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The presence of lignocellulose-derived microbial inhibitory compounds (LDMICs) in lignocellulosic biomass (LB) hydrolysates is a barrier to efficient conversion of LB hydrolysates to fuels and chemicals by fermenting microorganisms. Results from this study provide convincing evidence regarding the effectiveness of metabolically engineered

In this study, we explored the possibility of utilizing the succulent pulp of Spondias mombin (SM) as feedstock for the synthesis of biosurfactants by Pseudomonas spp. The cultures were composed of basic mineral medium amended with SM, SM + glucose, glucose (GLC), and nutrient broth (NB) as carbon sources. Biosurfactant production was determined by surface-active properties such as hemolysis, emulsification index (E24), drop collapse, oil-spreading assays, and reduction of surface tension. The stability of the biosurfactants was monitored across different temperature and pH regimes while chemical components of the extracted biosurfactants were determined by thin-layer chromatography. Biosurfactants synthesized from SM as sole substrate showed the highest emulsification index (56.35%), oil-spreading capacity (4.4 ± 1.31 cm), hemolysis (3.10 ± 0.02 cm), the shortest time for drop collapse (30 s), and surface tension reduction (24 mN/m). Biosurfactant concentrations ranged from 0.07 ± 0.01 in the NB to 2.08 ± 0.01 g/L in the media amended with SM. Chemical characterization revealed significant concentrations of carbohydrates and lipids in the biosurfactant produced from SM (1.2 ± 0.17 and 0.88 ± 0.04 g/L, respectively) when compared to SM + glucose (0.92 ± 0.05, and 0.62 ± 0.02 g/L, respectively), glucose (0.35 ± 0.04 and 0.13 ± 0.02 g/L, respectively), and nutrient broth (0.06 ± 0.03 and 0.01 ± 0.01 g/L, respectively). The biosurfactants were stable over a wide range of temperature while E24 increased with pH. Our results show the viability of SM fruit pulp as low-cost feedstock for industrial-scale production of biosurfactants using Pseudomonas spp.

Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in Clostridium beijerinckii NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of Escherichia coli RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GSCcpA). As negative controls, the ribozyme M1GSCcpA–Sc (constructed with a scrambled GSCcpA) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GSCcpA (C. beijerinckii_M1GSCcpA) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii_M1GSCcpA–Sc produced 50% more solvent than C. beijerinckii_pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GSCcpA–Sc could target the mRNA encoding DNA integrity scanning protein A (DisA), an expectation that was confirmed by a 53-fold knockdown in DisA mRNA levels. Therefore, M1GSCcpA–Sc was renamed M1GSDisA. Compared to C. beijerinckii_M1GSCcpA and _pMTL500E, C. beijerinckii_M1GSDisA exhibited a 7-fold decrease in the intracellular c-di-AMP level after 24 h of growth and a near-complete loss of viability upon exposure to DNA-damaging antibiotics. Alterations in c-di-AMP-mediated signaling and cell cycling likely culminate in a sporulation delay and the solvent production gains observed in C. beijerinckii_M1GSDisA. Successful knockdown of the CcpA and DisA mRNAs demonstrate the feasibility of using M1GS technology as a metabolic engineering tool for increasing butanol production in C. beijerinckii.

