Addition of cellulolytic enzymes and phytase for improving ethanol fermentation performance and oil recovery in corn dry grind process

Application of hydrolytic and other enzymes for improving fermentation performance and oil recovery in corn dry-grind process was optimized. Non-starch polysaccharide enzymes (BluZy-P XL; predominantly xylanase activity) were added at stages prior to fermentation at optimum conditions of 50 ◦C and pH 5.2 and compared with conventional fermentation (30 ◦C, pH 4.0). Enzyme applications resulted in faster ethanol production rates with a slight increase in yield compared to control. The thin stillage yield increased by 0.7–5% w/w wet basis with corresponding increase in solids content with enzyme treatment after liquefaction. The oil partitioned in thin stillage was at 67.7% dry basis after treatment with hydrolytic enzymes during fermentation. Further addition of protease and phytase during simultaneous saccharification and fermentation increased thin stillage oil partitioning to 77.8%. It also influenced other fermentation parameters, e.g., ethanol production rate increased to 1.16 g/g dry corn per hour, and thin stillage wet solids increased by 2% w/w. This study indicated that treatments with non-starch hydrolytic enzymes have potential to improve the performance of corn dry-grind process including oil partitioning into thin stillage. The novelty of this research is the addition of protease and phytase enzymes during simultaneous saccharification and fermentation of corn dry-grind process, which further improved ethanol yields and oil partitioning into thin stillage

Addition of cellulolytic enzymes and phytase for improving ethanol fermentation performance and oil recovery in corn dry grind process

Application of hydrolytic and other enzymes for improving fermentation performance and oil recovery in corn dry-grind process was optimized. Non-starch polysaccharide enzymes (BluZy-P XL; predominantly xylanase activity) were added at stages prior to fermentation at optimum conditions of 50 ◦C and pH 5.2 and compared with conventional fermentation (30 ◦C, pH 4.0). Enzyme applications resulted in faster ethanol production rates with a slight increase in yield compared to control. The thin stillage yield increased by 0.7–5% w/w wet basis with corresponding increase in solids content with enzyme treatment after liquefaction. The oil partitioned in thin stillage was at 67.7% dry basis after treatment with hydrolytic enzymes during fermentation. Further addition of protease and phytase during simultaneous saccharification and fermentation increased thin stillage oil partitioning to 77.8%. It also influenced other fermentation parameters, e.g., ethanol production rate increased to 1.16 g/g dry corn per hour, and thin stillage wet solids increased by 2% w/w. This study indicated that treatments with non-starch hydrolytic enzymes have potential to improve the performance of corn dry-grind process including oil partitioning into thin stillage. The novelty of this research is the addition of protease and phytase enzymes during simultaneous saccharification and fermentation of corn dry-grind process, which further improved ethanol yields and oil partitioning into thin stillage.This accepted article is published as Luangthongkam, P., Fang, L., Noomhorm, A., Lamsal, B.* 2015. Addition of hydrolytic enzymes and phytase for improving fermentation performance and oil recovery in dry-grind ethanol process, Industrial Crops and Products, 77: 803–808. DOI: 10.1016/j.indcrop.2015.09.060. Posted with permission.</p

Thermophilic production of poly(3-hydroxybutyrate-co-3-hydrovalerate) by a mixed methane-utilizing culture

The production of polyhydroxyalkanoates (PHAs) from methane is limited to mesophiles and thus suffers from high energy requirements for cooling. To address this issue, the use of thermophilic processes is gaining interest, as this strategy may deliver improved economic feasibility for PHA production. This study reports the first thermophilic PHA-producing culture grown on methane at 55 °C in fill-and-draw batch reactors. Harvested cells were incubated with various combinations of methane, propionic acid and valeric acid to assess their capacity for the synthesis of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Only PHB was produced when fed with methane alone. The addition of odd-carbon-number fatty acids resulted in higher PHA content with 3 HV fractions in the range of 15–99 mol%, depending on the types of fatty acids added. Acetic acid addition enhanced the synthesis of 3HB monomer, but not of 3 HV. On increasing the temperature to 58 °C, PHA productivity was not significantly affected

Comparison of spray-drying and freeze-drying for inoculum production of the probiotic Bacillus amyloliquefaciens strain H57

Spray and freeze drying practices for production of the probiotic Bacillus amyloliquefaciens H57 as a power for feed applications were investigated. The importance of inlet temperature, feed rate, solid content, and limestone, a heating protectant, concentration was demonstrated. Spray drying outlet temperature appeared to be crucial for survivability of B. amyloliquefaciens H57. Studying inactivation kinetics at 80, 90, and 100 °C revealed the highest D-value at 260.7 ± 47.3 min at 80 °C before gradually declining with increasing temperature. By considering the D-value, a high survival rate of 100%, which was far superior to freeze drying at 60 ± 4%, was obtained when the spray drying outlet temperature was maintained at 80 °C with 175 ± 2 °C inlet temperature, 20.9 ± 0.4 mL min−1 feed rate, 20% solid content, and 1:0.3 ratio of dry mass of the probiotic material to limestone. After 50-days of storage, significant differences in the viability of B. amyloliquefaciens H57 were observed between the powders obtained by the two drying methods. Storage temperature had a substantial impact on the probiotic stability. Best viability retention was spray dried powder stored at 4 °C. This work demonstrates that spray drying can be an effective method for producing a functional probiotic and highlights its potential for applications in feed supplement manufacturing.</p

