnesium-Calcite Crystal Formation Mediated by the Thermophilic Bacterium Geobacillus thermoglucosidasius Requires Calcium and Endospores.

Fresh Geobacillus thermoglucosidasius cells grown on soybean-casein digest nutrient agar were inoculated as a parent colony 1 cm in diameter on the surface of an agar gel containing acetate and calcium ions (calcite-promoting hydrogel) and incubated at 60 °C for 4 days, after which magnesium-calcite single crystals of 50-130 µm in size formed within the parent colony. Addition of EDTA, polyacrylic acid or N,N-dicyclohexylcarbodiimide to the calcite-forming hydrogel inhibited the parent colony from forming magnesium-calcite crystals. Inoculation of G. thermoglucosidasius on calcite-forming hydrogel containing 5 µM cadmium and 20 µM zinc resulted in a decrease in the sporulation rate from 55 to 7-8 %. Magnesium-calcite synthesis decreased relative to the sporulation rate. G. thermoglucosidasius exhibited higher adsorption/absorbance of calcium than other Geobacillus sp. that do not mediate calcite formation and higher levels of magnesium accumulation. Calcium ions contained in the calcite-promoting hydrogel and magnesium ions concentrated in G. thermoglucosidasius cells serve as the elements for magnesium-calcite synthesis. The observed decreases in sporulation rate and magnesium-calcite formation support the hypothesis that endospores act as nuclei for the synthesis of magnesium-calcite single crystals.

Bioprocess exploration for thermostable α-amylase production of a deep-sea thermophile Geobacillus sp. in high-temperature bioreactor.

Geobacillus sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40 U/ml. Then, a high-temperature batch culture of the thermophile in a 15 l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600 rpm and 15 l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79 U/ml and productivity of 19804 U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently.

Complete genome sequence, metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic, fast growing, xylose-utilizing bacterium.

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h(-1) on glucose and 1.52 h(-1) on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio's RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications.

A comparative study of fatty acid profile and formation of biofilm in Geobacillus gargensis exposed to variable abiotic stress.

Understanding bacterial fatty acid (FA) profile has a great taxonomic significance as well as clinical importance for diagnosis issues. Both the composition and nature of membrane FAs change under different nutritional, biotic and (or) abiotic stresses, and environmental stress. Bacteria produce both odd-carbon as well as branched-chain fatty acids (BCFAs). This study was designed to examine the effect of abiotic pressure, including salinity, temperature, pH, and oxinic stress on the growth, development, and FA profile in thermophilic Geobacillus gargensis. Under these stresses, 3 parametric ratios, 2-methyl fatty acids/3-methyl fatty acids (iso-/anteiso-FAs), BCFAs/straight-chain saturated fatty acids (SCSFA), and SCSFAs/straight-chain unsaturated fatty acids (SCUFA), in addition to total lipids affected by variable stresses were measured. Our results indicate that the ratio of total iso-/anteiso-FAs increased at the acidic pH range of 4.1-5.2 and decreased with increasing pH. The reverse was true for salt stress when iso-/anteiso-FAs ratio increased with salt concentration. The BCFAs/SCSFAs and SCSFAs/SCUFAs ratios increased at neutral and alkaline pH and high salt concentration, reduced incubation time, and comparatively high temperature (55-65 °C) of the growth medium. The bacterial total lipid percentage deceased with increasing salt concentration, incubation period, but it increased with temperature. The formation of extracellular polymeric substances was observed under all stress conditions and with the addition of sodium dodecyl sulfate (2 and 5 mmol/L) to the growth medium. The membrane phospholipid composition of the bacterium was analyzed by thin-layer chromatography.

Isobutanol production at elevated temperatures in thermophilic Geobacillus thermoglucosidasius.

The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50°C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3g/l of isobutanol was produced from glucose and 0.6g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48h at 50°C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature.

Extremophiles survival to simulated space conditions: an astrobiology model study.

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet?

The genus Geobacillus comprises endospore-forming obligate thermophiles. These bacteria have been isolated from cool soils and even cold ocean sediments in anomalously high numbers, given that the ambient temperatures are significantly below their minimum requirement for growth. Geobacilli are active in environments such as hot plant composts, however, and examination of their genome sequences reveals that they are endowed with a battery of sensors, transporters and enzymes dedicated to hydrolysing plant polysaccharides. Although they appear to be relatively minor members of the plant biomass-degrading microbial community, Geobacillus bacteria have achieved a significant population with a worldwide distribution, probably in large part due to adaptive features of their spores. First, their morphology and resistance properties enable them to be mobilized in the atmosphere and transported long distances. Second, their longevity, which in theory may be extreme, enables them to lie quiescent but viable for long periods of time, accumulating gradually over time to achieve surprisingly high population densities.

Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius.

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Šresolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

Characterization of enriched aerotolerant cellulose-degrading communities for biofuels production using differing selection pressures and inoculum sources.

