Enzymes and pathways in microbial production of 2,3-butanediol and 3-acetoin isomers.

2,3-Butanediol (BD) and acetoin (AC) are products of the non-oxidative metabolism of microorganisms, presenting industrial importance due to their wide range of applications and high market value. Their optical isomers have particular applications, justifying the efforts on the selective bioproduction. Each microorganism produces different isomer mixtures, as a consequence of having different butanediol dehydrogenase (BDH) enzymes. However, the whole scene of the isomer bioproduction, considering the several enzymes and conditions, has not been completely elucidated. Here we show the BDH classification as R, S or meso by bioinformatics analysis uncovering the details of the isomers production. The BDH was compared to diacetyl reductases (DAR) and the new enoyl reductases (ER). We observed that R-BDH is the most singular BDH, while meso and S-BDHs are similar and may be better distinguished through their stereo-selective triad. DAR and ER showed distinct stereo-triads from those described for BDHs, agreeing with kinetic data from the literature and our phylogenetic analysis. The ER family probably has meso-BDH like activity as already demonstrated for a single sequence from this group. These results are of great relevance, as they organize BD producing enzymes, to our known, never shown before in the literature. This review also brings attention to nontraditional enzymes/pathways that can be involved with BD/AC synthesis, as well as oxygen conditions that may lead to the differential production of their isomers. Together, this information can provide helpful orientation for future studies in the field of BD/AC biological production, thus contributing to achieve their production on an industrial scale.

Comparative performance and reusability studies of lipases on syntheses of octyl esters with an economic approach.

A suitable immobilized lipase for esters syntheses should be selected considering not only its cost. We evaluated five biocatalysts in syntheses of octyl caprylate, octyl caprate, and octyl laurate, in which conversions higher than 90% were achieved. Novozym®ï»¿ 435 and non-commercial preparations (including a dry fermented solid) were selected for short-term octyl laurate syntheses using different biocatalysts loadings. By increasing the biocatalyst's loading the lipase's reusability also raised, but without strict proportionality, which resulted in a convergence between the lowest biocatalyst loading and the lowest cost per batch. The use of a dry fermented solid was cost-effective, even using loadings as high as 20.0% wt/wt due to its low obtaining cost, although exhibiting low productiveness. The combination of biocatalyst's cost, esterification activity, stability, and reusability represents proper criteria for the choice. This kind of assessment may help to establish quantitative goals to improve or to develop new biocatalysts.

Influence of different PEG/salt aqueous two-phase system on the extraction of 2,3-butanediol.

The production of 2,3-butanediol (2,3-BDO), a dialcohol of great interest for the food, chemical, and pharmaceutical industry, through the fermentation of biomass, is a sustainable process strategic position for countries with abundant biomass generated by the agribusiness. However, the downstream process of 2,3-BDO is onerous due to the complexity of fermentation broth and the physical-chemical characteristics of the 2,3-BDO. This study investigated the feasibility of 2,3-BDO extraction from model aqueous solutions using aqueous two-phase systems (ATPS). A central composite rotational design (CCRD) was employed to evaluate different ATPS compositions and the influences on the 2,3-BDO recovery and partition coefficient. The polyethylene glycol (PEG) and different concentrations of sodium citrate, ammonium sulfate, and potassium phosphate were investigated. The concentration of salt and PEG in the ATPS was identified as the most significant factors influencing the recovery and partition coefficient of 2,3-BDO. The recovery of 2,3-BDO reached 94.5% and was obtained when the system was composed of 36.22% (w/w) of PEG 4000 and 4.47% (w/w) of potassium phosphate. The results indicate that ATPS based on PEG-salt has a high potential for industrial application, using mild conditions and a simple process for recovering and purifying the 2,3-BDO produced from microbiological synthesis.

Growth of Methylobacterium organophilum in Methanol for the Simultaneous Production of Single-Cell Protein and Metabolites of Interest.

