Continuous fermentation using high cell density cell recycle system for L-lactic acid production.

For complete utilization of high glucose at ∼100 g/L, a high cell density (HCD) continuous fermentation system was established using Lb. delbrueckii NCIM 2025 for the bioproduction of lactic acid (LA). An integrated membrane cell recycling system coupled with the continuous bioreactor, aided to achieve the highest 34.77 g/L h LA productivity and 0.94-0.98 g/g yield. ∼34 times higher productivity was observed (in comparison to batch fermentation conducted in this study), when the continuous operations were carried out at the maximum dilution rate and wet cell weight i.e. 0.36 h-1 and 230 g/L, respectively. These results show the potential of this method for large-scale lactic acid production because it not only produces high titers but also ensures that glucose is used effectively. The method's superior performance in comparison to earlier studies suggests it as an affordable and sustainable alternative for the production of LA.

High cell density continuous fermentation for L-lactic acid production from cane molasses.

Commercial production of lactic acid (LA) utilizes mostly glucose or lactose coupled with yeast extract (YE) as a supplement. With sugars, nitrogen, and vitamin supplementation being most of the LA production costs, the use of inexpensive molasses, a by-product of the sugar industry, can provide considerable cost savings. There are just a few publications on the production of LA from molasses; consequently, the present investigation was conducted using molasses supplemented with yeast extract. The research was done in a continuous-flow, high-cell-density (HCD) bioreactor with an external membrane microfiltration device for cell recycling. The system, run at 1 L with Lactobacillus delbrueckii NCIM 2025, produced a LA yield of 0.95-0.98 g/g from ∼100 g sugars/L when supplemented with 1 g/L YE. Dilution rates in the range of 0.04-0.36 h-1 resulted in volumetric lactic acid productivities in the range of 4.3-27.6 g/L h, which compares favorably with the highest values recorded in literature, for glucose in the presence of YE, which was as high as 30 g/L. The utilization of cane molasses has a significant impact on the economics of lactic acid production, as measured by a comparison of costs with commercial glucose.

Microbial conversion of lignin rich biomass hydrolysates to medium chain length polyhydroxyalkanoates (mcl-PHA) using Pseudomonas putida KT2440.

As world moves toward increasing number of products being produced from renewable lignocellulosic agricultural and forest residues, the major classes of products that will shift to greener routes on priority are energy, fuels, and materials in that order. In materials segment, polyhydroxyalkanoates are an emerging class of biopolyesters with several potential industrial uses. The present work investigates medium chain length polyhydroxyalkanoates (mcl-PHA) producing capabilities of Pseudomonas putida KT2440 from a mixture of compounds produced from lignocellulosic biomass deconstruction. The hydrolysates obtained from nitric acid pretreatment of lignin rich cotton stalk (CS) and palm empty fruit bunch (EFB) were used as substrates for production of mcl-PHA. Presence of 3-hydroxydecanoate and 3-hydroxyocytanoate observed on GC-MS confirmed PHA accumulation in the cells. PHA accumulation was estimated between 20% and 35% of cell dry weight when grown on both model substrates as well as biomass hydrolysates. PHA titers obtained on hydrolysates of CS and EFB were 0.24 g/L and 0.21 g/L, respectively.

Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate.

Catechol is an industrially relevant chemical with myriad applications. Its production via chemical route suffers from several drawbacks the major being a non-green and nonselective route. Currently, bio-based products using biocatalyst are gaining attention due to the growing environmental and health hazards concerns over the use of petroleum-derived feedstock. Lignocellulosic biomass serves as a promising feedstock. Lignin valorization is the demand of the current scenario which is complicated task by its complexity, heterogeneity and diversity of lignin structures posing limitations toward lignin valorization via chemical routes. There are several microorganisms that possess the ability to metabolize lignin monomers via their central metabolic pathways and this paves the way to the synthesis of a number of products. Pseudomonas putida KT2440 is one such organism and was chosen for genetic manipulations for catechol biosynthesis using lignin-derived model compounds and biomass hydrolysate stream comprising of various lignin monomers. Catechol production was engineered by diverting various lignin monomers and addressing the identified metabolic bottlenecks particularly vanillic acid accumulation toward catechol biosynthesis. The engineered strain could convert the model lignin monomers as well as monomers in the biomass hydrolysates to catechol and vanillic acid in more than 60% and 90% molar yields, respectively.

