Biochemical Production Research Articles

Engineering of Microbial Cell Factories for Enhanced Acetic Acid and Ethanol Production Via Heterologous Overexpression of the CODH Gene in CO2 Fermentation.

This study evaluates the performance of carbon monoxide dehydrogenase (codh)-embedded strains in bench-scale microbial electrochemical systems (MES) for CO2 reduction to biofuels and biochemicals. CO2 fermentation efficiency was evaluated by comparing the wild-type Clostridium acetobutylicum (Wild), a negative control E. coli strain lacking the codh gene (NC-BL21), and engineered E. coli strain (Eng) alone and with IPTG induction (Eng+IPTG). Four electrochemical systems were used, viz. Wild+E, NC-BL21+E, Eng+E, and Eng+IPTG+E, with a poised potential of -0.6 V applied to the working electrode. CO2 and bicarbonate were supplemented to a total inorganic carbon (IC) concentration of 40 g/L, with a retention time of 60 h. The engineered strain demonstrated enhanced metabolic performance compared to the wild-type and negative control strains, yielding maximum formic acid (2.1 g/L) and acetic acid (9.3 g/L) under the Eng+IPTG+E condition. Solventogenesis also influenced positively in the same system with the ethanol yield of 3.9 g/L, substantially exceeding biochemicals yield observed in the wild-type strain (2.4 g/L, acetic acid). The engineered strains exhibited superior cumulative yields (0.40 g/g), enhanced CODH-mediated charge flux stability (60 vs 5 in the wild type), and upregulated expression of key genes in the Wood-Ljungdahl pathway (WLP). Bioelectrochemical performance analysis demonstrated elevated reductive catalytic currents, enhanced CO2 reduction, and optimal charge transfer kinetics. This study highlights the synergistic potential of genetic engineering, specifically CODH overexpression, combined with electro-fermentation for enhanced biofuel and biochemical production from C1 gases.

Life cycle assessment of bioenergy and value-added biochemical production from Nizimudinia zanardini brown macroalgae.

Life cycle assessment of bioenergy and value-added biochemical production from Nizimudinia zanardini brown macroalgae.

Adaptive evolution and reverse engineering to explore the low pH tolerance mechanisms of Streptomyces albulus.

Streptomyces albulus is well-known as a cell factory for producing ε-poly-L-lysine (ε-PL), but its ability to produce effectively requires an environment with a pH of about 4.0. Unfortunately, prolonged exposure to low pH environment compromises the cellular integrity of S. albulus, leading to a decrease in the efficiency of ε-PL biosynthesis. To enhance the low pH tolerance of S. albulus and investigate its low pH tolerance mechanisms, we employed adaptive laboratory evolution (ALE) technology to evolve the S. albulus GS114 strain by progressively lowering the environmental pH. This process ultimately yielded the mutant strain ALE3.6, which exhibited significantly improved low pH tolerance at pH 3.6 and achieved a 37.9% increase in ε-PL production compared to the parental GS114 strain under the optimal fermentation condition. The physiological evaluation of the mutant strain ALE3.6 indicated a pronounced enhancement in the integrity of its cell membrane and cell wall under low pH conditions. To identify the key genes involved in low pH tolerance, we employed whole-genome resequencing and quantitative real-time PCR, which pinpointed desA, gatD, and mamU as critical contributors. We further validated the roles of these genes through reverse engineering, which improved both low pH tolerance and ε-PL production efficiency. Finally, we elucidated the response mechanisms of the S. albulus cell membrane and cell wall under low pH stress. This study enhances the understanding of low pH tolerance in the Streptomyces species, particularly regarding the production of valuable biochemical products under challenging environmental conditions.IMPORTANCEIn this study, we improved the viability and ε-poly-L-lysine production efficiency of Streptomyces albulus at low pH by staged adaptive laboratory evolution while simplifying the previously studied fed-batch fermentation strategy. We identified key genes associated with the mutant strains' cell membrane and cell wall phenotypes by utilizing whole-genome resequencing and reverse engineering. Subsequently, we validated the cell membrane and cell wall response mechanisms in S. albulus under low pH conditions.