Research background: This study aims to monitor the growth of the methylotrophic bacteria Methylobacterium organophilum in a culture medium with methanol as a carbon source and to verify the production of unicellular proteins and other biomolecules, such as carotenoids, exopolysaccharides and polyhydroxyalkanoates, making them more attractive as animal feed. Experimental approach: Bacterial growth was studied in shake flasks using different carbon/nitrogen (C:N) ratios to determine their best ratio for achieving the highest volumetric productivity of cells and substrate consumption rate. This optimal parameter was further used in a fed-batch operating bioreactor system to define the kinetic profile of cell growth. Methanol consumption was measured by HPLC analysis and the extracted pigments were analyzed by liquid chromatography/mass spectrometry. Chemical composition and rheological properties of the produced exopolysaccharides were also determined. Results and conclusions: The best experimental parameters were verified using an initial methanol concentration of 7 g/L in the culture medium. The same initial substrate concentration was used in the fed-batch operation and after 60 h of cultivation 5 g/L of biomass were obtained. The accumulation of carotenoids associated with cell growth was monitored, reaching a concentration of 1.6 mg/L at the end of the process. These pigments were then analyzed and characterized as a set of xanthophylls (oxidized carotenoids). In addition, two other product types were identified during the fed-batch operation: exopolysaccharides, which reached a concentration of 8.9 g/L at the end of the cultivation, and an intracellular granular structure that was detected by transmission electron microscopy (TEM), suggesting the accumulation of polyhydroxyalkanoate (PHA), most likely polyhydroxybutyrate. Novelty and scientific contribution: Methylobacterium organophilum demonstrated a unique ability to produce compounds of commercial interest. The distinct metabolic diversity of this bacterium makes room for its use in biorefineries.

New cost-effective bioconversion process of palm kernel cake into bioinsecticides based on Beauveria bassiana and Isaria javanica.

The present study aimed to add value to palm oil by-products as substrates to efficiently produce conidia of Beauveria bassiana and Isaria javanica (Hypocreales: Cordycipitaceae) for biological control of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), through a solid-state fermentation process using palm kernel cake and palm fiber as nutrient source and solid matrix, respectively. The optimum culture conditions yielded high concentrations of viable conidia after air-drying, when the fungi were grown on palm kernel cake (B. bassiana 7.65 × 109 and I. javanica 2.91 × 109 conidia g-1 dry substrate) after 6 days under optimal growth conditions set to 60% substrate moisture and 32 °C. Both fungal strains exhibited high efficacy against third-instar whitefly nymphs, inducing mortality up to 62.9 and 56.6% by B. bassiana and I. javanica, respectively, assessed after 9 days post-application in a screenhouse. Furthermore, we noted that insect mortality was strongly correlated with high atmospheric moisture, while B. bassiana appeared to require shorter accumulative hours under high moisture to kill whitefly nymphs compared to I. javanica. Our results underpin a feasible and cost-effective mass production method for aerial conidia, using palm kernel as the main substrate in order to produce efficacious fungal bioinsecticides against an invasive whitefly species in Brazil. Finally, our fermentation process may offer a sustainable and cost-effective means to produce eco-friendly mycoinsecticides, using an abundant agro-industrial by-product from Brazil that will ultimately assist in the integrated management of agricultural insect pests.

2,3-Butanediol production by the non-pathogenic bacterium Paenibacillus brasilensis.

2,3-Butanediol (2,3-BDO) is of considerable importance in the chemical, plastic, pharmaceutical, cosmetic, and food industries. The main bacterial species producing this compound are considered pathogenic, hindering large-scale productivity. The species Paenibacillus brasilensis is generally recognized as safe (GRAS) and is phylogenetically similar to P. polymyxa, a species widely used for 2,3-BDO production. Here, we demonstrate, for the first time, that P. brasilensis strains produce 2,3-BDO. Total 2,3-BDO concentrations for 15 P. brasilensis strains varied from 5.5 to 7.6 g/l after 8 h incubation at 32 °C in modified YEPD medium containing 20 g/l glucose. Strain PB24 produced 8.2 g/l of 2,3-BDO within a 12-h growth period, representing a yield of 0.43 g/g and a productivity of 0.68 g/l/h. An increase in 2,3-BDO production by strain PB24 was observed using higher concentrations of glucose, reaching 27 g/l of total 2,3-BDO in YEPD containing about 80 g/l glucose within a 72-h growth period. We sequenced the genome of P. brasilensis PB24 and uncovered at least six genes related to the 2,3-BDO pathway at four distinct loci. We also compared gene sequences related to the 2,3-BDO pathway in P. brasilensis PB24 with those of other spore-forming bacteria, and found strong similarity to P. polymyxa, P. terrae, and P. peoriae 2,3-BDO-related genes. Regulatory regions upstream of these genes indicated that they are probably co-regulated. Finally, we propose a production pathway from glucose to 2,3-BDO in P. brasilensis PB24. Although the gene encoding S-2,3-butanediol dehydrogenase (butA) was found in the genome of P. brasilensis PB24, only R,R-2,3- and meso-2,3-butanediol were detected by gas chromatography under the growth conditions tested here. Our findings can serve as a basis for further improvements to the metabolic capabilities of this little-studied Paenibacillus species in relation to production of the high-value chemical 2,3-butanediol.