In this study, we explored the possibility of utilizing the succulent pulp of Spondias mombin (SM) as feedstock for the synthesis of biosurfactants by Pseudomonas spp. The cultures were composed of basic mineral medium amended with SM, SM + glucose, glucose (GLC), and nutrient broth (NB) as carbon sources. Biosurfactant production was determined by surface-active properties such as hemolysis, emulsification index (E24), drop collapse, oil-spreading assays, and reduction of surface tension. The stability of the biosurfactants was monitored across different temperature and pH regimes while chemical components of the extracted biosurfactants were determined by thin-layer chromatography. Biosurfactants synthesized from SM as sole substrate showed the highest emulsification index (56.35%), oil-spreading capacity (4.4 ± 1.31 cm), hemolysis (3.10 ± 0.02 cm), the shortest time for drop collapse (30 s), and surface tension reduction (24 mN/m). Biosurfactant concentrations ranged from 0.07 ± 0.01 in the NB to 2.08 ± 0.01 g/L in the media amended with SM. Chemical characterization revealed significant concentrations of carbohydrates and lipids in the biosurfactant produced from SM (1.2 ± 0.17 and 0.88 ± 0.04 g/L, respectively) when compared to SM + glucose (0.92 ± 0.05, and 0.62 ± 0.02 g/L, respectively), glucose (0.35 ± 0.04 and 0.13 ± 0.02 g/L, respectively), and nutrient broth (0.06 ± 0.03 and 0.01 ± 0.01 g/L, respectively). The biosurfactants were stable over a wide range of temperature while E24 increased with pH. Our results show the viability of SM fruit pulp as low-cost feedstock for industrial-scale production of biosurfactants using Pseudomonas spp.

The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa. The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges. IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.

Lignocellulosic biomass (LB) is a cost-effective, readily available, and attractive substrate to produce 2,3-butanediol (2,3-BD) commercially. Fermentative conversion of non-detoxified wheat straw hydrolysate (WSH) to 2,3-BD was investigated using Paenibacillus polymyxa. The results show that P. polymyxa co-utilizes representative LB-derived sugars (glucose, xylose and arabinose). Comparatively, 2,3-BD fermentations with P. polymyxa in cultures containing glucose-xylose-arabinose in the ratio of 8:11:1 resulted in 1.03- and 1.18-fold increases in 2,3-BD yield and productivity, respectively, relative to glucose-only cultures (control). Subsequently, batch fermentations were conducted using 60 %, 80 %, and 100 % WSH and glucose-only control. P. polymyxa exhibited considerable tolerance to lignocellulose-derived microbial inhibitory compounds (LDMICs) in the 60 %, 80 % and 100 % WSH, as evidenced by growth, which increased 1.16-, 1.26- and 1.32-fold, respectively, when compared to the glucose-only control. The concentrations of 2,3-BD were 32, 31 and 23 g/L in 60 %, 80 % and 100 % WSH, respectively. These concentrations compare favorably to 32 g/L 2,3-BD produced with the glucose-only control without LDMICs. In addition, evaluation of the ability of P. polymyxa to transform LDMICs showed that P. polymyxa has the capacity to use HMF as a carbon source. These results underscore the potential of P. polymyxa for in situ detoxification and large-scale fermentation of WSH to 2,3-BD.

While production of biofuels from renewable resources is currently receiving increased attention globally, concerns on availability and sustainability of cheap substrates for their production are growing as well. Lignocellulose-derived sugars (LDS) remain underutilized and merit consideration as a key feedstock. Among other obstacles such as low yield and low solvent titer, mitigation of stresses stemming from lignocellulose-derived microbial inhibitory compounds (LDMICs) that severely impair cell growth and solvent production is a major area of research interest. In addition to attempts at developing LDMIC-tolerant strains via metabolic engineering to enhance utilization of LDS, unconventional approaches that elicit different metabolic perturbations in microorganisms to relieve solvent- and LDMIC-mediated stresses have been explored to increase solvent production from LDS. In this review, the impacts of metabolic perturbations including medium supplementation with glycerol; furfural and 5-hydroxymethyl furfural; allopurinol, an inhibitor of xanthine dehydrogenase; calcium (Ca(2+)) and zinc (Zn(2+)) ions); and artificial electron carriers, methyl viologen and neutral red, on butanol production are discussed. Although these approaches have brought about considerable increases in butanol production, both from LDS and defined glucose-based media, the modes of action for most of these perturbations have yet to be fully characterized. Better understanding of these mechanisms would likely inform development of LDMIC-tolerant, butanol-overproducing strains, as well as possible combinatorial application of these approaches for enhanced butanol production. Hence, delineating the underlying mechanisms of these perturbations deserves further attention.