Light-Emitting Diode Illumination Enhances Biomass, Pigment, and Lipid Production in Halotolerant Cyanobacterium Aphanothece halophytica

Light characteristics, including spectrum and intensity, significantly impact cyanobacterial biomass production, pigment biosynthesis, and cellular metabolism, influencing the composition of various biochemical compounds. This study aimed to investigate the effects of light-emitting diode (LED) illumination on biomass, pigment, and lipid production in the unicellular halotolerant cyanobacterium Aphanothece halophytica, cultivated in a suitable natural seawater (SNSW) medium. The results revealed that LED light outperformed fluorescent light, with blue LED light, particularly at an intensity of 60 μmol photons m−2 s−1, significantly enhancing growth, pigment synthesis, and lipid accumulation. This resulted in a maximum cell density of 68.96 ± 1.52 × 106 cells mL−1, a specific growth rate of 0.302 ± 0.002 day−1, and a lipid productivity of 56.81 ± 0.75 mg L−1 day−1. White LED light produced lipids suitable for biodiesel, whereas blue, green, and red LEDs promoted the accumulation of polyunsaturated fatty acids (PUFAs), beneficial for food supplements. These findings highlight the potential of LED-based cultivation strategies for optimizing biomass and biochemical compound production in A. halophytica.

Mathematical modelling for cellular processes

Biomanufacturing harnesses engineered cells for the large-scale production of biochemicals, biopharmaceuticals, biofuels, and biomaterials, playing a vital role in mitigating global environmental crises, achieving carbon peaking and neutrality, and driving the green transformation of the economy and society. The effective design and construction of these engineered cells require precise and comprehensive computational models. Recent technological breakthroughs including high-throughput sequencing, mass spectrometry, spectroscopy, and microfluidic devices, coupled with advances in data science, artificial intelligence, and automation, have enabled the rapid acquisition of large-scale biological datasets, thereby facilitating a deeper understanding of cellular dynamics and the construction of mechanism-based models with enhanced accuracy. This review systematically summarises the mathematical frameworks employed in cellular modelling. It begins by evaluating prevalent mathematical paradigms, such as network topology analyses, stochastic processes, and kinetic equations, critically assessing their applicability across various contexts. The discussion then categorises modelling strategies for specific cellular processes, including cellular growth and division, morphogenesis, DNA replication, transcriptional regulation, metabolism, signal transduction, and quorum sensing. We also examine the recent progress in developing whole-cell models through the integration of diverse cellular processes. The review concludes by addressing key challenges such as data scarcity, unknown mechanisms, multi-dimensional data integration, and exponentially escalating computational complexity. Overall, this work consolidates the mathematical models for the precise simulation of cellular processes, thereby enhancing our understanding of the molecular mechanisms governing cellular functions and contributing to the future design and optimisation of engineered organisms.

Valorizing Bio-Waste and Residuals.

The circular bioeconomy connects waste recycling with utilizing organic biomass waste for bioenergy, bio-based materials, and biochemical production. This integration promotes efficient resource utilization, reduced greenhouse gas emissions, and sustainable economic growth. Several technologies such as composting, anaerobic digestion, biochar production, gasification, pyrolysis, pelletization, and advanced thermal conversion technologies are utilized to manage agricultural waste efficiently. Waste-to-energy systems and food waste valorization techniques are employed to convert agro-waste into renewable energy sources such as bioethanol, biodiesel, and biogas through fermentation, transesterification, and anaerobic digestion. These biofuels offer renewable alternatives to fossil fuels, reducing greenhouse gas emissions and dependence on non-renewable resources. Rice husk, a globally abundant agricultural waste, can be utilized for energy production through technologies like direct combustion and fast pyrolysis. Biobutanol, synthesized from acetone-butanol-ethanol fermentation of agricultural residues like orange peel, presents a promising fuel option. Agricultural waste can also serve as feedstock for bio-based chemicals like organic acids, solvents, and polymers, reducing reliance on petroleum-based chemicals. Agro-waste materials like grass, garlic peel, and rice bran have shown potential for dye adsorption in wastewater treatment applications. Moreover, agricultural waste can be repurposed as animal feed, contributing to waste reduction and providing sustainable nutrition for livestock. Plant seeds and green biomass offer sustainable protein sources, while residues like straw and sawdust can be used for mushroom cultivation. Agro-waste biopolymers like starch and cellulose can be transformed into biodegradable plastics and biocomposites, offering eco-friendly alternatives. Additionally, agro-waste materials like straw, rice husks, and bamboo can be processed into construction materials, reducing environmental impact in building projects. Extracts from plant residues and fruit pomace can be utilized in pharmaceuticals, nutraceuticals, and cosmetics. Valorizing agro-waste for food, feed, fibers, and fuel offers opportunities to minimize waste and maximize resource efficiency, resulting in high-value products.