Simultaneous lipase production and immobilization: morphology and physiology study of Penicillium simplicissimum in submerged and solid-state fermentation with polypropylene as an inert support.

The influence of different carbon sources (glucose (G), olive oil (O), and a combination of both (GO)) in the physiology (biomass and lipase production) and morphology (light and environmental and scanning electron microscopy) of the fungus Penicillium simplicissimum by applying submerged (SmF) and solid-state (SSF) fermentations was investigated. The cultivation was carried out using polypropylene as hydrophobic inert support in SmF and SSF to understand better the influence of a support for the fungus growth and also provides the immobilization of lipases during its production. Micrographs show different morphologies: in SSF, the fungus grows on and inside the inert support independent of the media; in SmF, the formation of high-density spherical pellets obtained in medium GO leads to the best productivity and specific product yield Yp/x..Conidiation is observed mainly in SSF, a few in SmF with polypropylene as inert support and not in SmF, which may indicate a stress condition in SSF. Possibly, the morphology acquired by the fungus under stressful conditions may be the key to the higher biomass and lipase productivity at SSF. The developed process with simultaneous production and immobilization of lipase leads to a new promissory biocatalyst once it can be directly applied with no need for downstream processes.

Sustainable production of biopesticides for common bean root rot control within the biorefinery approach: a Technology Readiness Level 3 experimental proof of concept.

Biopesticides are recognized as an efficient alternative to synthetic pesticides for pest and disease crop management. However, their commercial production processes use grains, generating large amounts of organic waste, even when agriculture waste or byproducts are the feedstock of choice. Frequently, these organic wastes are rich in nutrients that, after adequate treatment, can be used as nitrogen and carbohydrate sources for secondary metabolite production produced by microorganisms during submerged fermentation. In this sense, this study aimed to prove the concept that biopesticides could be produced under a full biorefinery process, using the entire biomass of an underexplored agroindustrial waste-damaged bean-as the main feedstock. A combination of sequential processes, including solid state fermentation, hydrolysis, and submerged fermentation, were designed for the production of two biopesticides (conventional-fungal conidia and second-generation secondary metabolite-cerulenin) from a high potential biological control agent strain Sarocladium oryzae BRM 59907. The combined processes, using damaged common bean grain as the main feedstock, provided biopesticides and organic fertilizer production that successfully controlled common bean root rot disease. This work proved to be possible the biopesticide production using a full biorefinery concept, inside the same productive chain, contributing to a sustainable environment and economy, together with animal and human health safety.

Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria.