Microbial consortia adaptation to substrate changes in anaerobic digestion.

Renewable natural gas (RNG) produced from anaerobic digestion (AD) of agricultural residues is emerging a serious biofuel alternative. Complex nature of lignocellulosic biomass residues coupled with complex biochemical transformations involving a large spectrum of microbial communities make anaerobic digestion of biomass difficult to understand and control. The present work aims at studying adaptation of microbial consortia in AD to substrates changes and correlating these to biogas generation. The double edged study deals with (a) using a common starting culture inoculum on different fractions of pretreated lignocellulosic biomass (LBM) fractions; and (b) using different starter inocula for gas generation from simple glucose substrate. Taxonomic analysis using 16S amplicon sequencing is shown to highlight changes in microbial community structure and predominance, majorly in hydrolytic bacterial populations. Observed variations in the rate of digestion with different starter inocula could be related to differences in microbial community structure and relative abundance. Results with different treated biomass fractions as substrates indicated that AD performance could be related to abundance of substrate-specific microbial communities. The work is a step to a deeper understanding of AD processes that may lead to better control and operation of AD for super-scale production of RNG from biomass feedstocks.

Protocatechuic acid production from lignin-associated phenolics.

Biobased chemicals are gaining popularity and market in attempts to mitigate the deteriorating environmental and sustainability issues. Components of renewable agricultural and forest biomass residues are projected to serve as abundant precursors to synthesis of expanding range of products. Agroindustrial wastes comprises of several phenolic compounds associated with lignin via ether linkages such as ferulic acid, p-coumaric, syringic acid and vanillin. These aromatic chemicals have myriad industrial applications. In this study, p-coumaric acid and ferulic acid were found to be two major components in corn bran derived lignin hydrolysate. Engineered Pseudomonas putida KT2440 was constructed and found to convert p-coumaric acid and vanillic acid to protocatechuic acid in >90% and >50% yields, respectively. Engineering the strain included deletion of the gene encoding protocatechuate 3,4-dioxygenase, and overexpression of vanillate-O-demethylase gene from Acinetobacter sp. ADP1.

2,3-Butanediol production using soy-based nitrogen source and fermentation process evaluation by a novel isolate of Bacillus licheniformis BL1.

2,3-Butanediol (2,3-BDO) has varied applications in chemical, pharmaceutical, & food industry. Microorganisms belonging to Klebsiella, Enterobacter & Serratia genera are well-known producers of 2,3-BDO. However, they have limited usage in industrial-scale owing to their pathogenic nature. A nonpathogenic soil isolate identified as Bacillus licheniformis (BL1) was thus investigated for 2,3-BDO production. Soy flakes, soy flour, defatted soy, and soybean meal-based hydrolysates replaced yeast extract and peptone as nitrogen sources. Defatted soy flakes and soybean meal hydrolysate led to an equivalent 2,3-BDO yield and productivity as compared to that of Yeast Extract and peptone. The pH and oxygen variation influenced the proportion of various products of the mixed acid-butanediol pathway. Further, the batch mode fermentation with soy hydrolysate and optimized process parameter resulted in 2,3-BDO titer, yield and productivity of 11.06 g/L, 0.43 g/g and 0.48 g/L h respectively. Glucose concentration above 5% was inhibitory and led to reduction in the specific growth rate of BL1 in batch cultivation. Intermittent glucose feeding in fed-batch mode overcame this substrate limitation resulting in increased titers (49.8 g/L) and productivity (0.62 g/L h). Modified medium containing soy hydrolysate as nitrogen source with fermentation process optimization resulted in 67% decrease in medium cost for 2,3-BDO production.

Preparative separation of nebivolol isomers by improved throughput reverse phase tandem two column chromatography.