Isolation, Identification, and Physiological Characterization of Indigenous Yeast Species Capable of Efficiently Utilizing Sugarcane Molasses as a Carbon Source

Molasses, a byproduct of sugar production, contains sugars, ash, and inhibitors, limiting its microbial use. This study screened yeast species for efficient molasses utilization and inhibitor tolerance. Samples from four Khyber Pakhtunkhwa districts yielded 33 yeast strains after scrutiny. Following initial characterization, the strains were identified based on both morphological features and molecular methods involving the amplification of Internal Transcribed Spacer (ITS) regions. By the BLAST analysis, the ITS sequences for Candida tropicalis, Pichia kudriavzevii, Saccharomyces cerevisiae, Torulaspora delbrueckii, Trichosporon asahii, and Wickerhamomyces anomalus demonstrated 100% identity, whereas the sequence for Aspergillus fumigatus exhibited a maximum identity of 99.79% with the same species. In the phylogenetic analysis, these sequences were clustered with their respective corresponding species. Since molasses contain sucrose in major quantity, the physiological characterization of these isolated species in synthetic media containing sucrose as a sole carbon source reveals the higher growth efficiency of Torulaspora delbrueckii (OD600nm 5.24, μmax 0.0058 h-1) with second best performance of Trichosporon asahii (OD600nm 4.4, μmax 0.0049 h-1). The lowest grower was Saccharomyces cerevisiae (OD600nm 1.78 μmax 0.00016 h-1) while the remaining species i.e., Aspergillus fumigatus, Candida tropicalis, Pichia kudriavzevii, and Wickerhamomyces anomalus were of intermediate level (OD600nm 3.44, 3.89, 3.81, and 3.77, μmax was 0.0045 h-1, 0.0042 h-1, 0.0042 h-1, 0.0042 h-1 respectively). The isolated yeast species, known for utilizing non-molasses carbon sources, expand our understanding of substrate usage. Their potential as biofactories or genetic resources from natural evolution could aid in engineering industrial yeast strains for biofuel and biochemical production.

High Impact Biomass Valorization for Second Generation Biorefineries in India: Recent Developments and Future Strategies for Sustainable Circular Economy

India’s rapidly growing automobile industry has intensified the need for sustainable fuel alternatives to reduce dependency on imported fossil fuels and mitigate greenhouse gas (GHG) emissions. This study examines the potential of second-generation biorefineries as a comprehensive solution for efficient biomass valorization in India. With a projected bioethanol demand of 10,160 million liters by 2025 for India’s 20% ethanol blending target, there is an urgent need to develop sustainable production pathways. The biorefinery approach enables simultaneous production of multiple valuable products, including bioethanol, biochemicals, and bioproducts, from the same feedstock, thereby enhancing economic viability through additional revenue streams while minimizing waste. This paper systematically analyzes available biomass resources across India, evaluates integrated conversion technologies (biochemical, thermochemical, and synergistic approaches), and examines current policy frameworks supporting biorefinery implementation. Our findings reveal that second-generation biorefineries can significantly contribute to reducing GHG emissions by up to 2.7% of gross domestic product (GDP) by 2030 while creating rural employment opportunities and strengthening energy security. However, challenges in supply chain logistics, technological optimization, and policy harmonization continue to hinder large-scale commercialization. The paper concludes by proposing strategic interventions to overcome these barriers and accelerate the transition toward a sustainable circular bioeconomy in India.

Enhanced Enzymatic Hydrolysis of Tobacco Stalk via Simultaneous Deconstruction and Modification through Triton X-100-Mediated Organosolv Pretreatment.

