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.

Biobased volatile fatty acids (VFA) production via anaerobic acidogenesis of sugar processing industry effluent.

Rapid industrialization and unscientific disposal of industrial wastewaters have resulted in the pollution of water bodies and deterioration of water quality all over the globe. Valorization of industrial wastewaters will help in reducing the negative impact on the environment and will add value to the waste. The present study targets utilization of sugar processing industrial effluent for bio-based production of Volatile fatty acids (VFA) through anaerobic acidogenesis. Batch studies conducted to determine the VFA production potential of sugar processing industry effluent resulted in the VFA yield of 0.70 g/g COD utilized. Further continuous VFA production system was developed and optimization of Organic loading rate (OLR) (2-22 g COD/L·day) was carried out with constant Hydraulic retention time (HRT) of 1 day. The continuous reactors studies resulted in a maximum VFA yield of 0.72 g/g COD utilized and productivity of 11.04 g COD/L·day at OLR of 14 g COD/L·day and 22 g COD/L·day, respectively. The developed process will provide an environmentally safe and efficient method for the conversion of complex industrial wastes to valuable products such as VFA.

Heterologous mannitol-1-phosphate dehydrogenase gene over-expression in Parachlorella kessleri for enhanced microalgal biomass productivity.

BACKGROUND: Microalgae have tremendous potential in CO2 sequestration, bioenergy, biofuels, wastewater treatment, and high-value metabolites production. However, large-scale production of microalgae is hampered due to photo-inhibition in outdoor cultivation. Mannitol, as an osmolyte, is known to relieve the stress produced under different abiotic stress conditions during the growth of a photosynthetic organism. RESULTS: In the present study, Mannitol-1-phosphate 5-dehydrogenase (Mt1D) was over-expressed to study the effect of mannitol over-production in Parachlorella kessleri under high-light induced stress. Over-expression of Mt1D led to 65% increased mannitol content in the transformed P. kessleri compared to that of wild type. Mannitol transformant demonstrated > 20-fold reduction in reactive oxygen species generation and 15% higher biomass productivity when grown in outdoor cultivation with high-light irradiance of 1200 µmol photons m-2 s-1. CONCLUSIONS: The current study establishes that a higher mannitol concentration provides stress shielding and leads to better acclimatization of transgenic microalgae against high-light generated stress. It also led to reduced ROS generation and improved growth of microalga under study. Thus, overexpression of the Mt1D gene in microalgae can be a suitable strategy to combat high-light stress.

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.

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.

Isolation and optimization of a novel thraustochytrid strain for DHA rich and astaxanthin comprising biomass as aquafeed supplement.

A marine organism, belonging to the Thraustochytrids family was isolated from mangroves of Mumbai, India. The isolated strain was identified as Aurantiochytrium limacinum by internal transcribed spacer sequence analysis. Optimization of process parameters yielded 14.47 g/L dry cell weight containing 55-58% oil in 3.5 days' cultivation on glucose, yeast extract, and peptone in the bioreactor. Docosahexaenoic acid (DHA) was found to be the dominant long-chain polyunsaturated fatty acid, accounting for 32-35% of total fatty acid content. The process parameter was tweaked to simultaneously synthesize astaxanthin along with DHA. The concurrent synthesis of DHA and astaxanthin-containing biomass establishes the isolated strain as a perfect choice for aquafeed. Accession number: NCBI accession number MN046792.

Integration of continuous-high cell density-fed-batch fermentation for Aurantiochytrium limacinum for simultaneous high biomass, lipids and docosahexaenoic acid production.

Docosahexaenoic acid (DHA) rich oil or biomass is currently being produced by fermentation of thraustochytrids by repeated fed-batch. Continuous cultivation has not been successful for DHA production because of excess carbon and limited nitrogen conditions requirement. The present study describes an alternative integrative fermentation strategy to simultaneously produce high cell density, lipids and DHA in continuous mode for Aurantiochytrium limacinum. The high cell density system (≥120 g/L DCW basis) on carbon feeding led to DHA productivity of 0.508 g/L.h on poultry waste based medium with a process time of 48-54 h. The strategy integrates the advantages of repeated fed-batch for high cell densities and DHA content in continuous cultivation.

Organic waste streams as feedstock for the production of high volume-low value products.

Valorisation of organic wastes to produce industrially relevant commodity products is a sustainable, cost-effective and viable alternative providing a green platform for chemical production while simultaneously leading to waste disposal management. In the present study, organic wastes such as agricultural residue-derived sugars, oilseed meals, poultry waste and molasses were used for substituting expensive organic fermentation medium components. Moorella thermoacetica and Aurantiochytrium limacinum were adapted on these waste-derived hydrolysates to produce high volume-low value products such as bio-acetic acid (80% theoretical yields) and oil-rich fish/animal feed (more than 85% dry cell weight as compared with conventional nutrient sources) respectively. Use of these waste-derived nutrients led to ~ 75% and ~ 90% reduction in media cost for acetic acid and oil-rich biomass production respectively as compared with that of traditionally used high-priced medium components. The strategy will assist in the cost reduction for high volume-low value products while also ensuring waste recovery.

