Nanoscale characteristics of conditioning film development on photobioreactor materials: influence on the initial adhesion and biofilm formation by a cyanobacterium.

Adsorption of conditioning films on a solid surface is the first step in the development of biofilms. With the goal of understanding the preliminary adhesion mechanisms of cyanobacteria on photobioreactor (PBR) materials to prevent biofouling, the physical changes occurring on PBR materials were investigated during the initial adhesion and biofilm formation by Anabaena sp. PCC 7120, a cyanobacterium that is genetically modified to produce linalool. Atomic force microscopy (AFM) revealed that the conditioning film deposition was in the form of spike-like structures on all the materials except PVC. The average heights (in the range 9 - 16 nm) of the conditioning films deposited on glass, PMMA, PC and HDPE were 11 to 20 times higher than on PVC at 96 h. The time dependent change in thickness of conditioning films correlated well with Anabaena cell attachment to the PBR materials. The rapid and significant colonization of Anabaena on glass within 48 h was consistent with the increase in thickness of the conditioning film within this time period. Lack of the conditioning film spike structures and no change in thickness of the conditioning films with time on the PVC together with comparatively delayed cell attachment and conditioning-film protein deposition on this material, indicated that the nanoscale spike structures on the other PBR materials may be accelerating the cell attachment process but are not a prerequisite for cell attachment. These results suggest that PVC should be explored further as an antifouling material for photobioreactors. The thickness of the conditioning films on glass measured by a scratch and scan method was in good agreement with the thickness values measured by an adhesive tape method, indicating that both these methods can be used for fast and reliable AFM thickness determination of bacterial conditioning films.

Conditioning film formation and its influence on the initial adhesion and biofilm formation by a cyanobacterium on photobioreactor materials.

Although cyanobacteria are a common group of microorganisms well-suited to utilization in photobioreactors (PBRs), studies of cyanobacteria fouling and its prevention are scarce. Using a cyanobacterium, Anabaena sp. PCC 7120, which had been genetically modified to enhance linalool production, the formation of conditioning films and the effects of these on the physico-chemical surface properties of various PBR materials during initial adhesion and biofilm formation were investigated. The adhesion assay revealed that the overall attachment of Anabaena was substratum dependent and no correlation between the hydrophobicity/roughness of clean material and cell attachment was found. Surface hydrophilicity/hydrophobicity of all the materials changed within 12 h due to formation of conditioning films. ATR-FTIR spectroscopy revealed that the fractional change in protein deposition between 12 to 96 h was consistent with Anabaena cell attachment but polysaccharide deposition was material specific and did not correlate with cell attachment on the PBR materials. Also, the delay in conditioning film proteins on PVC and PTFE indicated that components other than proteins may be responsible for the decrease in contact angles on these surfaces within 12 h. This indicates the important role of the chemical nature of adsorbed conditioning films in determining the initial attachment of Anabaena to PBR materials. The lower rate of attachment of Anabaena on the hydrophilic surfaces (glass and PMMA) between 72 h to 96 h (regime 3) showed that these surfaces could potentially have low fouling characteristics at extended time scales and should be considered for further research.

Classification of bacterial nanowire proteins using Machine Learning and Feature Engineering model.

Nanowires (NW) have been extensively studied for Shewanella spp. and Geobacter spp. and are mostly produced by Type IV pili or multiheme c-type cytochrome. Electron transfer via NW is the most studied mechanism in microbially induced corrosion, with recent interest in application in bioelectronics and biosensor. In this study, a machine learning (ML) based tool was developed to classify NW proteins. A manually curated 999 protein collection was developed as an NW protein dataset. Gene ontology analysis of the dataset revealed microbial NW is part of membranal proteins with metal ion binding motifs and plays a central role in electron transfer activity. Random Forest (RF), support vector machine (SVM), and extreme gradient boost (XGBoost) models were implemented in the prediction model and were observed to identify target proteins based on functional, structural, and physicochemical properties with 89.33%, 95.6%, and 99.99% accuracy. Dipetide amino acid composition, transition, and distribution protein features of NW are key important features aiding in the model's high performance.

