Mixed Culture Cultivation in Microbial Bioprocesses.

Mixed culture cultivation is well renowned for industrial applications due to its technological and economic benefits in bioprocess, food processing, and pharmaceutical industries. A mixed consortium encompasses to achieve growth in unsterile conditions, robustness to environmental stresses, perform difficult functions, show better substrate utilization, and increase productivity. Hence, mixed cultures are being valorized currently and has also augmented our understanding of microbial activities in communities. This chapter covers a wide range of discussion on recent improvements in mixed culture cultivation for microbial bioprocessing and multifarious applications in different areas. The history of microbial culture, microbial metabolism in mixed culture, biosynthetic pathway studies, isolation and identification of strains, along with the types of microbial interactions involved during their production and propagation, are meticulously detailed in the current chapter. Besides, parameters for evaluating mixed culture performance, large-scale production, and challenges associated with it are also discussed vividly. Microbial community, characteristics of single and mixed culture fermentation, and microbe-microbe interactions in mixed cultures have been summarized comprehensively. Lastly, various challenges and opportunities in the area of microbial mixed culture that are obligatory to improve the current knowledge of microbial bioprocesses are projected.

Enhanced activity of hyperthermostable Pyrococcus horikoshii endoglucanase in superbase ionic liquids.

OBJECTIVES: Ionic liquids (ILs) that dissolve biomass are harmful to the enzymes that degrade lignocellulose. Enzyme hyperthermostability promotes a tolerance to ILs. Therefore, the limits of hyperthemophilic Pyrococcus horikoschii endoglucanase (PhEG) to tolerate 11 superbase ILs were explored. RESULTS: PhEG was found to be most tolerant to 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) in soluble 1% carboxymethylcellulose (CMC) and insoluble 1% Avicel substrates. At 35% concentration, this IL caused an increase in enzyme activity (up to 1.5-fold) with CMC. Several ILs were more enzyme inhibiting with insoluble Avicel than with soluble CMC. Km increased greatly in the presence ILs, indicating significant competitive inhibition. Increased hydrophobicity of the IL cation or anion was associated with the strongest enzyme inhibition and activation. Surprisingly, PhEG activity was increased 2.0-2.5-fold by several ILs in 4% substrate. Cations exerted the main role in competitive inhibition of the enzyme as revealed by their greater binding energy to the active site. CONCLUSIONS: These results reveal new ways to design a beneficial combination of ILs and enzymes for the hydrolysis of lignocellulose, and the strong potential of PhEG in industrial, high substrate concentrations in aqueous IL solutions.

Enhanced Biobutanol Production in Folic Acid-Induced Medium by Using Clostridium acetobutylicum NRRL B-527.

The conventional acetone-butanol-ethanol fermentation process suffers from several key hurdles viz. low solvent titer, insufficient yield and productivity, and solvent intolerance which largely affect butanol commercialization. To counteract these issues, the effect of stimulator, namely, folic acid was investigated in the present study to improve butanol titer. Folic acid is involved in biosynthesis of a diverse range of cellular components, which subsequently alter the amino acid balance. Therefore, different concentrations of folic acid were screened, and 10 mg/L supplementation resulted in a maximum butanol production of 10.78 ± 0.09 g/L with total solvents of 18.91 ± 0.21 g/L. Folic acid addition at different time intervals was also optimized to get additional improvements in final butanol concentration. Overall, folic acid supplementation resulted in two-fold increase in butanol concentration and thus could be considered as a promising strategy to enhance solvent titers.

Strategic intensification in butanol production by exogenous amino acid supplementation: Fermentation kinetics and thermodynamic studies.

Amino acids are vital precursors in many biochemical production pathways in addition to efficient nitrogen source which could enhance microbial growth yields. Therefore, in present study, the effect of amino acids from aliphatic and aromatic family was comprehensively evaluated in batch and integrated fed batch fermentation system. Clostridium acetobutylicum NRRL B-527 was able to utilize 54.15 ±â€¯1.0 g/L glucose to produce 12.43 ±â€¯0.10 g/L butanol under batch cultivation. Interestingly, a significant step up in butanol titer (20.82 ±â€¯0.33 g/L) was achieved by using fed-batch fermentation process integrated with liquid-liquid extraction module. Besides, mathematical modeling studies demonstrated the best fitting of experimental data with first order reaction kinetics. Overall, an enhancement in solvent titer by induction of essential cellular components coupled with advance bioprocess strategy was successfully utilized in this study for its further applications.