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae.

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.

Metabolic Engineering of Chlamydomonas reinhardtii for Enhanced ß-Carotene and Lutein Production.

The metabolic engineering of Chlamydomonas reinhardtii, one of the fastest-growing microalgae, is a potential alternative for enhanced carotenoid productivity. CrtYB (phytoene-ß-carotene synthase - PBS) gene from red yeast Xanthophyllomyces dendrorhous encodes for a bifunctional enzyme that harbours both phytoene synthase (psy) and lycopene cyclization (lcyb) activities. Heterologous expression of this bifunctional PBS gene led to 38% enhancement in ß-carotene along with 60% increase in the lutein yields under low light conditions of 75 µmol photons m-2 s-1. Short Duration-High Light induction strategy led to overall 72% and 83% increase in ß-carotene and lutein yield reaching up to 22.8 mg g-1 and 8.9 mg g-1, respectively. This is the first report of expression of heterologous bifunctional PBS gene resulting in simultaneous enhancement in ß-carotene and lutein content in phototrophic engineered cells. Graphical Abstract.

Synergistic biorefinery of Scenedesmus obliquus and Ulva lactuca in poultry manure towards sustainable bioproduct generation.

Exploiting solar energy for growing algal biomass in waters enriched with farm manures is a holistic method of waste management. The proposed cultivation strategy termed SAR'CENA ('Synergistic Algal Refinery for Circular Economy using Nutrient Analogues), involves integrated cultivation of microalga, Scenedesmus obliquus and marine macroalga, Ulva lactuca in litter to harness biorefinery products. From various litters tested, poultry litter manure (PLM) was most amenable for growth. The microalga yielded 410 ± 6.2 g·DW· m-2· d-1 of biomass with total nitrogen (TN) concentration of 70 mg·L-1 in the media, while the macroalgae yielded 334 ± 9.9 g DW m-2 d-1 of biomass with TN concentration of 17.5 mg·L-1. The nutrient uptake efficiency was observed to be >60% with uncompromised biomass composition. Thus, SAR'CENA is projected as an ideal farming solution incorporating efficient waste management and feedstock generation thereby establishing a circular economy towards clean energy.

Production of Pentaerythritol Monoricinoleate (PEMR) by immobilized Candida antarctica lipase B.

In the present study, green synthesis of pentaerythritol monoricinoleate (PEMR) was carried out using Candida antarctica lipase B immobilized on hydrophobic adsorbent via interfacial activation. Various reaction parameters such as reaction time, organic solvent, molar ratio, the enzyme load, and presence of molecular sieves on pentaerythritol (PE) ester synthesis were systematically studied to yield selective monoester of PE. The strategies (smart use of substrate molar ratio and polar organic solvent) were employed to suppress dimerization of ricinoleic acid (RA) to avoid by-product formation and hence to obtain superior mono-ester yield. Under optimized conditions viz. substrate molar ratio of 4 (PE):1 (RA) with 2% enzyme load and 200 g/L molecular sieves in the presence of tert-butanol, 93% substrate molar conversion in 24 h reaction time was obtained. The synthesized PEMR was also characterized using FT-IR and Mass spectroscopy. To the best of our knowledge, this is the first report describing the enzymatic synthesis of PEMR.

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.

N-terminal domain replacement changes an archaeal monoacylglycerol lipase into a triacylglycerol lipase.

BACKGROUND: Lipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C2-C12) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from Archaeglobus fulgidus. Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil-water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon Thermococcus onnurineus (strain NA1) into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase-Thermomyces lanuginosus (TLIP). RESULTS: TON-LPL and rc-TGL were cloned and overexpressed in E. coli, and the proteins were purified by Ni-NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 °C in its optimum temperature (Topt). The far UV-CD spectrums were consistent with a well-folded α/ß-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates. CONCLUSIONS: Here, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals.

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.

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.

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.

Synthesis of designer triglycerides by enzymatic acidolysis.

Enzymatic acidolysis process was developed for modification of fully hydrogenated soybean oil (FHSO) by incorporation of caprylic acid, a medium chain fatty acid. Immobilized sn-1,3 specific lipase PyLip was used to modify FHSO to produce a new fat with improved physico-chemical and functional properties. PyLip mediated acidolysis resulted in 88% reduction of substrate triglycerides and 45.16% incorporation of caprylic acid in FHSO at molar ratio of 1:3 of FHSO and caprylic acid in 60 min reaction time. HPLC analysis revealed formation of mono-substituted and di-substituted TAGs post enzymatic acidolysis. Physical properties of synthesized lipid were studied using DSC and XRD and considerable change was observed in the final product compared to the starting material. The present study reports a faster acidolysis process in the presence of solvent enhancing the modification of FHSO with caprylic acid and having no side products formation (monoglycerides and diglycerides) making the entire process highly efficient and commercially attaractive.

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.