High production of acetoin from glycerol by Bacillus subtilis 35.

Acetoin is a high-value volatile compound widely applied in the chemical, food, and pharmaceutical industries. Despite the promising use of waste glycerol as a substrate in several microbial syntheses, acetoin production by natural microorganisms from glycerol as a sole carbon source has never been reported. The present study investigates the innate ability of Bacillus subtilis 35 (DSM 113,620) to convert glycerol into acetoin and 2,3-butanediol. The fermentation was directed towards acetoin production by medium selection and process parameter optimization using response surface design methodology. Thus, the fed batch conducted under optimized conditions received 77.9 g/L acetoin with a productivity of 0.85 g/L h and a yield of 0.36 g/g. The obtained acetoin concentration is the highest from glycerol reported to date, comparable to the highest values gained from glucose. Transcription analysis of the gene cluster glpPFKD showed that all four genes responsible for the utilization of glycerol were expressed. This natural ability of the strain, along with its non-pathogenic nature, defines B. subtilis 35 as a very promising candidate for acetoin production from glycerol on an industrial scale. KEY POINTS: • The highest microbial production of acetoin from glycerol. • Process parameter optimization directs glycerol conversion to acetoin production. • B. subtilis 35 is promising for industrial acetoin production from glycerol.

Multiple cry Genes in Bacillus thuringiensis Strain BTG Suggest a Broad-Spectrum Insecticidal Activity.

The properties of Bacillus thuringiensis strains as a biopesticide with potent action against moths, beetles, and mosquitoes have been known for decades, with individual subspecies showing specific activity against a particular pest. The aim of the present work is to characterize strains that can be used for broad-spectrum pest control in agriculture. Twenty strains of B. thuringiensis were isolated from Bulgarian soil habitats. The strains were screened for genes encoding 12 different crystal (Cry) endotoxins by PCR with specific primer pairs. Seven of the isolates contained cry genes in their genomes. B. thuringiensis strains PL1, PL3, and PL20 contained at least three different cry genes, while B. thuringiensis serovar galleriae BTG contained at least four. Moreover, scanning electron microscopy (SEM) investigation revealed the production of bipyramidal (PL1, PL3, PL20), polygonal (PL1), cubic (BTG), and spherical crystals (BTG and PL20). Potentially containing the most cry genes, the BTG genome was sequenced and annotated. It comprises 6,275,416 base pairs, does not contain plasmids, has a GC content of 35.05%, and contained 7 genes encoding crystal toxins: cry1Ab35, cry1Db, cry1Fb, cry1Ib, cry2Ab, cry8Ea1, and cry9Ba. This unique combination would possibly enable the simultaneous pesticidal action against pest species from orders Lepidoptera, Coleoptera, Diptera, and Hemiptera, as well as class Gastropoda. Whole-genome sequencing provided accurate information about the presence, localization, and classification of Cry toxins in B. thuringiensis BTG, revealing the great potential of the strain for the development of new broad-spectrum bio-insecticides.

Influence of pH on Inulin Conversion to 2,3-Butanediol by Bacillus licheniformis 24: A Gene Expression Assay.

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA, sacB, sacC, levB, and fruA. Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.

In Vitro Production of Galactooligosaccharides by a Novel ß-Galactosidase of Lactobacillus bulgaricus.

ß-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of ß-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the ß-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other ß-galactosidases of this species. The molecular model revealed that the new ß-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The ß-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme's temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, ß-Gal produced a large amount of GOS with DP3 containing ß-(1→3) and ß-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of ß-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

Archaeal and Bacterial Content in a Two-Stage Anaerobic System for Efficient Energy Production from Agricultural Wastes.