Development of preparative methods for the isolation of chiral molecules has been considered challenging by conventional unit operations due to their identical physical and chemical properties. This has evolved chiral stationary phases for the separation of chiral components using chromatography technique. However, separation method using chiral adsorbents requires high pressure, are expensive, and have low productivity. Generation of bulk quantities purified nebivolols using the available high pressure chiral separation methods is impractical and operating cost-intensive. Thus, there is a need to develop economical methods using nonchiral adsorbents for the purification of nebivolols or similar active ingredients. The present work demonstrates a unique and scalable tandem two-column method for the separation of isomers of nebivolol using inexpensive reverse phase adsorbents. The first column of the scheme causes removal of charged and nonisomeric impurities whereas tandem operation of second column increases resolution of d-nebivolol and l-nebivolol. The maximization of separation due to tandem operation of second column causes enhancement of the throughput of the process. The developed preparative process produces >98% purity of both d-nebivolol and l-nebivolol with overall loading capacity of 56 g (L of adsorbent)-1 and productivity of 20 g L-1 day-1 .

Identification of Glyceryl MonoRicinoleate (GMR) isomers using RP-HPLC and regio-specificity of lipases.

In the present study, we report a reverse-phase high-performance liquid chromatography (RP-HPLC) method for separation of the regio-isomers of Glyceryl MonoRicinoleate (GMR) identified using position specificity of lipases. The approaches explored to identify these regio-isomers include LC-mass spectrometry, UV spectroscopy, and selective hydrolysis with lipases. A distinct UV absorption spectrum and λmax values for each isomer were noted, and mass spectral analysis further revealed their molecular weight. Lastly, the purified regio-isomers were subjected to hydrolysis with two distinctive regio-specific lipases to identified as sn-2 and sn-1(3) GMR. The current methodology of using analytic tool and enzyme specificity provides a useful platform for identifying regio-isomers for structured lipid synthesis.

Biotransformation of corn bran derived ferulic acid to vanillic acid using engineered Pseudomonas putida KT2440.

Ferulic acid is a fraction of the phenolics present in cereals such as rice and corn as a component of the bran. Substantial amounts of waste bran are generated by the grain processing industry and this can be valorized via extraction, purification and conversion of phenolics to value added chemical products. Alkaline alcohol based extracted and purified ferulic acid from corn bran was converted to vanillic acid using engineered Pseudomonas putida KT2440. The strain was engineered by rendering the vanAB gene nonfunctional and obtaining the mutant defective in vanillic acid metabolism. Biotransformation of ferulic acid using resting Pseudomonas putida KT2440 mutant cells resulted in more than 95 ± 1.4% molar yield from standard ferulic acid; while the corn bran derived ferulic acid gave 87 ± 0.38% molar yield. With fermentation time of less than 24 h the mutant becomes a promising candidate for the stable biosynthesis of vanillic acid at industrial scale.

Influence of nitrogen source on photochemistry and antenna size of the photosystems in marine green macroalgae, Ulva lactuca.

Ulva lactuca is regarded as a prospective energy crop for biorefinery owing to its affluent biochemical composition and high growth rate. In fast-growing macroalgae, biomass development strictly depends on external nitrogen pools. Additionally, nitrogen uptake rates and photosynthetic pigment content vary with type of nitrogen source and light conditions. However, the combined influence of nitrogen source and light intensity on photosynthesis is not widely studied. In present study, pale green phenotype of U. lactuca was obtained under high light (HL) condition when inorganic nitrogen (nitrate) in the media was substituted with organic nitrogen (urea). Further, pale green phenotype survived the saturating light intensities in contrast to the normal pigmented control which bleached in HL. Detailed analysis of biochemical composition and photosynthesis was performed to understand functional antenna size and photoprotection in pale green phenotype. Under HL, urea-grown cultures exhibited increased growth rate, carbohydrate and lipid content while substantial reduction in protein, chlorophyll content and PSII antenna size was observed. Further, in vivo slow and polyphasic chlorophyll a (Chl a) fluorescence studies revealed reduction in excitation pressure on PSII along with low non-photochemical quenching thus, transmitting most of the absorbed energy into photochemistry. The results obtained could be correlated to previous report on cultivation of U. lactuca through saturating summer intensities (1000 µmole photons m-2 s-1) in urea based: poultry litter extract (PLE). Having proved critical role of urea in conforming photoprotection, the application PLE was authenticated for futuristic, sustainable and year-round biomass cultivation.

Modulation in light utilization by a microalga Asteracys sp. under mixotrophic growth regimes.