BACKGROUND: Bacillus sp. H2O-1, isolated from the connate water of a Brazilian reservoir, produces an antimicrobial substance (denoted as AMS H2O-1) that is active against sulfate reducing bacteria, which are the major bacterial group responsible for biogenic souring and biocorrosion in petroleum reservoirs. Thus, the use of AMS H2O-1 for sulfate reducing bacteria control in the petroleum industry is a promising alternative to chemical biocides. However, prior to the large-scale production of AMS H2O-1 for industrial applications, its chemical structure must be elucidated. This study also analyzed the changes in the wetting properties of different surfaces conditioned with AMS H2O-1 and demonstrated the effect of AMS H2O-1 on sulfate reducing bacteria cells. RESULTS: A lipopeptide mixture from AMS H2O-1 was partially purified on a silica gel column and identified via mass spectrometry (ESI-MS). It comprises four major components that range in size from 1007 to 1049 Da. The lipid moiety contains linear and branched ß-hydroxy fatty acids that range in length from C13 to C16. The peptide moiety contains seven amino acids identified as Glu-Leu-Leu-Val-Asp-Leu-Leu.Transmission electron microscopy revealed cell membrane alteration of sulfate reducing bacteria after AMS H2O-1 treatment at the minimum inhibitory concentration (5 µg/ml). Cytoplasmic electron dense inclusions were observed in treated cells but not in untreated cells. AMS H2O-1 enhanced the osmosis of sulfate reducing bacteria cells and caused the leakage of the intracellular contents. In addition, contact angle measurements indicated that different surfaces conditioned by AMS H2O-1 were less hydrophobic and more electron-donor than untreated surfaces. CONCLUSION: AMS H2O-1 is a mixture of four surfactin-like homologues, and its biocidal activity and surfactant properties suggest that this compound may be a good candidate for sulfate reducing bacteria control. Thus, it is a potential alternative to the chemical biocides or surface coating agents currently used to prevent SRB growth in petroleum industries.

Rhamnolipid production: effect of oxidative stress on virulence factors and proteome of Pseudomonas aeruginosa PA1.

Under specific environmental conditions, Pseudomonas aeruginosa produces a biodegradable surfactant rhamnolipid. Evidences suggest that this biosurfactant is involved in protecting cells against oxidative stress; however, the effects of oxidative stress on its production and other virulence factors are still unclear. Here we show that rhamnolipid production is dependent on the aeration surface when P. aeruginosa is cultured in shaken flasks, as well as in production of elastases and alkaline proteases. The production of alginate, lipase, and pyocyanin was not detected in our shaken-flask experiments. P. aeruginosa was treated with hydrogen peroxide to trigger its oxidative stress response, and the proteome profile was analyzed. We identified 14 proteins that were expressed differently between samples that were treated and not treated with peroxide; these proteins are potentially involved in the rhamnolipid production/secretion pathway and oxidative stress.

Revisiting Jatropha curcas Monomeric Esterase: A Dienelactone Hydrolase Compatible with the Electrostatic Catapult Model.

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.

Benchmarking recombinant Pichia pastoris for 3-hydroxypropionic acid production from glycerol.

The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3-hydroxypropionic acid (3-HP) from glycerol. 3-HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum-based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3-HP from glycerol through the malonyl-CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14-fold increase in the production of 3-HP compared to the initial strain. The best strain (PpHP6) was tested in a fed-batch culture, achieving a final concentration of 3-HP of 24.75 g l-1 , a product yield of 0.13 g g-1 and a volumetric productivity of 0.54 g l-1  h-1 , which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3-HP.

Bacillus velezensis H2O-1 surfactin efficiently maintains its interfacial properties in extreme conditions found in post-salt and pre-salt oil reservoirs.

Biosurfactants are molecules with surfactant properties produced by microorganisms, and can be used in various industrial sectors, e.g., the oil industry. These molecules can be used in enhanced oil recovery (EOR) in the pre-salt and post-salt reservoirs, where conditions of temperature, pressure, and salinity are quite varied, requiring a study of the stability of these molecules under these conditions. Bacillus velezensis H2O-1 produces five different surfactin homologs with a fatty-acid chain ranging from C11 to C16 and with a high capacity to reduce surface (24.8 mN.m-1) and interfacial tensions (1.5 and 0.8 8 mN.m-1 using light, medium oil and n-hexadecane, respectively). The critical micellar concentration (CMC) was 38.7 mg.L-1. Inversion wettability tests were carried out under the salinity conditions found in the post-salt (35 g.L-1) and pre-salt (70 g.L-1) reservoirs, in which it was observed that the surfactin reversed 100 % of the wettability of the calcite impregnated with light and medium oil. Using a central composite rotatable design, we demonstrated that surfactin maintained its interfacial properties when subjected simultaneously to extreme conditions of pressure, temperature and salinity commonly found in the post-salt (70 °C, 70 g.L-1 and 27.58 MPa) and pre-salt (100 °C, 150 g.L-1 and 48.2 MPa) layers. The results presented here highlight the efficiency and stability of H2O-1 surfactin in environmental conditions found in pre-salt and post-salt oil reservoirs.