Molecular regulation of conditioning film formation and quorum quenching in sulfate reducing bacteria.

Sensing surface topography, an upsurge of signaling biomolecules, and upholding cellular homeostasis are the rate-limiting spatio-temporal events in microbial attachment and biofilm formation. Initially, a set of highly specialized proteins, viz. conditioning protein, directs the irreversible attachment of the microbes. Later signaling molecules, viz. autoinducer, take over the cellular communication phenomenon, resulting in a mature microbial biofilm. The mandatory release of conditioning proteins and autoinducers corroborated the existence of two independent mechanisms operating sequentially for biofilm development. However, both these mechanisms are significantly affected by the availability of the cofactor, e.g., Copper (Cu). Generally, the Cu concentration beyond threshold levels is detrimental to the anaerobes except for a few species of sulfate-reducing bacteria (SRB). Remarkably SRB has developed intricate ways to resist and thrive in the presence of Cu by activating numerous genes responsible for modifying the presence of more toxic Cu(I) to Cu(II) within the periplasm, followed by their export through the outer membrane. Therefore, the determinants of Cu toxicity, sequestration, and transportation are reconnoitered for their contribution towards microbial adaptations and biofilm formation. The mechanistic details revealing Cu as a quorum quencher (QQ) are provided in addition to the three pathways involved in the dissolution of cellular communications. This review articulates the Machine Learning based data curing and data processing for designing novel anti-biofilm peptides and for an in-depth understanding of QQ mechanisms. A pioneering data set has been mined and presented on the functional properties of the QQ homolog in Oleidesulfovibrio alaskensis G20 and residues regulating the multicopper oxidase properties in SRB.

Enhancement of Methane Catalysis Rates in Methylosinus trichosporium OB3b.

Particulate methane monooxygenase (pMMO), a membrane-bound enzyme having three subunits (α, ß, and γ) and copper-containing centers, is found in most of the methanotrophs that selectively catalyze the oxidation of methane into methanol. Active sites in the pMMO of Methylosinus trichosporium OB3b were determined by docking the modeled structure with ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene. The docking energy between the modeled pMMO structure and ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene was -5.2, -5.7, -4.2, and -3.8 kcal/mol, respectively, suggesting the existence of more than one active site within the monomeric subunits due to the presence of multiple binding sites within the pMMO monomer. The evaluation of tunnels and cavities of the active sites and the docking results showed that each active site is specific to the radius of the substrate. To increase the catalysis rates of methane in the pMMO of M. trichosporium OB3b, selected amino acid residues interacting at the binding site of ethylbenzene, toluene, 1,3-dibutadiene, and trichloroethylene were mutated. Based on screening the strain energy, docking energy, and physiochemical properties, five mutants were downselected, B:Leu31Ser, B:Phe96Gly, B:Phe92Thr, B:Trp106Ala, and B:Tyr110Phe, which showed the docking energy of -6.3, -6.7, -6.3, -6.5, and -6.5 kcal/mol, respectively, as compared to the wild type (-5.2 kcal/mol) with ethylbenzene. These results suggest that these five mutants would likely increase methane oxidation rates compared to wild-type pMMO.

Two new exopolysaccharides from a thermophilic bacterium Geobacillus sp. WSUCF1: Characterization and bioactivities.