This study evaluated the feasibility of concomitant nutrient removal, cleaner water recovery, and improved ethanol production via glucose fermentation in the liquid fraction of anaerobic digestate (ADE) by Saccharomyces cerevisiae. The 25%, 50%, and 100% (v/v) ADE supported the growth of S. cerevisiae, glucose utilization (~100 g/L) and ethanol production (up to 50.4 ± 6.4 g/L). After a 144 h fermentation in the 50% ADE, the concentrations of ammonia, total nitrogen, phosphate, and total phosphorus decreased 1000-, 104.43-, 1.94-, and 2.20-fold, respectively. Notably, only 0.40 ± 0.61 mg/L ammonia was detected in the 50% ADE post-fermentation. Similarly, the concentrations of aluminum, copper, magnesium, manganese, molybdenum, potassium, sodium, iron, sulfur, zinc, chloride, and sulfate decreased significantly in the ADE. Further analysis suggests that the nitrogen (ammonia and protein), phosphate, and the metal contents of the digestate work in tandem to promote growth and ethanol production. Among these, ammonia and protein appear to exert considerable effects on S. cerevisiae. These results represent a significant first step towards repurposing ADE as a resource in bio-production of fuels and chemicals, whilst generating effluent that is economically treatable by conventional wastewater treatment technologies.

Biochar can be an inexpensive pH buffer and source of mineral and trace metal nutrients in acetone-butanol-ethanol (ABE) fermentation. This study evaluated the feasibility of replacing expensive 4-morpholineethanesulfonic acid (MES) P2 buffer and mineral nutrients with biochar made from switchgrass (SGBC), forage sorghum (FSBC), redcedar (RCBC) and poultry litter (PLBC) for ABE fermentation. Fermentations using Clostridium beijerinckii ATCC 51743 in glucose and non-detoxified switchgrass hydrolysate media were performed at 35 °C in 250 mL bottles for 72 h. Medium containing buffer and minerals without biochar was the control. Similar ABE production (about 18.0 g/L) in glucose media with SGBC, FSBC and RCBC and control was measured. However in non-detoxified switchgrass hydrolysate medium, SGBC, RCBC and PLBC produced more ABE (about 18.5 g/L) than the control (10.1 g/L). This demonstrates that biochar is an effective buffer and mineral supplement for ABE production from lignocellulosic biomass without costly detoxification process.

Formation of Exopolysaccharides (EPS) during 2,3-butanediol (2,3-BD) fermentation byPaenibacillus polymyxadecreases 2,3-BD yield, increases medium viscosity and impacts 2,3-BD downstream processing. Therefore, additional purification steps are required to rid the fermentation broth of EPS prior to 2,3-BD purification, which adds to the production cost. To eliminate EPS production during 2,3-BD fermentation, we explored a metabolic engineering strategy to disable the EPS production pathway ofP. polymyxa, thereby increasing 2,3-BD yield and productivity. The levansucrase gene which encodes levansucrase, the enzyme responsible for EPS biosynthesis inP. polymyxa, was successfully disrupted. The resultingP. polymyxalevansucrase null mutant showed 34% and 54% increases in growth with 6.4- and 2.4-folds decrease in EPS formation in sucrose and glucose cultures, respectively. The observed decrease in EPS formation by the levansucrase null mutant may account for the 27% and 4% increase in 2,3-BD yield, and 4% and 128% increases in 2,3-BD productivity when grown on sucrose and glucose media, respectively. Genetic stability of the levansucrase null mutant was further evaluated. Interestingly, the levansucrase null mutant remained genetically stable over fifty generations with no observable decrease in growth and 2,3- BD formation with or without antibiotic supplementations. Collectively, our results show thatP. polymyxalevansucrase null mutant has potential for improving 2,3-BD yield, and ultimately, the economics of large-scale microbial 2,3-BD production.