Ethanol production from direct cellulose fermentation has mainly been described as a strictly anaerobic process. The use of air-tolerant organisms or consortia for this process would reduce the need for prereduction of the medium and also permit continuous feed of aerobic feedstock. To this end, moderately thermophilic (60 °C) consortia of fermentative, cellulolytic bacteria were enriched from 3 distinct environments (manure, marsh, and rotten wood) from a farm in southeast Saskatchewan, Canada. Community phenotypic and metabolic profiles were characterized. Selection methods included direct plating under an aerobic atmosphere and repeated passaging; the methods were designed to select for robust, stable aerotolerant cellulose-degrading communities. Several of the isolated communities exhibited an increase in total cellulose degradation and total ethanol yield when compared with a monoculture of Clostridium thermocellum DSMZ 1237. Owing to stringent selection conditions, low diversity enrichments were found, and many appeared to be binary cultures via density gradient gel electrophoresis analysis. On the basis of 16S rRNA gene sequencing, aerobic conditions selected for a mix of organisms highly related to C. thermocellum and Geobacillus species, while anaerobic conditions led to the development of consortia containing strains related to C. thermocellum with strains from either the genus Geobacillus or the genus Thermoanaerobacter. The presence of a Geobacillus-like species appeared to be a prerequisite for aerotolerance of the cellulolytic enrichments, a highly desired phenotype in lignocellulosic consolidated bioprocessing.

Enhancing the cellulose-degrading activity of cellulolytic bacteria CTL-6 (Clostridium thermocellum) by co-culture with non-cellulolytic bacteria W2-10 (Geobacillus sp.).

The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of W2-10 resulted in a balanced medium pH, and may provide the required anaerobic environment for CTL-6. Overall, addition of W2-10 was beneficial to CTL-6 growth in the adverse environment of the PCS medium. In co-culture with W2-10, the CTL-6 cellulose degradation efficiency of filter paper and alkaline-treated wheat straw significantly increased up to 72.45 and 37.79 %, respectively. The CMCase activity and biomass of CTL-6 also increased from 0.23 U ml(-1) and 45.1 µg ml(-1) (DNA content) up to 0.47 U ml(-1) and 112.2 µg ml(-1), respectively. In addition, co-culture resulted in accumulation of acetate and propionate up to 4.26 and 2.76 mg ml(-1). This was a respective increase of 2.58 and 4.45 times, in comparison to the monoculture with CTL-6.

Construction and evaluation of an exopolysaccharide-producing engineered bacterial strain by protoplast fusion for microbial enhanced oil recovery.

Enterobacter cloacae strain JD, which produces water-insoluble biopolymers at optimal temperature of 30°C, and a thermophilic Geobacillus strain were used to construct an engineered strain for exopolysaccharide production at high temperatures by protoplast fusion. The obtained fusant strain ZR3 produced exopolysaccharides at up to 45°C with optimal growth temperature at 35°C. The fusant produced exopolysaccharides of approximately 7.5 g/L or more at pH between 7.0 and 9.0. The feasibility of the enhancement of crude oil recovery with the fusant was tested in a sand-packed column at 40°C. The results demonstrated that bioaugmentation of the fusant was promising approach for MEOR. Mass growth of the fusant was confirmed in fermentor tests.

Lipid and fatty acid profile of Geobacillus kaustophilus in response to abiotic stress.

Geobacillus kaustophilus is an important food-borne, spore-forming, thermotolerant bacterium. It has a good potential for biotransformation of steroid hormones, such as progesterone and testosterone. In this study, we report G. kaustophilus membrane lipid modifications in response to temperature shock, salinity, incubation time, and pH. Total lipids significantly increased in response to increasing temperature, incubation time, and salt concentration. However, the bacterium presented a significant decrease in the accumulation of total lipids in response to pH shock. The ratio of branched-chain fatty acids/straight-chain fatty acids decreased significantly under all stress conditions. With an increase in temperature, incubation time, and salt concentration, the ratio of iso-fatty acids/anteiso-fatty acids increased significantly, while this ratio remained unaffected by changes in the pH of the growth medium. Our results suggest a modification occurs in the bacterial membrane structure in response to temperature, salinity, incubation time, and pH shock. The variable abiotic stress resulted in a multiple increase in odd-numbered-carbon and low-melting-point anteiso-branched-chain fatty acids, helping the membrane keep its integrity, fluidity, and function for growth of the bacteria under abiotic stress conditions.

Cd, Cu, Ni, Mn and Zn resistance and bioaccumulation by thermophilic bacteria, Geobacillus toebii subsp. decanicus and Geobacillus thermoleovorans subsp. stromboliensis.

Bioaccumulation and heavy metal resistance of Cd(2+), Cu(2+), Ni(2+), Zn(2+) and Mn(2+) ions by thermophilic Geobacillus toebii subsp. decanicus and Geobacillus thermoleovorans subsp. stromboliensis were investigated. The metal resistance from the most resistant to the most sensitive was found as Mn > Ni > Cu > Zn > Cd for both Geobacillus thermoleovorans subsp. stromboliensis and Geobacillus toebii subsp. decanicus. It was determined that the highest metal bioaccumulation was performed by Geobacillus toebii subsp. decanicus for Zn (36,496 µg/g dry weight cell), and the lowest metal bioaccumulation was performed by Geobacillus toebii subsp. decanicus for Ni (660.3 µg/g dry weight cell). Moreover, the dead cells were found to biosorbe more metal in their membranes compared to the live cells. In the presence of 7.32 mg/l Cd concentration, the levels of Cd absorbed in live and dead cell membranes were found as 17.44 and 46.2 mg/g membrane, respectively.

Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture.

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.