The production, characterization and bioactivities of exopolysaccharides (EPSs) from a thermophilic bacterium Geobacillus sp. strain WSUCF1 were investigated. Using glucose as a carbon source 525.7 mg/L of exoproduct were produced in a 40-L bioreactor at 60 °C. Two purified EPSs were obtained: EPS-1 was a glucomannan containing mannose and glucose in a molar ratio of 1:0.21, while EPS-2 was composed of mannan only. The molecular weights of both EPSs were estimated to be approximately 1000 kDa, their FTIR and NMR spectra indicated the presence of α-type glycosidic bonds in a linear structure, and XRD analysis indicated a low degree of crystallinity of 0.11 (EPS-1) and 0.27 (EPS-2). EPS-1 and EPS-2 demonstrated high degradation temperatures of 319 °C and 314 °C, respectively, and non-cytotoxicity to HEK-293 cells at 2 and 3 mg/mL, respectively. In addition, both showed antioxidant activities. EPSs from strain WSUCF1 may expand the applications of microorganisms isolated from extreme environments and provide a valuable resource for exploitation in biomedical fields such as drug delivery carriers.

Extremophilic Exopolysaccharides: Biotechnologies and Wastewater Remediation.

Various microorganisms thrive under extreme environments, like hot springs, hydrothermal vents, deep marine ecosystems, hyperacid lakes, acid mine drainage, high UV exposure, and more. To survive against the deleterious effect of these extreme circumstances, they form a network of biofilm where exopolysaccharides (EPSs) comprise a substantial part. The EPSs are often polyanionic due to different functional groups in their structural backbone, including uronic acids, sulfated units, and phosphate groups. Altogether, these chemical groups provide EPSs with a negative charge allowing them to (a) act as ligands toward dissolved cations as well as trace, and toxic metals; (b) be tolerant to the presence of salts, surfactants, and alpha-hydroxyl acids; and (c) interface the solubilization of hydrocarbons. Owing to their unique structural and functional characteristics, EPSs are anticipated to be utilized industrially to remediation of metals, crude oil, and hydrocarbons from contaminated wastewaters, mines, and oil spills. The biotechnological advantages of extremophilic EPSs are more diverse than traditional biopolymers. The present review aims at discussing the mechanisms and strategies for using EPSs from extremophiles in industries and environment bioremediation. Additionally, the potential of EPSs as fascinating biomaterials to mediate biogenic nanoparticles synthesis and treat multicomponent water contaminants is discussed.

Metagenomics and Culture Dependent Insights into the Distribution of Firmicutes across Two Different Sample Types Located in the Black Hills Region of South Dakota, USA.

Firmicutes is almost a ubiquitous phylum. Several genera of this group, for instance, Geobacillus, are recognized for decomposing plant organic matter and for producing thermostable ligninolytic enzymes. Amplicon sequencing was used in this study to determine the prevalence and genetic diversity of the Firmicutes in two distinctly related environmental samples-South Dakota Landfill Compost (SDLC, 60 °C), and Sanford Underground Research Facility sediments (SURF, 45 °C). Although distinct microbial community compositions were observed, there was a dominance of Firmicutes in both the SDLC and SURF samples, followed by Proteobacteria. The abundant classes of bacteria in the SDLC site, within the phylum Firmicutes, were Bacilli (83.2%), and Clostridia (2.9%). In comparison, the sample from the SURF mine was dominated by the Clostridia (45.8%) and then Bacilli (20.1%). Within the class Bacilli, the SDLC sample had more diversity (a total of 11 genera with more than 1% operational taxonomic unit, OTU). On the other hand, SURF samples had just three genera, about 1% of the total population: Bacilli, Paenibacillus, and Solibacillus. With specific regard to Geobacillus, it was found to be present at a level of 0.07% and 2.5% in SURF and SDLC, respectively. Subsequently, culture isolations of endospore-forming Firmicutes members from these samples led to the isolation of a total of 117 isolates. According to colony morphologies, and identification based upon 16S rRNA and gyrB gene sequence analysis, we obtained 58 taxonomically distinct strains. Depending on the similarity indexes, a gyrB sequence comparison appeared more useful than 16S rRNA sequence analysis for inferring intra- and some intergeneric relationships between the isolates.

Bioelectrochemical approach for enhancing lignocellulose degradation and biofilm formation in Geobacillus strain WSUCF1.