Non-self contact between fungi elicits strong morphological and biochemical reactions in the mycelia of interacting species. Although these reactions appear to be species- and interaction-specific, some responses such as pigmentation, increased secretion of phenol-oxidases, barrage formation and sealing of the mycelia front are common responses in most interactions. Hence, some species recruit similar molecular machineries in response to non-self. Increasing number of fully sequenced and annotated fungal genomes and advances in genome-wide and global proteome analytical tools now allow researchers to use techniques such as RNA sequencing, micro and macroarray analysis, 2-dimensional protein gel profiling, and differential display of mRNA to probe the underlying molecular mechanisms of combative mycelial interactions. This review provides an overview of the genes and proteins found to be differentially expressed in conflicting fungal mycelia by the use of 'omics' tools. Connections between observed gene and protein repertoires of competing mycelia and the attendant morphological and biochemical changes are presented.

Abrus precatorius seed oil is not edible and possesses lethal toxicological properties; hence, alternative use as low cost feedstock for biodiesel production was investigated. The n-hexane extracted oil was characterized for its chemical and physical properties, and subsequently transesterified using 1% sodium hydroxide at 60°C to produce biodiesel. The biodiesel produced had acceptable quality following characterization of its fuel properties. The relative density was found to be 0.889 with kinematic viscosity of 3.34 mm2/s within limits of Thailand biodiesel fuel standard. The acid value was 0.281 mg KOH/g, iodine value 52.43 mgI2/g, peroxide value 3.45 mEq/kg, saponification value 227.8 mg KOH/g and 2.87% free fatty acid content. The flash point and the cetane number were 137°C and 58.3, respectively while the heat of combustion was 38.28 MJ/Kg. The low temperature operability properties of A. precatorius seed biodiesel determined by parameters such as cloud point, pour point and cold-filter plugging point were - 2, 1 and - 4°C, respectively. The sulfated ash value and refractive index were found to be 0.09% and 1.457, respectively. GC analysis of the fatty acid methyl esters profile revealed 50.86 and 49.1%, saturated and unsaturated fatty acid methyl esters (FAME), respectively. A percentage FAME yield of 86.1%; with higher content of methyl palmitoleate (31.94%) and a lowest value for methyldecanoate (1.27%) was obtained. Although a n-hexane oil yield was low (2.52% w/w), the results show that Abrus seed oil derived biodiesel has commercially acceptable fuel properties and may be suitable as fuel for internal combustion engine.Keywords: Biodiesel, Abrus precatorius, oil, non-edible oil, fatty acid methyl esterAfrican Journal of Biotechnology, Vol 13(44) 4226-4235

Background and Aim Persea americana is a plant used by traditional medicine practitioners to treat ailments including diarrhoea and diabetes mellitus in Nigeria. Hence, the chloroform and the methanol fractions of the chloroform–methanol extract of the seeds of P. americana were evaluated for their acute toxicity as well as anti-diarrhoeal effects in Wistar rats to substantiate this claim. Methods The chloroform and methanol fractions [at graded doses of 100 and 200 mg/kg body weight (b.w) of each] were studied for their anti-diarrhoeal effects in terms of the reductions in the wetness of faeces and the frequency of defaecation of castor oil-induced diarrhoea. To understand the mechanism of their anti-diarrhoeal effects, their actions were further evaluated on castor oil-induced enteropooling (intestinal fluid accumulation). Results The median lethal dose (LD 50 ) of the chloroform and methanol fractions were each found to be less than 5000 mg/kg b.w. At the two doses, the chloroform and the methanol fractions showed dose-dependent significant ( p p p Conclusion Experimental findings show that the chloroform–methanol extract of the seeds of P. americana possesses significant anti-diarrhoeal effect and may be a potent source of anti-diarrhoeal drug(s) in future.