Anaerobic digestion (AD) is a microbially-driven process enabling energy production. Microorganisms are the core of anaerobic digesters and play an important role in the succession of hydrolysis, acidogenesis, acetogenesis, and methanogenesis processes. The diversity of participating microbial communities can provide new information on digester performance for biomass valorization and biofuel production. In this study anaerobic systems were used, operating under mesophilic conditions that realized biodegradation processes of waste wheat straw pretreated with NaOH-a renewable source for hydrogen and methane production. These processes could be managed and optimized for hydrogen and methane separately but combining them in a two-stage system can lead to higher yields and a positive energy balance. The aim of the study was to depict a process of biohydrogen production from lignocellulosic waste followed by a second one leading to the production of biomethane. Archaeal and bacterial consortia in a two-stage system operating with wheat straw were identified for the first time and the role of the most important representatives was elucidated. The mixed cultures were identified by the molecular-biological methods of metagenomics. The results showed that biohydrogen generation is most probably due to the presence of Proteiniphilum saccharofermentans, which was 28.2% to 45.4% of the microbial community in the first and the second bioreactor, respectively. Archaeal representatives belonging to Methanobacterium formicicum (0.71% of the community), Methanosarcina spelaei (0.03%), Methanothrix soehngenii (0.012%), and Methanobacterium beijingense (0.01%) were proven in the methane-generating reactor. The correlation between substrate degradation and biogas accumulation was calculated, together with the profile of fatty acids as intermediates produced during the processes. The hydrogen concentration in the biogas reached 14.43%, and the Methane concentration was 69%. Calculations of the energy yield during the two-stage process showed 1195.89 kWh·t-1 compared to a 361.62 kWh·t-1 cumulative yield of energy carrier for a one-stage process.

Safe Sialidase Production by the Saprophyte Oerskovia paurometabola: Gene Sequence and Enzyme Purification.

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed ß-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.

Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis.

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.

High lactic acid and fructose production via Mn2+-mediated conversion of inulin by Lactobacillus paracasei.

Lactobacillus paracasei DSM 23505 is able to produce high amounts of lactic acid (LA) by simultaneous saccharification and fermentation (SSF) of inulin. Aiming to obtain the highest possible amounts of LA and fructose, the present study is devoted to evaluate the impact of bivalent metal ions on the process of inulin conversion. It was shown that Mn2+ strongly increases the activity of the purified key enzyme ß-fructosidase. In vivo, batch fermentation kinetics revealed that the high Mn2+ concentrations accelerated inulin hydrolysis by raise of the inulinase activity, and increased sugars conversion to LA through enhancement of the whole glycolytic flux. The highest LA concentration and yield were reached by addition of 15 mM Mn2+-151 g/L (corresponding to 40% increase) and 0.83 g/g, respectively. However, the relative quantification by real-time reverse transcription assay showed that the presence of Mn2+ decreases the expression levels of fosE gene encoding ß-fructosidase. Contrariwise, the full exclusion of metal ions resulted in fosE gene expression enhancement, blocked fructose transport, and hindered fructose conversion thus leading to huge fructose accumulation. During fed-batch with optimized medium and fermentation parameters, the fructose content reached 35.9% (w/v), achieving yield of 467 g fructose from 675 g inulin containing chicory flour powder (0.69 g/g). LA received in course of the batch fermentation and fructose gained by the fed-batch are the highest amounts ever obtained from inulin, thus disclosing the key role of Mn2+ as a powerful tool to guide inulin conversion to targeted bio-chemicals.

2,3-butanediol production from starch by engineered Klebsiella pneumoniae G31-A.

2,3-Butanediol (2,3-BD) is an organic compound, which is widely used as a fuel and fuel additive and applied in chemical, food, and pharmaceutical industries. Contemporary strategies for its economic synthesis include the development of microbial technologies that use starch as cheap and renewable feedstock. The present work encompasses the metabolic engineering of the excellent 2,3-BD producer Klebsiella pneumoniae G31. In order to perform direct starch conversion into 2,3-BD, the amyL gene encoding quite active, liquefying α-amylase in Bacillus licheniformis was cloned under lac promoter control in the recombinant K. pneumoniae G31-A. The enhanced extracellular over-expression of amyL led to the highest extracellular amylase activity (68 U/ml) ever detected in Klebsiella. The recombinant strain was capable of simultaneous saccharification and fermentation (SSF) of potato starch to 2,3-BD. In SSF batch process by the use of 200 g/l starch, the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3-BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversion rate. The presented results are the first to show successful starch conversion to 2,3-BD by K. pneumoniae in a one-step process.

Glucose Catabolite Repression Participates in the Regulation of Sialidase Biosynthesis by Antarctic Strain Penicillium griseofulvum P29.

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.

Microbial Detoxification of Residual Pesticides in Fermented Foods: Current Status and Prospects.