Investigations on microbial electrocatalysis as a strategy for enhancing the rates of substrate utilization leading to enhanced yield of biomass and enhanced biofilm formation are reported. A thermophilic Geobacillus sp. strain WSUCF1 (60 °C), a potential lignocellulose degrading microorganism was used as the electrocatalyst. Glucose, cellulose, and corn stover were used as the feedstocks. The results of this investigation showed that applying the oxidation potential of -0.383 mV (vs PRE) increased the glucose utilization and COD removal by 25.5% and 29.7% respectively. The bioelectrocatalysis strategy also increased the biomass yield by 81.2, 42.1, and 49.5% in the case of systems fed with glucose, cellulose, and corn stover, respectively, when compared with the systems without applied oxidation potential. This is the first work reporting the effects of applied oxidation potential on increasing the rates of degradation of lignocellulosic biomass and enhanced biofilm formation.

Adaptive Enrichment of a Thermophilic Bacterial Isolate for Enhanced Enzymatic Activity.

The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain.

Extremophilic exopolysaccharides: A review and new perspectives on engineering strategies and applications.

Numerous microorganisms inhabiting harsh niches produce exopolysaccharides as a significant strategy to survive in extreme conditions. The exopolysaccharides synthesized by extremophiles possess distinctive characteristics due to the varied harsh environments which stimulate the microorganisms to produce these biopolymers. Despite many bioprocesses have been designed to yield exopolysaccharides, the production of exopolysaccharides by extremophiles is inefficient compared with mesophilic and neutrophilic exopolysaccharide producers. Meanwhile, the industrial development of novel extremophilic exopolysaccharides remains constrained due to the lack of exploration. In this review, we summarize the structure and properties of various exopolysaccharides produced by extremophiles, and also discuss potential metabolic and genetic engineering strategies for enhanced yield and modified structure of extremophilic exopolysaccharides. Special focus is given to the applications of extremophilic exopolysaccharides in the areas of biomedicine, food industry, and biomaterials via nano-techniques, casting and electrospinning.

Genome analysis of a thermophilic exopolysaccharide-producing bacterium - Geobacillus sp. WSUCF1.

Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.

Thermophiles for biohydrogen production in microbial electrolytic cells.

Thermophiles are promising options to use as electrocatalysts for bioelectrochemical applications including microbial electrolysis. They possess several interesting characteristics such as ability to catalyze a broad range of substrates at better rates and over a broad range of operating conditions, and better electrocatalysis/electrogenic activity over mesophiles. However, a very limited number of investigations have been carried out to explore the microbial reactions/pathways and the molecular mechanisms that contribute to better electrocatalysis/electrolysis in thermophiles. Here, we review the electroactive characteristics of thermophiles, their electron transfer mechanisms, and molecular insights behind the choice of thermophiles for bioelectrochemical/electrolytic processes.

"MINES" method for genomic DNA extraction from deep biosphere biofilms.

Successful and efficient extraction of high quality, high molecular weight genomic DNA from the environmental samples is an essential primary step to understand the genetic, metabolic and evolutionary characteristics of the microbial communities. Deep mine biofilm samples that contain high amounts of mucoid exopolysaccharide often pose difficulties to obtaining refined community DNA. To circumvent this hindrance, we report our "MINES" method which we developed for optimal biofilm DNA recovery suitable for all types of high-resolution downstream applications. The method is also suitable for samples collected from landfill compost, kitchen digest (KD), and for Gram-positive Geobacillus sp. strain WSUCF1 and Gram-negative E. coli DH5α strains. In one form of the method, use of a gentle preprocessing technique to loosen the mucoid layer, combined with a multi-lytic polyzyme treatment to maximize yields from all cell types in the biofilm sample, yielded >1 µg of high molecular weight DNA (16-20 kb) per gram of the biofilm sample, with an A260/280 and A260/230 ratio of about 2. Furthermore, amplification of 16S rRNA genes as well as restriction digestion with BamHI and HindIII suggest that the newly developed method can minimize any inhibitory effects of contaminants. Results indicate that it is an appropriate methodology for the extraction of total genomic DNA for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging. IMPORTANCE: Our present knowledge of microorganisms and their enzymes from deep mine subsurfaces is based largely on laboratory studies of pure microbial cultures. These methods tend only to hit nearly 1% of the entire microbial community. In this regard, metagenomics, has emerged as a strategic approach to explore unculturable microbes through the sequencing and analysis of DNA extracted from the environmental samples. This research paper discusses our "MINES" method for genomic DNA extraction from deep biosphere biofilm samples.