Tobacco stalks (TS) present substantial potential for biofuel and biochemical production; however, their complex lignin structures and tightly bound carbohydrates pose significant challenges for enzymatic hydrolysis due to high recalcitrance. This study explores Triton-X 100-mediated 1,4-butanediol combined with AlCl3 pretreatment for TS fractionation towards improving enzymatic hydrolysis. Optimized pretreatment conditions achieved a significant removal of 87.8 % of hemicellulose and 81.0 % of lignin while maintaining a high cellulose retention of 90.1 %. Subsequently, the pretreated biomass recorded 91.2 % glucose yield after enzymatic hydrolysis at 10 % w/w solid with 12 FPU/g enzyme loadings, substantially outperforming controls. The presence of Triton-X 100 in pretreatment reduced enzyme requirements by up to 33.3 %. Structural characterization of the pretreated TS indicated effective disruption of lignin-carbohydrate complexes and an increase in biomass porosity by 1.2-2.3 folds, contributing to improved cellulose accessibility and enzymatic hydrolysis efficiency. Moreover, structural characterization of lignin revealed that Triton-X 100 grafted onto lignin by etherification, yielding a 21 % reduction in phenolic hydroxyl content and enhancing surface negative charge. These modifications effectively weaken both hydrogen bonding and electrostatic interactions between lignin and cellulase, thereby improving enzymatic hydrolysis efficiency. Overall, the proposed pretreatment presents a promising strategy for efficient fractionation and hydrolysis of TS biomass.

Diversifying product portfolio of syngas fermentation in addition to ethanol production by using Clostridium species.

Diversifying product portfolio of syngas fermentation in addition to ethanol production by using Clostridium species.

Freshwater algal species consortia exhibit optimal biomass and biodiesel production under calcium chloride and magnesium sulphate stress

ABSTRACT Microalgae have been pioneering microbial cell factories for sustainable bioenergy production and other high-value product extraction. The present study affirms using mixed algal species (MAS) as a consortium for escalated biomass yield and lipid production under different calcium chloride and magnesium sulphate concentrations toward biofuel production. Of the different concentrations of Ca and Mg concentrations tested, 75 mg/L of calcium and 125 mg/L of magnesium displayed maximal biomass yield, cell density, and biomass productivity. The optimization revealed 18 days of day-light incubation for enhanced biomass and biochemical production. Further, a higher lipid content of 24.55% was noticed at 0 mg/L Ca, while a maximum lipid content of 22–23.2% was observed from the MAS grown in 100 and 125 mg/L Mg concentrations. Regarding the biodiesel yield from MAS, 0 mg/L Ca showed a higher biodiesel yield of 68.45%, while 65.62–68.33% was obtained from 75 mg/L and 125 mg/L Mg concentrations. In addition, the biodiesel produced from the high Ca concentration showed higher unsaturated fatty acids of 55.13%, whereas biodiesel obtained from a higher Mg – amended consortium presented high saturated fatty acids of 53.12%. The present work will enable environmental scientists to combinatorial benefits for utilizing microalgal consortium for circular bionomy aspects and attaining sustainable development goals. Moreover, the present attempt aims to assess the microalgal consortium-based – biodiesel production in accomplishing Sustainable Development Goal – 7 (clean and affordable energy) as enlisted by the UNSDG 2030 agenda.

Biochemical Methane Production Potential of Different Industrial Wastes: The Impact of the Food-to-Microorganism (F/M) Ratio

In this study, five distinct industrial waste streams, encompassing bakery processing and kitchen waste (BP plus KW) mixture, fat, oil, and grease (FOG), ultrafiltered milk permeate (UFWP), powder whey (PW), and pulp and paper (PP) compost, underwent mesophilic biochemical methane potential (BMP) assays at F/M ratios of 1, 2, 4, and 6 g COD/g VSS. An F/M ratio of 1 g COD/g VSS showed the highest methane yield across the investigated feedstocks. In the case of UFPW and PW, an F/M ratio of 2 produced identical results to an F/M ratio of 1 despite their relatively high carbohydrate content which is easily acidified to VFAs. Increasing the F/M ratio to 2 decreased the biodegradability of both BP plus KW and FOG by 63%. Increasing the F/M ratio of the PP did not show as much of a significant impact on biodegradability compared to the other feedstocks as methane yields decreased from 135 to 92 mL CH4/g COD, a decrease of 32%.

Improving Bacillus subtilis as Biological Chassis Performance by the CRISPR Genetic Toolkit.