Stabilization of cutinase by covalent attachment on magnetic nanoparticles and improvement of its catalytic activity by ultrasonication.

This paper reports on stabilization of serine cutinase activity by immobilizing it through cross linking with glutaraldehyde on magnetic nanoparticles (Fe-NPs) and intensification of catalytic activity by ultrasonic treatment. The optimum parameters were cross linking with 10.52 mM glutaraldehyde for 90 min using 1:2 (w/w) ratio of enzyme:Fe-NPs. The characterization of cutinase-Fe-NPs was done by different instrumental analysis. Ultrasonic power showed a beneficial effect on the activity of free and immobilized cutinase at 5.76 and 7.63 W, respectively, after 12 min. Immobilization and ultrasonic treatment led to increments in kinetic parameters (Km and Vmax) along with noticeable changes in the secondary structural fractions of cutinase. Cutinase-Fe-NPs showed augmented pH (4-8) and thermal stability (40-60 °C). Considerably higher thermal inactivation kinetic constants (kd, t1/2 and D-value) and thermodynamic constants (Ed, ΔH°, ΔG° and ΔS°) highlighted superior thermostability of cutinase-Fe-NPs. Cutinase-Fe-NPs and ultrasound treated cutinase-Fe-NPs retained 61.88% and 38.76% activity during 21-day storage, and 82.82 and 80.69% activity after fifth reusability cycle, respectively.

In Situ Bioprocessing of Bacterial Cellulose with Graphene: Percolation Network Formation, Kinetic Analysis with Physicochemical and Structural Properties Assessment.

The understanding of microbial growth dynamics during in situ fermentation and production of bacterial cellulose (BC) with impressive properties mimicking artificial nacre, suitable for commodity applications remains fundamentally challenging. Fabrication of BC/graphene films through a single step in situ fermentation with improved properties provides a sustainable replacement to the conventional chemical-based modification using toxic compounds. This work reports the effect of reduced graphene oxide (RGO) on in situ fermentation kinetics and demonstrates the formation of percolated-network in BC/RGO nanostructures. The evaluation of kinetic parameters shows that the specific growth rate reaches optimal values at 3 wt % RGO loadings, with mixed growth associated BC production behavior. The two-dimensional graphene sheets uniformly dispersed into a three-dimensional matrix of BC nanofibers via hydrogen-bonded interactions along with in situ reductions of RGO sheets, as confirmed from spectroscopic studies. This study also demonstrates the presence of percolated network-like structures between BC fibers and RGO platelets, which resulted in the formation of nanostructures with exceptional mechanical robustness and electrical conductivity. The physicochemical and structural properties of fabricated BC/RGO films were found to significantly depend upon the RGO compositions as well as fermentation conditions. We envision that the proposed ecofriendly and scalable technology for the formation of BC/RGO films with excellent inherent properties and performance will attract great interest for its prospective applications in flexible electronics.

Role of Trace Elements as Cofactor: An Efficient Strategy toward Enhanced Biobutanol Production.

Metabolic engineering has the potential to steadily enhance product titers by inducing changes in metabolism. Especially, availability of cofactors plays a crucial role in improving efficacy of product conversion. Hence, the effect of certain trace elements was studied individually or in combinations, to enhance butanol flux during its biological production. Interestingly, nickel chloride (100 mg L-1) and sodium selenite (1 mg L-1) showed a nearly 2-fold increase in solvent titer, achieving 16.13 ± 0.24 and 12.88 ± 0.36 g L-1 total solvents with yields of 0.30 and 0.33 g g-1, respectively. Subsequently, the addition time (screened entities) was optimized (8 h) to further increase solvent production up to 18.17 ± 0.19 and 15.5 ± 0.13 g L-1 by using nickel and selenite, respectively. A significant upsurge in butanol dehydrogenase (BDH) levels was observed, which reflected in improved solvent productions. Additionally, a three-dimensional structure of BDH was also constructed using homology modeling and subsequently docked with substrate, cofactor, and metal ion to investigate proper orientation and molecular interactions.

Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization.

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.

Cauliflower waste utilization for sustainable biobutanol production: revelation of drying kinetics and bioprocess development.