Enzymes produced by solid state fermentation of agro-industrial by-products release ferulic acid in bioprocessed whole-wheat breads.

Solid-state fermentation (SSF) presents low cost and the possibility of adding value to waste by generating products rich in enzymes. The production of enzymes by SSF and its application in bakery have been previously reported separately in the literature. However, very few studies combine both approaches to evaluate the feasibility of applying enzymes produced by SSF to bread processing. The objective of this study was to use cocoa bean shell (CBS), wheat bran (WB) and brewer's spent grain (BSG) for enzyme production by SSF, and to evaluate their addition in breads. Three breads were produced: control bread (CB), bioprocessed bread added with fermented wheat bran (WBB) and bioprocessed bread added with fermented BSG (BSGB). Feruloyl esterase highest activities were 1,730 mU/g for WB fermented for 24 h and 1,128 mU/g for BSG fermented for 72 h. Xylanase highest activities were 547.9 U/g for BSG fermented for 48 h and 868.1 U/g for WB fermented for 72 h. CBS showed the lowest enzymatic activities. Bioprocessing breads with fermented WB and BSG led to an increase in soluble ferulic acid of 159% and 198%, respectively. The combination of SSF enzyme production and bread enzymatic bioprocessing strategies proved to be an effective green option for the valorization of agro-industrial by-products and the production of breads with enhanced ferulic acid content.

Application of Rhizomucor miehei lipase-displaying Pichia pastoris whole cell for biodiesel production using agro-industrial residuals as substrate.

This work aimed the application of a new biocatalyst for biodiesel production from residual agro-industrial fatty acids. A recombinant Pichia pastoris displaying lipase from Rhizomucor miehei (RML) on the cell surface, using the PIR-1 anchor system, were prepared using glycerol as the carbon source. The biocatalyst, named RML-PIR1 showed optimum temperature of 45 °C (74.0 U/L). The stability tests resulted in t1/2 of 3.49 and 2.15 h at 40 and 45 °C, respectively. RML-PIR1 was applied in esterification reactions using industrial co-products as substrates, palm fatty acid distillate (PFAD) and soybean fatty acid distillate (SFAD). The highest productivity was observed for SFAD after 48 h presenting 79.1% of conversion using only 10% of biocatalyst and free-solvent system. This is about ca. eight times higher than commercial free RML in the same conditions. The stabilizing agents study revealed that the treatment using glutaraldehyde (GA) and poly(ethylene glycol) (PEG) enabled increased stability and reuse of biocatalyst. It was observed by SEM analysis that the treatment modified the cell morphology. RML-PIR1-GA presented 87.9% of the initial activity after 6 reuses, whilst the activity of unmodified RML-PIR decreased by 40% after the first use. These results were superior to those obtained in the literature, making this new biocatalyst promising for biotechnological applications, such as the production of biofuels on a large scale.

Antibiofilm effect of mono-rhamnolipids and di-rhamnolipids on carbon steel submitted to oil produced water.

The objective of this study was to compare the potential of mono-rhamnolipids (mono-RML) and di-rhamnolipids (di-RML) against biofilm formation on carbon steel coupons submitted to oil produced water for 14 days. The antibiofilm effect of the RML on the coupons was analyzed by scanning electron microscopy (SEM) and by epifluorescence microscopy, and the contact angle was measured using a goniometer. SEM analysis results showed that all RML congeners had effective antibiofilm action, as well as preliminary anticorrosion evaluation confirmed that all RML congeners prevented the metal deterioration. In more detail, epifluorescence microscopy showed that mono-RML were the most efficient congeners in preventing microorganism's adherence on the carbon steel metal. Image analyses indicate the presence of 15.9%, 3.4%, and <0.1% of viable particles in di-RML, mono/di-RML and mono-RML pretreatments, respectively, in comparison to control samples. Contact angle results showed that the crude carbon steel coupon presented hydrophobic character favoring hydrophobic molecules adhesion. We calculated the theoretical polarity of the RML congeners and verified that mono-RML (log P = 3.63) presented the most hydrophobic character. This had perfect correspondence to contact angle results, since mono-RML conditioning (58.2°) more significantly changed the contact angle compared with the conditioning with one of the most common surfactants used on oil industry (29.4°). Based on the results, it was concluded that rhamnolipids are efficient molecules to be used to avoid biofilm on carbon steel metal when submitted to oil produced water and that a higher proportion of mono-rhamnolipids is more indicated for this application.