The treatment of agricultural areas with pesticides is an indispensable approach to improve crop yields and cannot be avoided in the coming decades. At the same time, significant amounts of pesticides remain in food and their ingestion causes serious damage such as neurological, gastrointestinal, and allergic reactions; cancer; and even death. However, during the fermentation processing of foods, residual amounts of pesticides are significantly reduced thanks to enzymatic degradation by the starter and accompanying microflora. This review concentrates on foods with the highest levels of pesticide residues, such as milk, yogurt, fermented vegetables (pickles, kimchi, and olives), fruit juices, grains, sourdough, and wines. The focus is on the molecular mechanisms of pesticide degradation due to the presence of specific microbial species. They contain a unique genetic pool that confers an appropriate enzymological profile to act as pesticide detoxifiers. The prospects of developing more effective biodetoxification strategies by engaging probiotic lactic acid bacteria are also discussed.

Novel sialidase from non-pathogenic bacterium Oerskovia paurometabola strain O129.

Bacterial sialidases are enzymes that are involved in a number of vital processes in microorganisms and in their interaction with the host or the environment. Their wide application for scientific and applied purposes requires the search for highly effective and non-pathogenic producers. Here, we report the first description of sialidase from Oerskovia paurometabola. The extracellular enzyme preparation was partially purified. The presence of sialidase was confirmed in native PAGE treated with the fluorogenic substrate 4MU-Neu5Ac. Maximum enzyme activity was registered at 37 °C and in the pH range of 4.0-5.5. The influence of metal ions and EDTA was examined. It was demonstrated that EDTA, Mn2+ and Ba2+ ions inhibit the sialidase activity to different extent, while Cd2+, Fe2+ and Fe3+ have stimulating effect on it. These features are studied for the first time concerning sialidase of Oerskovia representative. Cell bound sialidase and sialate aldolase were also established.

Whole-genome sequence of Bacillus velezensis strain R22 isolated from Oryza sativa rhizosphere in Bulgaria.

Bacillus velezensis R22 was isolated from a rice rhizosphere in Bulgaria. Its genome (assembled into 14 scaffolds) has a size of 4.08 Mbp and a G + C content of 46.35%. Nine full biosynthetic clusters for antimicrobials were predicted, among them two new gene clusters probably encoding polyketides named macrolactin R22 and velezensin.

Multifunctional Nutraceutical Composition Based on Fermented Spirulina, Apple Cider Vinegar, Jerusalem Artichoke, and Bovine Colostrum.

The main purpose of this experiment was to develop a multifunctional nutraceutical composition based on ingredients of different origins (Spirulina powder (SP), bovine colostrum (BC), Jerusalem artichoke powder (JAP), and apple cider vinegar (ACV)) which possess different health benefits through their different mechanisms of action. In order to improve the functional properties of Spirulina and bovine colostrum, fermentation with the Pediococcus acidilactici No. 29 and Lacticaseibacillus paracasei LUHS244 strains, respectively, was carried out. These LAB strains were chosen due to their good antimicrobial properties. The following parameters were analysed: for Spirulina (non-treated and fermented)-pH, colour coordinates, fatty acid profile, and contents of L-glutamic and GABA acids; for bovine colostrum (non-treated and fermented)-pH, colour coordinates, dry matter, and microbiological parameters (total LAB, total bacteria, total enterobacteria, Escherichia coli, and mould/yeast counts); for the produced nutraceuticals-hardness, colour coordinates, and overall acceptability. It was established that fermentation reduced the pH of the SP and BC and affected their colour coordinates. Fermented SP contained a greater concentration of gamma-aminobutyric and L-glutamic acids (by 5.2 times and 31.4% more, respectively), compared to the non-treated SP and BC. In addition, the presence of gamma-linolenic and omega-3 fatty acids was observed in fermented SP. Fermentation of BC reduces Escherichia coli, total bacteria, total enterobacteria, and total mould/yeast counts in samples. The obtained three-layer nutraceutical (I layer-fermented SP; II-fermented BC and JAP; III-ACV) demonstrated a high overall acceptability. Finally, our finding suggest that the selected nutraceutical combination has immense potential in the production of a multifunctional product with improved functionality and a high acceptability.

Biogas Production Potential of Thermophilic Anaerobic Biodegradation of Organic Waste by a Microbial Consortium Identified with Metagenomics.