Efficient yet economic production of biofuel(s) using varied second-generation feedstock needs to be explored in the current scenario to cope up with global fuel demand. Hence, the present study was performed to reveal the use of cauliflower waste for acetone-butanol-ethanol (ABE) production using Clostridium acetobutylicum NRRL B 527. The proximate analysis of cauliflower waste demonstrated to comprise 17.32% cellulose, 9.12% hemicellulose, and 5.94% lignin. Drying of cauliflower waste was carried out in the temperature range of 60-120 °C to investigate its effect on ABE production. The experimental drying data were simulated using moisture diffusion control model. The cauliflower waste dried at 80 °C showed maximum total sugar yield of 26.05 g L-1. Furthermore, the removal of phenolics, acetic acid, and total furans was found to be 90-97, 10-40, and 95-97%, respectively. Incidentally, maximum ABE titer obtained was 5.35 g L-1 with 50% sugar utilization.

New Insight into Sugarcane Industry Waste Utilization (Press Mud) for Cleaner Biobutanol Production by Using C. acetobutylicum NRRL B-527.

In the present study, press mud, a sugar industry waste, was explored for biobutanol production to strengthen agricultural economy. The fermentative production of biobutanol was investigated via series of steps, viz. characterization, drying, acid hydrolysis, detoxification, and fermentation. Press mud contains an adequate amount of cellulose (22.3%) and hemicellulose (21.67%) on dry basis, and hence, it can be utilized for further acetone-butanol-ethanol (ABE) production. Drying experiments were conducted in the temperature range of 60-120 °C to circumvent microbial spoilage and enhance storability of press mud. Furthermore, acidic pretreatment variables, viz. sulfuric acid concentration, solid to liquid ratio, and time, were optimized using response surface methodology. The corresponding values were found to be 1.5% (v/v), 1:5 g/mL, and 15 min, respectively. In addition, detoxification studies were also conducted using activated charcoal, which removed almost 93-97% phenolics and around 98% furans, which are toxic to microorganisms during fermentation. Finally, the batch fermentation of detoxified press mud slurry (the sample dried at 100 °C and pretreated) using Clostridium acetobutylicum NRRL B-527 resulted in a higher butanol production of 4.43 g/L with a total ABE of 6.69 g/L.

Sustainable biobutanol production from pineapple waste by using Clostridium acetobutylicum B 527: Drying kinetics study.

Present investigation explores the use of pineapple peel, a food industry waste, for acetone-butanol-ethanol (ABE) production using Clostridium acetobutylicum B 527. Proximate analysis of pineapple peel shows that it contains 35% cellulose, 19% hemicellulose, and 16% lignin on dry basis. Drying experiments on pineapple peel waste were carried out in the temperature range of 60-120°C and experimental drying data was modeled using moisture diffusion control model to study its effect on ABE production. The production of ABE was further accomplished via acid hydrolysis, detoxification, and fermentation process. Maximum total sugar release obtained by using acid hydrolysis was 97g/L with 95-97% and 10-50% removal of phenolics and acetic acid, respectively during detoxification process. The maximum ABE titer obtained was 5.23g/L with 55.6% substrate consumption when samples dried at 120°C were used as a substrate (after detoxification).

Chaotropicity: a key factor in product tolerance of biofuel-producing microorganisms.

Fermentation products can chaotropically disorder macromolecular systems and induce oxidative stress, thus inhibiting biofuel production. Recently, the chaotropic activities of ethanol, butanol and vanillin have been quantified (5.93, 37.4, 174kJ kg(-1)m(-1) respectively). Use of low temperatures and/or stabilizing (kosmotropic) substances, and other approaches, can reduce, neutralize or circumvent product-chaotropicity. However, there may be limits to the alcohol concentrations that cells can tolerate; e.g. for ethanol tolerance in the most robust Saccharomyces cerevisiae strains, these are close to both the solubility limit (<25%, w/v ethanol) and the water-activity limit of the most xerotolerant strains (0.880). Nevertheless, knowledge-based strategies to mitigate or neutralize chaotropicity could lead to major improvements in rates of product formation and yields, and also therefore in the economics of biofuel production.

Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

Enzymatic hydrolysis of hardwood and softwood harvest residue fibers released by sulfur dioxide-ethanol-water fractionation.

The enzymatic hydrolysis of hardwood and softwood harvest residues treated by SO2-ethanol-water (SEW) fractionation was studied. The target was to convert these fibers with high yield into glucose monomers which could be further converted into biofuel by a subsequent fermentation stage. Hardwood biomass residues were efficiently digested at low enzyme dosage (5 FPU/g cellulose) whereas the softwood residues required notably higher enzyme dosage (20 FPU) for sufficient conversion. However, cellulase dosage of softwood could be reduced mannanase supplementation. Especially the high lignin content of softwood biomass pulps impairs the digestibility and thereby, improved delignification could notably enhance the hydrolysis yields. It was shown that inferior delignification of SW biomass is due to persistent polyphenolic acids present in coniferous bark, whereas no evidence of the negative effect of inorganics and acetone extractives was observed. Additionally, SW hydrolyzate was successfully converted into a mixture of butanol, acetone and ethanol through ABE fermentation.