Bioaccessibility and gut metabolism of phenolic compounds of breads added with green coffee infusion and enzymatically bioprocessed.

This study aimed at investigating two strategies to enhance the bioaccessibility of phenolic compounds from whole-wheat breads: enzymatic bioprocessing and addition of green coffee infusion. Although both strategies had a significant effect on increasing the contents of total soluble phenolic compounds in breads, the addition of green coffee infusion was much more relevant (19.1-fold) than enzymatic bioprocessing (1.8-fold). The phenolic compounds present as soluble forms were completely released from all breads' matrix already at the oral phase of digestion. While gastric digestion did not promote the release of insoluble phenolic compounds, intestinal conditions led to a slight release. All bread samples showed maximum phenolic compounds bioaccessibility after 4 h of gut fermentation. Upon the end of in vitro digestion and gut fermentation, the difference between the strategies was that enzymatic bioprocessing accelerated ferulic acid release, while the addition of green coffee infusion increased 10.4-fold the overall phenolic compounds bioaccessibility.

A newly isolated Enterobacter sp. strain produces 2,3-butanediol during its cultivation on low-cost carbohydrate-based substrates.

2,3-Butanediol (BDO) is an important platform chemical with a wide range of applications in various industries. In the present study, a newly isolated wild Enterobacter sp. strain (FMCC-208) was evaluated towards its ability to produce BDO on media composed of sugars derived from sucrose refinery plant. Optimum values of temperature and pH as well as substrate inhibition were determined through batch experiments. The ability of the strain to convert various monosaccharides was also investigated. Maximum BDO concentrations of 90.3 and 10 g l-1 of acetoin were obtained during a fed-batch bioreactor experiment with cane molasses and sucrose employed as substrates. A high volumetric productivity was noted in a fed-batch experiment using molasses and sucrose as carbon sources at T = 37°C, in which 73.0 g l-1 of BDO together with 12.4 g l-1 of acetoin was produced where 1.15 g l-1 h-1 of diol/acetoin was produced. In previously pasteurized media, 70.0 g l-1 of BDO and 5.0 g l-1 of acetoin were produced (yield = 0.39 g g-1). Finally, besides BDO production, growth on molasses was accompanied by non-negligible decolorization (25-35%) of the residue. Therefore, the strain is a promising candidate for the conversion of sucrose-based materials into BDO.

Fire Ant Venom Alkaloids Inhibit Biofilm Formation.

Biofilm formation on exposed surfaces is a serious issue for the food industry and medical health facilities. There are many proposed strategies to delay, reduce, or even eliminate biofilm formation on surfaces. The present study focuses on the applicability of fire ant venom alkaloids (aka 'solenopsins', from Solenopsis invicta) tested on polystyrene and stainless steel surfaces relative to the adhesion and biofilm-formation by the bacterium Pseudomonas fluorescens. Conditioning with solenopsins demonstrates significant reduction of bacterial adhesion. Inhibition rates were 62.7% on polystyrene and 59.0% on stainless steel surfaces. In addition, solenopsins drastically reduced cell populations already growing on conditioned surfaces. Contrary to assumptions by previous authors, solenopsins tested negative for amphipathic properties, thus understanding the mechanisms behind the observed effects still relies on further investigation.

Increase of Candida antarctica lipase B production under PGK promoter in Pichia pastoris: effect of multicopies.

The effect of gene dosage on the production of Candida antarctica lipase B (CalB) in the methylotrophic yeast Komagataella phaffii, at high densities in a simple medium containing crude glycerin as the sole carbon source, is described. The use of crude glycerin, the main by-product of biodiesel production from vegetable oils, will reduce the production cost of the bioprocess. Two K. phaffii strains were constructed with one or three copies of LipB, an optimized version of the gene encoding CalB under the control of the constitutive PPGK1 promoter. These two constructs were tested and compared on batches using minimal-salts medium with crude glycerin. The strain with three copies achieved a higher enzyme yield (48,760 U/L, 2.3-fold higher than the one-copy strain), with 42 g/L biomass, with no effects on growth.