Anaerobic digestion (AD) is a widespread biological process treating organic waste for green energy production. In this study, wheat straw and corn stalks without any harsh preliminary treatment were collected as a renewable source to be employed in a laboratory-scale digester to produce biogas/biomethane. Processes parameters of temperature, pH, total solids, volatile solid, concentration of volatile fatty acids (VFA), and cellulose concentration, were followed. The volume of biogas produced was measured. The impact of organic loading was stated, showing that the process at 55 °C tolerated a higher substrate load, up to 45 g/L. Further substrate increase did not lead to biogas accumulation increase, probably due to inhibition or mass transfer limitations. After a 12-day anaerobic digestion process, cumulative volumes of biogas yields were 4.78 L for 1 L of the bioreactor working volume with substrate loading 30 g/L of wheat straw, 7.39 L for 40 g/L and 8.22 L for 45 g/L. The degree of biodegradation was calculated to be 68.9%, 74% and 72%, respectively. A fast, effective process for biogas production was developed from native wheat straw, with the highest quantity of daily biogas production occurring between day 2 and day 5. Biomethane concentration in the biogas was 60%. An analysis of bacterial diversity by metagenomics revealed that more than one third of bacteria belonged to class Clostridia (32.9%), followed by Bacteroidia (21.5%), Betaproteobacteria (11.2%), Gammaproteobacteria (6.1%), and Alphaproteobacteria (5%). The most prominent genera among them were Proteiniphilum, Proteiniborus, and Pseudomonas. Archaeal share was 1.37% of the microflora in the thermophilic bioreactor, as the genera Methanocorpusculum, Methanobacterium, Methanomassiliicoccus, Methanoculleus, and Methanosarcina were the most abundant. A knowledge of the microbiome residing in the anaerobic digester can be further used for the development of more effective processes in conjunction with theidentified consortium.

The Complex Role of Lactic Acid Bacteria in Food Detoxification.

Toxic ingredients in food can lead to serious food-related diseases. Such compounds are bacterial toxins (Shiga-toxin, listeriolysin, Botulinum toxin), mycotoxins (aflatoxin, ochratoxin, zearalenone, fumonisin), pesticides of different classes (organochlorine, organophosphate, synthetic pyrethroids), heavy metals, and natural antinutrients such as phytates, oxalates, and cyanide-generating glycosides. The generally regarded safe (GRAS) status and long history of lactic acid bacteria (LAB) as essential ingredients of fermented foods and probiotics make them a major biological tool against a great variety of food-related toxins. This state-of-the-art review aims to summarize and discuss the data revealing the involvement of LAB in the detoxification of foods from hazardous agents of microbial and chemical nature. It is focused on the specific properties that allow LAB to counteract toxins and destroy them, as well as on the mechanisms of microbial antagonism toward toxigenic producers. Toxins of microbial origin are either adsorbed or degraded, toxic chemicals are hydrolyzed and then used as a carbon source, while heavy metals are bound and accumulated. Based on these comprehensive data, the prospects for developing new combinations of probiotic starters for food detoxification are considered.

Curcumin and Capsaicin-Loaded Ag-Modified Mesoporous Silica Carriers: A New Alternative in Skin Treatment.

Biologically active substances of natural origin offer a promising alternative in skin disease treatment in comparison to synthetic medications. The limiting factors for the efficient application of natural compounds, such as low water solubility and low bioavailability, can be easily overcome by the development of suitable delivery systems. In this study, the exchange with the template procedure was used for the preparation ofa spherical silver-modified mesoporous silica nanocarrier. The initial and drug-loaded formulations are fully characterized by different physico-chemical methods. The incipient wetness impregnation method used to load health-promoting agents, curcumin, and capsaicin in Ag-modified carriers separately or in combinationresulted in high loading efficiency (up to 33 wt.%). The interaction between drugs and carriers was studied by ATR-FTIR spectroscopy. The release experiments of both active substances from the developed formulations were studied in buffers with pH 5.5, and showed improved solubility. Radical scavenging activity and ferric-reducing antioxidant power assays were successfully used for the evaluation of the antiradical and antioxidant capacity of the curcumin or/and capsaicin loaded on mesoporous carriers. Formulations containing a mixture of curcumin and capsaicin were characterized bypotentiation of their antiproliferative effect against maligning cells, and it was confirmed that the system for simultaneous delivery of both drugs has lower IC50 values than the free substances.The antibacterial tests showed better activity of the obtained delivery systems in comparison with the pure curcumin and capsaicin. Considering the obtained results, it can be concluded that the obtained delivery systems are promising for potential dermal treatment.

How to outwit nature: Omics insight into butanol tolerance.