This study is the first to explore the influence of incident light intensity on the photosynthetic responses under mixotrophic growth of microalga Asteracys sp. When grown mixotrophically, there was an enhanced regulation of non-photochemical quenching (NPQ) of the excited state of chlorophyll (Chl) a within the cells in response to white cool fluorescent high light (HL; 600 µmol photons m-2 s-1). Simultaneous measurement of reactive oxygen species (ROS) production as malondialdehyde (MDA) and ascorbate peroxidase (APX), an ROS scavenger, showed improved management of stress within mixotrophic cells under HL. Despite the observed decrease in quantum yield of photosynthesis measured through the Chl a fluorescence transient, no reduction in biomass accumulation was observed under HL for mixotrophy. However, biomass loss owing to photoinhibition was observed in cells grown phototrophically under the same irradiance. The measurements of dark recovery of NPQ suggested that "state transitions" may be partly responsible for regulating overall photosynthesis in Asteracys sp. The partitioning of photochemical and non-photochemical processes to sustain HL stress was analysed. Collectively, this study proposes that mixotrophy using glucose leads to a change in the photosynthetic abilities of Asteracys sp. while enhancing the adaptability of the alga to high irradiances.

Activation of alternative metabolic pathways diverts carbon flux away from isobutanol formation in an engineered Escherichia coli strain.

OBJECTIVE: Metabolic engineering efforts are guided by identifying gene targets for overexpression and/or deletion. Isobutanol, a biofuel candidate, is biosynthesized using the valine biosynthesis pathway and enzymes of the Ehrlich pathway. Most reported studies for isobutanol production in Escherichia coli employ multicopy plasmids, an approach that suffers from disadvantages such as plasmid instability, increased metabolic burden, and use of antibiotics to maintain selection pressure. Cofactor imbalance is another issue that may limit production of isobutanol, as two enzymes of the pathway utilize NADPH as a cofactor. RESULTS: To address these issues, we constructed E. coli strains with chromosomally-integrated, codon-optimized isobutanol pathway genes (ilvGM, ilvC, kivd, adh) selected on the basis of their cofactor preferences. Genes involved in diverting pyruvate flux toward fermentation byproducts were deleted. Metabolite analyses of the constructed strains revealed extracellular accumulation of significant amounts of isobutyraldehyde, a pathway intermediate, and the overflow metabolites 2,3-butanediol and acetol. CONCLUSIONS: These results demonstrate that the genetic modifications carried out led to activation of alternative pathways that diverted carbon flux toward formation of unwanted metabolites. The present study highlights how precursor metabolites can be metabolized through enzymatic routes that have not been considered important in previous studies due to the different strategies employed therein. The insights gained from the present study will allow rational genetic modification of host cells for production of metabolites of interest.

Production of trans-free interesterified fat using indigenously immobilized lipase.

Enzymatic interesterification was carried out between high-oleic canola oil and fully hydrogenated soybean oil using indigenously immobilized Thermomyces lanuginosus lipas substrate concentration, moisture content of enzyme, and enzyme load. Interesterification resulted in a decrease in the concentration of tri-unsaturated and trisaturated TAG and an increase of mono- and di-saturated TAG as observed by reversed-phase HPLC. The alteration in TAG composition and the presence of new TAG species after interesterification was correlated with extended plasticity characterized by lower slip melting point with a significant change in functionality and consistency of the interesterified product. Thermal and structural properties of the blends before and after interesterification were assessed by differential scanning calorimetry (DSC), X-ray diffraction and polarized light microscopy. Trans-fat analysis indicated the absence of any trans fatty acid in the final interesterified product. The resultant interesterified products with varying slip melting points can be used in the formulation of healthier fat and oil products and address a critical industrial demand for trans free formulations for base-stocks of spreads, margarines, and confectionary fats.

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation.

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54 ± 0.023 g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48 hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74 ± 0.06 g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.