Enhanced stability of alcohol dehydrogenase by non-covalent interaction with polysaccharides.

Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase-gum Arabic complex formed under optimized conditions showed 93% retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase-gum Arabic complex within the temperature range of 45 to 60 °C implied its better stability. Half-life of alcohol dehydrogenase-gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants (K(m) and V(max)) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement.

The two stage immobilized column reactor with an integrated solvent recovery module for enhanced ABE production.

The production of acetone, butanol, and ethanol (ABE) by fermentation is a process that had been used by industries for decades. Two stage immobilized column reactor system integrated with liquid-liquid extraction was used with immobilized Clostridium acetobutylicum DSM 792, to enhance the ABE productivity and yield. The sugar mixture (glucose, mannose, galactose, arabinose, and xylose) representative to the lignocellulose hydrolysates was used as a substrate for continuous ABE production. Maximum total ABE solvent concentration of 20.30 g L(-1) was achieved at a dilution rate (D) of 0.2h(-1), with the sugar mixture as a substrate. The maximum solvent productivity (10.85 g L(-1)h(-1)) and the solvent yield (0.38 g g(-1)) were obtained at a dilution rate of 1.0 h(-1). The maximum sugar mixture utilization rate was achieved with the present set up which is difficult to reach in a single stage chemostat. The system was operated for 48 days without any technical problems.

Continuous two stage acetone-butanol-ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313.

The objective of this study was to optimize continuous acetone-butanol-ethanol (ABE) fermentation using a two stage chemostat system integrated with liquid-liquid extraction of solvents produced in the first stage. This minimized end product inhibition by butanol and subsequently enhanced glucose utilization and solvent production in continuous cultures of Clostridium acetobutylicum B 5313. During continuous two-stage ABE fermentation, sugarcane bagasse was used as the cell holding material for the both stages and liquid-liquid extraction was performed using an oleyl alcohol and decanol mixture. An overall solvent production of 25.32g/L (acetone 5.93g/L, butanol 16.90g/L and ethanol 2.48g/L) was observed as compared to 15.98g/L in the single stage chemostat with highest solvent productivity and solvent yield of 2.5g/Lh and of 0.35g/g, respectively. Maximum glucose utilization (83.21%) at a dilution rate of 0.051/h was observed as compared to 54.38% in the single stage chemostat.

Improved poly-ε-lysine biosynthesis using Streptomyces noursei NRRL 5126 by controlling dissolved oxygen during fermentation.

The growth kinetics of Streptomyces noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 l stirred tank fermenter. Poly-epsilon-lysine biosynthesis, cell mass formation, and glycerol utilization rates were affected markedly by both aeration and agitation. An agitation speed of 300 rpm and aeration rate at 2.0 vvm supported better yields of 1,622.81 mg/l with highest specific productivity of 15 mg/l.h. Fermentation kinetics performed under different aeration and agitation conditions showed poly- epsilon-lysine fermentation to be a growth-associated production. A constant DO at 40% in the growth phase and 20% in the production phase increased the poly-epsilon-lysine yield as well as cell mass to their maximum values of 1,992.35 mg/l and 20.73 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates (qO2) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.

Optimization of poly-epsilon-lysine production by Streptomyces noursei NRRL 5126.

Poly-epsilon-lysine (epsilon-PL) is a non-toxic biopolymer with antimicrobial properties. The production of epsilon-PL by Streptomyces noursei NRRL 5126 shake-flask culture was optimized by identifying the most significant medium components which affect epsilon-PL production (glycerol, proteose peptone and ammonium sulphate) by Placket-Burman design and by application of an evolutionary operation (EVOP) to determine the optimal concentrations of these components. The epsilon-PL yield increased from 41.81 g/l in basal medium to 98.07 g/l in the EVOP-optimized medium containing 3% glycerol, 1% proteose peptone and 0.8% ammonium sulphate. Further improvements in media composition and culture conditions will be required to obtain yields comparable to those obtained with current commercial strains such as Streptomyces albulus.