The energy crisis, depletion of oil reserves, and global climate changes are pressing problems of developed societies. One possibility to counteract that is microbial production of butanol, a promising new fuel and alternative to many petrochemical reagents. However, the high butanol toxicity to all known microbial species is the main obstacle to its industrial implementation. The present state of the art review aims to expound the recent advances in modern omics approaches to resolving this insurmountable to date problem of low butanol tolerance. Genomics, transcriptomics, and proteomics show that butanol tolerance is a complex phenomenon affecting multiple genes and their expression. Efflux pumps, stress and multidrug response, membrane transport, and redox-related genes are indicated as being most important during butanol challenge, in addition to fine-tuning of global regulators of transcription (Spo0A, GntR), which may further improve tolerance. Lipidomics shows that the alterations in membrane composition (saturated lipids and plasmalogen increase) are very much species-specific and butanol-related. Glycomics discloses the pleiotropic effect of CcpA, the role of alternative sugar transport, and the production of exopolysaccharides as alternative routes to overcoming butanol stress. Unfortunately, the strain that simultaneously syntheses and tolerates butanol in concentrations that allow its commercialization has not yet been discovered or produced. Omics insight will allow the purposeful increase of butanol tolerance in natural and engineered producers and the effective heterologous expression of synthetic butanol pathways in strains hereditary butanol-resistant up to 3.2 - 4.9% (w/v). Future breakthrough can be achieved by a detailed study of the membrane proteome, of which 21% are proteins with unknown functions.

Butanol Tolerance of Lactiplantibacillus plantarum: A Transcriptome Study.

Biobutanol is a promising alternative fuel with impaired microbial production thanks to its toxicity. Lactiplantibacillus plantarum (L. plantarum) is among the few bacterial species that can naturally tolerate 3% (v/v) butanol. This study aims to identify the genetic factors involved in the butanol stress response of L. plantarum by comparing the differential gene expression in two strains with very different butanol tolerance: the highly resistant Ym1, and the relatively sensitive 8-1. During butanol stress, a total of 319 differentially expressed genes (DEGs) were found in Ym1, and 516 in 8-1. Fifty genes were upregulated and 54 were downregulated in both strains, revealing the common species-specific effects of butanol stress: upregulation of multidrug efflux transporters (SMR, MSF), toxin-antitoxin system, transcriptional regulators (TetR/AcrR, Crp/Fnr, and DeoR/GlpR), Hsp20, and genes involved in polysaccharide biosynthesis. Strong inhibition of the pyrimidine biosynthesis occurred in both strains. However, the strains differed greatly in DEGs responsible for the membrane transport, tryptophan synthesis, glycerol metabolism, tRNAs, and some important transcriptional regulators (Spx, LacI). Uniquely upregulated in the butanol-resistant strain Ym1 were the genes encoding GntR, GroEL, GroES, and foldase PrsA. The phosphoenolpyruvate flux and the phosphotransferase system (PTS) also appear to be major factors in butanol tolerance.

New Exopolysaccharides Produced by Bacillus licheniformis 24 Display Substrate-Dependent Content and Antioxidant Activity.

Bacillus licheniformis is a soil bacterium with many industrial applications. In addition to enzymes, platform chemicals, antibiotics and phytohormones, the species produces exopolysaccharides (EPSs) of various biological activities. This study revealed that Bulgarian isolate B. licheniformis 24 produced EPSs consisting of galactose, glucose and mannose with substrate-dependent ratio. From glucose, B. licheniformis 24 secreted EPS1, consisting of 54% galactose, 39% glucose and 7% mannose. From fructose, the strain formed EPS2, containing 51% glucose, 30% mannose and 19% galactose. Batch cultivation in flasks yielded 2.2-2.6 g/L EPS1 and 1.90-2.11 g/L EPS2. Four to five times higher yields of EPS were obtained from both substrates during batch and fed-batch processes in a fermenter at 37.8 °C, pH 6.2 and aeration 3.68 vvm. The batch process with 200 g/L of starting substrates received 9.64 g/L EPS1 and 6.29 g/L EPS2, reaching maximum values at the 33rd and 24th h, respectively. Fed-batch fermentation resulted in the highest yields, 12.61 g/L EPS1 and 7.03 g/L EPS2. In all processes, EPSs were produced only in the exponential growth phase. Both EPSs exhibited antioxidant activity, but EPS2 was much more potent in this regard, reaching 811 µM Vitamin C Equivalent Antioxidant Capacity (versus 135 µM for EPS1). EPS1 displayed antibacterial activity against a non-O1 strain of Vibrio cholerae.