SGNH hydrolase-type esterase domain containing Cbes-AcXE2: a novel and thermostable acetyl xylan esterase from Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium, is rich in hydrolytic and accessory enzymes that can degrade untreated biomass, but the precise role of many these enzymes is unknown. One of such enzymes is a predicted GDSL lipase or esterase encoded by the locus Athe_0553. In this study, this probable esterase named as Cbes-AcXE2 was overexpressed in Escherichia coli. The Ni-NTA affinity purified enzyme exhibited an optimum pH of 7.5 at an optimum temperature of 70 °C. Cbes-AcXE2 hydrolyzed p-nitrophenyl (pNP) acetate, pNP-butyrate, and phenyl acetate with approximately equal efficiency. The specific activity and K M for the most preferred substrate, phenyl acetate, were 142 U/mg and 0.85 mM, respectively. Cbes-AcXE2 removed the acetyl group of xylobiose hexaacetate and glucose pentaacetate like an acetyl xylan esterase (AcXE). Bioinformatics analyses suggested that Cbes-AcXE2, which carries an SGNH hydrolase-type esterase domain, is a member of an unclassified carbohydrate esterase (CE) family. Moreover, Cbes-AcXE2 is evolutionarily and biochemically similar to an unclassified AcXE, Axe2, of Geobacillus stearothermophilus. Thus, we proposed a novel family of carbohydrate esterase for both Cbes-AcXE2 and Axe2.

A shortened, two-enzyme pathway for 2,3-butanediol production in Escherichia coli.

The platform chemical 2,3-butanediol (2,3-BDO) is produced by a number of microorganisms via a three-enzyme pathway starting from pyruvate. Here, we report production of 2,3-BDO via a shortened, two-enzyme pathway in Escherichia coli. A synthetic operon consisting of the acetolactate synthase (ALS) and acetoin reductase (AR) genes from Enterobacter under control of the T7 promoter was cloned in an episomal plasmid. E. coli transformed with this plasmid produced 2,3-BDO and the pathway intermediate acetoin, demonstrating that the shortened pathway was functional. To assemble a synthetic operon for inducer- and plasmid-free production of 2,3-BDO, ALS and AR genes were integrated in the E. coli genome under control of the constitutive ackA promoter. Shake flask-level cultivation led to accumulation of ~1 g/L acetoin and ~0.66 g/L 2,3-BDO in the medium. The novel biosynthetic route for 2,3-BDO biosynthesis described herein provides a simple and cost-effective approach for production of this important chemical.

Importance of cellulase cocktails favoring hydrolysis of cellulose.

Depolymerization of lignocellulosic biomass is catalyzed by groups of enzymes whose action is influenced by substrate features and the composition of cellulase preparation. Cellulases contain a mixture of variety of enzymes, whose proportions dictate the saccharification of biomass. In the current study, four cellulase preparation varying in their composition were used to hydrolyze two types of alkali-treated biomass (aqueous ammonia-treated rice straw and sodium hydroxide-treated rice straw) to study the effect on catalytic rate, saccharification yields, and sugar release profile. We found that substrate features affected the extent of saccharification but had minimal effect on the sugar release pattern. In addition, complete hydrolysis to glucose was observed with enzyme preparation having at least a cellobiase units (CBU)/carboxymethyl cellulose (CMC) ratio (>0.15), while a modified enzyme ratio can be used for oligosaccharide synthesis. Thus, cellulase preparation with defined ratios of the three main enzymes can improve the saccharification which is of utmost importance in defining the success of lignocellulose-based economies.

Preparation of cellulase concoction using differential adsorption phenomenon.

Controlled depolymerization of cellulose is essential for the production of valuable cellooligosaccharides and cellobiose from lignocellulosic biomass. However, enzymatic cellulose hydrolysis involves multiple synergistically acting enzymes, making difficult to control the depolymerization process and generate desired product. This work exploits the varying adsorption properties of the cellulase components to the cellulosic substrate and aims to control the enzyme activity. Cellulase adsorption was favored on pretreated cellulosic biomass as compared to synthetic cellulose. Preferential adsorption of exocellulases was observed over endocellulase, while ß-glucosidases remained unadsorbed. Adsorbed enzyme fraction with bound exocellulases when used for hydrolysis generated cellobiose predominantly, while the unadsorbed enzymes in the liquid fraction produced cellooligosaccharides majorly, owing to its high endocellulases activity. Thus, the differential adsorption phenomenon of the cellulase components can be used for the controlling cellulose hydrolysis for the production of an array of sugars.

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B.

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2 h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.