Lipase-catalyzed interfacial polymerization of omega-pentadecalactone in aqueous biphasic medium: A mechanistic study

The synthetic activity of lipases in biphasic o/w systems was investigated with respect to their use in the synthesis of polyester chains via transesterification reactions. Lipase-catalyzed ring-opening polymerization (ROP) of pentadecalactone (omega-PDL) dispersed in water was used as a model reaction to understand the synthetic activity of lipases in biphasic o/w system. We conducted a systematic investigation of the influence of reaction conditions on the macromolecular characteristics of oligo(omega-PDL) encompassing chemical, thermophysical and colloidal properties of the reaction medium. A model was proposed assuming Michaelis-Menten interfacial kinetics followed by chain extension via lipase-catalyzed linear polycondensation. The solidification of oligo(omega)-PDL) chains with a degree of polymerization of approximately three was identified as a major factor limiting the molecular weight of obtained oligomers to similar to 870 g mol(-1), despite the fast reaction rate and complete conversion of omega-PDL. The addition of toluene into the dispersed phase at a volumetric ratio of 0.3-0.5 of toluene to omega-PDL allowed us to circumvent this problem and increase the molecular weight of obtained oligomers up to 1460 g mol(-1). The molecular weight of polymer product according to this model was thus inversely related to the weight ratio percentage of interfacial lipase PS to omega-PDL per droplet and correspondingly correlated with the activity of lipase. Taking into account all these parameters allowed increasing the molar mass of oligo(omega-PDL) from 870 g mol(-1) to 3507 g mol(-1)

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris

Background: Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains. Results: Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter. Discussion: The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.</p

Improved Corn Ethanol Fermentation and Oil Distribution by Using Polysaccharide Hydrolyzing Enzymes

We determined the effects of a commercial proprietary formulation of polysaccharide hydrolyzing enzymes on ethanol fermentation performance, oil partitioning and recovery, and quality of dried distillers grains with solubles (DDGS) on a 1.5-L and 50-L fermentation scale. The enzyme was added at the start of fermentation. Whole beer was subjected to beer well incubation, distillation, and separation of thin stillage from the wet cake. The enzyme promoted faster ethanol production without affecting the final ethanol yield. The enzyme treatments resulted in 8–18% higher wet yield of thin stillage than the control, 13–21% of oil increase in thin stillage, and 11% fiber reduction in DDGS. Free oil recovery from thin stillage was improved by the enzyme treatments (13–53% increase). The present study shows that the use of the polysaccharide hydrolyzing enzymes can add benefits to ethanol plants by increasing corn oil yield and producing fermentation co-products with increased nutritional value and potentially broader applications in animal feedsThis article is from Journal of Bioprocess Engineering and Biorefinery, 2014; 3(4); 323-331. Doi: 10.1166/jbeb.2014.1106. Posted with permission. </p

The effect of methane and odd-chain fatty acids on 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) synthesis by a Methylosinus-dominated mixed culture

A methanotrophic community was enriched in a semi-continuous reactor under non-aseptic conditions with methane and ammonia as carbon and nitrogen source. After a year of operation, Methylosinus sp., accounted for 80% relative abundance of the total sequences identified from potential polyhydroxyalkanoates (PHAs) producers, dominated the methane-fed enrichment. Prior to induction of PHA accumulation, cells harvested from the parent reactor contained low level of PHA at 4.0 ± 0.3\ua0wt%. The cells were later incubated in the absence of ammonia with various combinations of methane, propionic acid, and valeric acid to induce biosynthesis of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Previous studies reported that methanotrophic utilization of odd-chain fatty acids for the production of PHAs requires reducing power from methane oxidation. However, our findings demonstrated that the PHB-containing methanotrophic enrichment does not require methane availability to generate 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV)—when odd-chain fatty acids are presented. The enrichment yielded up to 14\ua0wt% PHA with various mole fractions of 3HV monomer depending on the availability of methane and odd-fatty acids. Overall, the addition of valeric acid resulted in a higher PHA content and a higher 3HV fraction. The highest 3HV fraction (up to 65\ua0mol%) was obtained from the methane–valeric acid experiment, which is higher than those previously reported for PHA-producing methanotrophic mixed microbial cultures.[Figure not available: see fulltext.]

Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

Background: Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results: In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions: Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.</p