Understanding the capacity of Paenibacillus polymyxa DSM 365 to tolerate increasing concentrations of 2,3-butanediol (2,3-BD) is critical to engineering a 2,3-BD-overproducing strain. Hence, we investigated the response of P. polymyxa to high 2,3-BD concentrations. In fed-batch cultures (6-L bioreactor) 2,3-BD was accumulated to a maximum concentration of 47g/L despite the presence of residual 13g/L glucose in the medium. Concomitantly, accumulation of acetoin, the precursor of 2,3-BD increased after maximum 2,3-BD concentration was reached, suggesting that 2,3-BD was reconverted to acetoin after the concentration tolerance threshold of 2,3-BD was exceeded. Cultures of P. polymyxa were then challenged with levo-2,3-BD (20, 40 and 60g/L) at 0h in a glucose medium, and a concentration dependent growth inhibition response to levo-2,3-BD was observed. The growth of P. polymyxa was completely inhibited by 60g/L levo-2,3-BD. Furthermore, P. polymyxa was challenged with incremental 2,3-BD concentrations (20, 40 and 60g/L at 12, 24 and 36h, respectively) to mimic 2,3-BD accumulation during fermentation. Interestingly, 2,3-BD was reconverted to acetoin when its concentration reached 60g/L, possibly to alleviate 2,3-BD toxicity. Collectively, our findings indicate that 2,3-BD-mediated toxicity is a major metabolic impediment to 2,3-BD overproduction, thus, making it an important metabolic engineering target towards rational design of a 2,3-BD-overproducing strain.

Studies were performed on the effect of CaCO3 and CaCl2 supplementation to fermentation medium for ethanol production from xylose, glucose, or their mixtures using Scheffersomyces (Pichia) stipitis. Both of these chemicals were found to improve maximum ethanol concentration and ethanol productivity. Use of xylose alone resulted in the production of 20.68 ± 0.44 g L(-1) ethanol with a productivity of 0.17 ± 0.00 g L(-1) h(-1), while xylose plus 3 g L(-1) CaCO3 resulted in the production of 24.68 ± 0.75 g L(-1) ethanol with a productivity of 0.21 ± 0.01 g L(-1) h(-1). Use of xylose plus glucose in combination with 3 g L(-1) CaCO3 resulted in the production of 47.37 ± 0.55 g L(-1) ethanol (aerobic culture), thus resulting in an ethanol productivity of 0.39 ± 0.00 g L(-1) h(-1). These values are 229 % of that achieved in xylose medium. Supplementation of xylose and glucose medium with 0.40 g L(-1) CaCl2 resulted in the production of 44.84 ± 0.28 g L(-1) ethanol with a productivity of 0.37 ± 0.02 g L(-1) h(-1). Use of glucose plus 3 g L(-1) CaCO3 resulted in the production of 57.39 ± 1.41 g L(-1) ethanol under micro-aerophilic conditions. These results indicate that supplementation of cellulosic sugars in the fermentation medium with CaCO3 and CaCl2 would improve economics of ethanol production from agricultural residues.

While chiral 2,3-Butanediol (2,3-BD) is currently receiving remarkable attention because of its numerous industrial applications in the synthetic rubber, bioplastics, cosmetics, and flavor industries, 2,3-BD-mediated feedback inhibition of Paenibacillus polymyxa DSM 365 limits the accumulation of higher concentrations of 2,3-BD in the bioreactor during fermentation. The Box-Behnken design, Plackett-Burman design (PBD), and response surface methodology were employed to evaluate the impacts of seven factors including tryptone, yeast extract, ammonium acetate, ammonium sulfate, glycerol concentrations, temperature, and inoculum size on 2,3-butanediol (2,3-BD) production by Paenibacillus polymyxa DSM 365. Results showed that three factors; tryptone, temperature, and inoculum size significantly influence 2,3-BD production (p < 0.05) by P. polymyxa. The optimal levels of tryptone, inoculum size, and temperature as determined by the Box-Behnken design and response surface methodology were 3.5 g/L, 9.5%, and 35 °C, respectively. The optimized process was validated in batch and fed-batch fermentations in a 5-L Bioflo 3000 Bioreactor, and 51.10 and 68.54 g/L 2,3-BD were obtained, respectively. Interestingly, the production of exopolysaccharides (EPS), an undesirable co-product, was reduced by 19% when compared to the control. These results underscore an interplay between medium components and fermentation conditions, leading to increased 2,3-BD production and decreased EPS production by P. polymyxa. Collectively, our findings demonstrate both increased 2,3-BD titer, a fundamental prerequisite to the potential commercialization of fermentative 2,3-BD production using renewable feedstocks, and reduced flux of carbons towards undesirable EPS production.