Bacillus subtilis is the model Gram-positive and industrial chassis bacterium; it has blossomed as a robust and promising host for enzyme, biochemical, or bioflocculant production. However, synthetic biology and metabolic engineering technologies of B. subtilis have lagged behind the most widely used industrial chassis Saccharomyces cerevisiae and Escherichia coli. CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) enables efficient, site-specific, and programmable DNA cleavage, which has revolutionized the manner of genome editing. In 2016, CRISPR technology was first introduced into B. subtilis and has been intensely upgraded since then. In this Review, we discuss recently developed key additions to CRISPR toolkit design in B. subtilis with gene editing, transcriptional regulation, and enzyme modulation. Second, advances in the B. subtilis chassis of efficient biochemicals and proteins with CRISPR engineering are discussed. Finally, we conclude with perspectives on the challenges and opportunities of CRISPR-based biotechnology in B. subtilis, wishing that B. subtilis can be comparable to traditional industrial microorganisms such as E. coli and S. cerevisiae someday soon.

A short-term cross-sectional retrospective study on procalcitonin as a diagnostic aid for various infectious diseases

BackgroundProcalcitonin (PCT) was first described in the early 1960s as a precursor of calcitonin that is synthesized mainly in the thyroid and lung tissues. It is an immediate biosynthetic product in response to bacterial toxins and the pro-inflammatory cytokines, which in turn makes it a distinctive biomarker for bacterial infections. The present analysis deals with the study on the biochemical characteristics, production pathways, and clinical uses of PCT as a diagnostic tool.MethodsDuring March and June 2024, a cross-sectional study was performed, among 357 patients with the proper characteristics who were admitted to the hospital and got their PCT levels, there were those who had respiratory, cardiac, gastrointestinal, and systemic infections. The exclusion criteria were the hospital stay of less than 24 h and a few noninfectious diseases.ResultsThe higher PCT levels (> 0.5 ng/mL) reliably informed about the presence of bacterial infections like pneumonia, endocarditis, urinary tract infections, and sepsis. PCT levels demonstrated trivial increase in viral as well as fungal infections. The study ratified the significant relationship between PCT levels and the severity of bacterial infections, thus backing its usefulness in the diagnosis and treatment control.ConclusionProcalcitonin is increasingly emerging as a trustworthy biomarker for bacterial infections, thus helping in the early diagnosis, guiding targeted antibiotic therapy, and reducing inappropriate antibiotic use. Its high specificity for bacterial etiology has largely contributed to the success and potency of antibiotic stewardship programs.

Harnessing organelle engineering to facilitate biofuels and biochemicals production in yeast.

Microbial biosynthesis using yeast species offers numerous advantages to produce industrially relevant biofuels and biochemicals. Conventional metabolic engineering approaches in yeast focus on biosynthetic pathways in the cytoplasm, but these approaches are disturbed by various undesired factors including metabolic crosstalk, competing pathways and insufficient precursors. Given that eukaryotic cells contain subcellular organelles with distinct physicochemical properties, an emerging strategy to overcome cytosolic pathway engineering bottlenecks is through repurposing these organelles as specialized microbial cell factories for enhanced production of valuable chemicals. Here, we review recent progress and significant outcomes of harnessing organelle engineering for biofuels and biochemicals production in both conventional and non-conventional yeasts. We highlight key engineering strategies for the compartmentalization of biosynthetic pathways within specific organelles such as mitochondria, peroxisomes, and endoplasmic reticulum; involved in engineering of signal peptide, cofactor and energy enhancement, organelle biogenesis and dual subcellular engineering. Finally, we discuss the potential and challenges of organelle engineering for future studies and propose an automated pipeline to fully exploit this approach.

Resource recovery from wastewater by directing microbial metabolism toward production of value-added biochemicals.

Resource recovery from wastewater by directing microbial metabolism toward production of value-added biochemicals.

Evaluating value-added biochemical and biodiesel production from Chlorococcum humicolo algal biomass in phycoremediation of textile dye effluents.

Evaluating value-added biochemical and biodiesel production from Chlorococcum humicolo algal biomass in phycoremediation of textile dye effluents.

Exploring lipid remodeling and antioxidant responses in Chlorella pyrenoidosa exposed to streptomycin sulfate stress.

Exploring lipid remodeling and antioxidant responses in Chlorella pyrenoidosa exposed to streptomycin sulfate stress.

Developing a Bacillus licheniformis platform for de novo production of γ-aminobutyric acid and other glutamate-derived chemicals.

Developing a Bacillus licheniformis platform for de novo production of γ-aminobutyric acid and other glutamate-derived chemicals.

Enhanced anaerobic digestion of waste activated sludge using magnetite-modified sludge ceramsite: Performance and microbial dynamics.

Enhanced anaerobic digestion of waste activated sludge using magnetite-modified sludge ceramsite: Performance and microbial dynamics.