Rewiring the microbe-electrode interfaces with biologically reduced graphene oxide for improved bioelectrocatalysis.

The aim of this work was to study biologically reduced graphene oxide (RGO) for engineering the surface architecture of the bioelectrodes to improve the performance of Bioelectrochemical System (BES). Gluconobacter roseus mediates the reduction of graphene oxide (GO). The RGO modified bioelectrodes produced a current density of 1 mA/cm2 and 0.69 mA/cm2 with ethanol and glucose as substrates, respectively. The current density of RGO modified electrodes was nearly 10-times higher than the controls. This study, for the first time, reports a new strategy to improve the yield as well as efficiency of the BES by wrapping and wiring the electroactive microorganisms to the electrode surfaces using RGO. This innovative wrapping approach will decrease the loss of electrons in the microbe-electrolyte interfaces as well as increase the electron transfer rates at the microorganism-electrode interfaces.

Single pot bioconversion of prairie cordgrass into biohydrogen by thermophiles.

The aim of the present work was to use a thermophilic consortium for H2 production using lignocellulosic biomass in a single pot. The thermophilic consortium, growing at 60 °C utilized both glucose and xylose, making it an ideal source of microbes capable of utilizing and fermenting both hexose and pentose sugars. The optimization of pH, temperature, and substrate concentration increased the H2 production from 1.07 mmol H2/g of prairie cordgrass (PCG) to 2.2 mmol H2/g PCG by using the thermophilic consortium. A sequential cultivation of a thermostable lignocellulolytic enzyme producing strain Geobacillus sp. strain WSUCF1 (aerobic) with the thermophilic consortium (anaerobic) further increased H2 production with PCG 3-fold (3.74 mmol H2/g PCG). A single pot sequential culturing of aerobic and anaerobic microbes can be sustainable and advantageous for industrial scale production of biofuels.

Biohydrogen production from space crew's waste simulants using thermophilic consolidated bioprocessing.

Human waste simulants were for the first time converted into biohydrogen by a newly developed anaerobic microbial consortium via thermophilic consolidated bioprocessing. Four different BioH2-producing consortia (denoted as C1, C2, C3 and C4) were isolated, and developed using human waste simulants as substrate. The thermophilic consortium C3, which contained Thermoanaerobacterium, Caloribacterium, and Caldanaerobius species as the main constituents, showed the highest BioH2 production (3.999 mmol/g) from human waste simulants under optimized conditions (pH 7.0 and 60 °C). The consortium C3 also produced significant amounts of BioH2 (5.732 mmol/g and 2.186 mmol/g) using wastewater and activated sludge, respectively. The developed consortium in this study is a promising candidate for H2 production in space applications as in situ resource utilization.

Impact-induced solidlike behavior and elasticity in concentrated colloidal suspensions.

Modified drop weight impact tests were performed on SiO_{2}-ethylene glycol concentrated suspensions. Counterintuitive impact-induced solidlike behavior and elasticity, causing significant deceleration and rebound of the impactor, were observed. We provide evidence that the observed large deceleration force on the impactor mainly originates from the hydrodynamic force, and that the elasticity arises from the short-range repulsive force of a solvation layer on the particle surface. This study presents key experimental results to help understand the mechanisms underlying various stress-induced solidification phenomena.