Sustainable Bioprocesses Research Articles

Biovalorization of lignin-derived substrates to vanillylamine via a self-sufficient amino donor and cofactor recycling whole-cell platform

Lignin valorization through bioconversion to high-value chemicals is crucial for sustainable bioprocessing. Vanillin (VN), a primary lignin derivative, can be transaminated into vanillylamine (VM), a key precursor for capsaicin and pharmaceuticals. This study established a novel self-sufficient redox-complementary whole-cell system, facilitating the recycling of L-alanine and cofactors for efficient VM biosynthesis. Ammonium formate (AF) was employed as amino donor and co-substrate. Recombinant E. coli strain, co-expressing ω-transaminase (CvTA), L-alanine dehydrogenase (ALD), and formate dehydrogenase (FDH), showed higher yield in shorter reaction time compared to the strain expressing only CvTA and ALD. Intermittent feeding strategy was developed to mitigate VN cytotoxicity problem and a remarkable yield of 97.3 ± 1.0% was achieved of VM from 60 mM VN under optimized biotransamination conditions (37 °C, pH 8.0, VN:AF = 1:5, and 1.5 mM NAD+). Notably, a double-plasmid E. coli recombinant harboring CvTA, ALD, FDH, and aromatic dioxygenase (ADO) was constructed to convert isoeugenol into VM with a 73.2 ± 1.1% yield. This efficient biotransamination platform not only offers a sustainable route to VM for capsaicin production but also promotes lignin valorization for a greener bioeconomy.

From Knallgas Bacterium to Promising Biomanufacturing Host: The Evolution of Cupriavidus necator.

The expanding field of synthetic biology requires diversification of microbial chassis to expedite the transition from a fossil fuel-dependent economy to a sustainable bioeconomy. Relying exclusively on established model organisms such as Escherichia coli and Saccharomyces cerevisiae may not suffice to drive the profound advancements needed in biotechnology. In this context, Cupriavidus necator, an extraordinarily versatile microorganism, has emerged as a potential catalyst for transformative breakthroughs in industrial biomanufacturing. This comprehensive book chapter offers an in-depth review of the remarkable technological progress achieved by C. necator in the past decade, with a specific focus on the fields of molecular biology tools, metabolic engineering, and innovative fermentation strategies. Through this exploration, we aim to shed light on the pivotal role of C. necator in shaping the future of sustainable bioprocessing and bioproduct development.

Advances in microalgae-based lutein production and extraction: enhancing bioavailability and applications in health and industry

BackgroundNatural lutein, a bioactive xanthophyll, is widely used in pharmaceuticals, nutraceuticals, aquaculture, and cosmetics due to its broad health benefits, especially in protecting ocular health and chronic and acute syndromes. Microalgae have become a preferred sustainable platform for high-value lutein production, surpassing marigold petals in content (∼2 mg g−1) and consistency. MethodsThis review highlights advancements in upstream lutein production and downstream extraction techniques, focusing on scalable and sustainable bioprocesses. Under optimal conditions, microalgae cultivation in open and closed reactors for 2–4 weeks is examined. Comparative yields among studies are analyzed, detailing which technologies and their combinations offer superior yields for commercial production. Significant FindingsMicroalgae demonstrate high lutein content (∼10–17 mg g−1) and productivity (2–7 mg L−1d−1). The review identifies key advancements in smart delivery systems and bioavailability enhancement strategies, which are novel aspects of lutein research. The review aims to expand lutein distribution and its health benefits by addressing production challenges, market potential, and commercial opportunities. Aligning with Sustainable Development Goals (SDGs), the review promotes good health (SDG 3), economic growth (SDG 8), industry innovation (SDG 9), and climate action (SDG 13).

Unlocking xylan’s potential: Coffee husk-derived xylanolytic blend for sustainable bioprocessing

Unlocking xylan’s potential: Coffee husk-derived xylanolytic blend for sustainable bioprocessing

Conformational Selection in Enzyme-Catalyzed Depolymerization of Bio-based Polyesters.

Enzymatic degradation of polymers holds promise for advancing towards a bio-based economy. However, bulky polymers presents challenges in accessibility for biocatalysts, hindering depolymerization reactions. Beyond the impact of crystallinity, polymer chains can reside in different conformations affecting binding efficiency to the enzyme. We previously showed that the gauche and trans chain conformers associated with crystalline and amorphous regions of the synthetic polyethylene terephthalate (PET) display different affinity to PETase, thus affecting the depolymerization rate. However, structural-function relationships for biopolymers remain poorly understood in biocatalysis. In this study, we explored biodegradation of by-us previously synthesized bio-polyesters made from a rigid bicyclic chiral terpene-based diol and copolymerized with various renewable diesters. Herein, four of those polyesters spanning from semi-aromatic to aliphatic were subjected to enzymatic degradations in concert with induced-fit docking (IFD) analyses. Our findings demonstrate the importance of conformational selection in enzymatic depolymerization of biopolymers. A straight or twisted conformation of the polymer chain is crucial in biocatalytic degradation by showing different affinities to enzyme ground-state conformers. This work highlights the importance of considering the conformational match between the polymer and the enzyme to optimize the biocatalytic degradation efficiency of biopolymers, providing valuable insights for the development of sustainable bioprocesses.

Integration of ultrasound and microwave pretreatments with solid-state fermentation enhances the release of sugars, organic acids, and phenolic compounds in wheat bran

Wheat bran (WB), a byproduct of milling, is rich in bioactive compounds with significant health benefits. This study aimed to enhance the release of phenolic compounds, sugars, and organic acids from WB by integrating ultrasound (UsP) and microwave (MWP) pretreatments with solid-state fermentation (SSF). UsP and MWP disrupted WB cell walls, followed by SSF with Aspergillus niger. UsP increased total phenolic content by 21.30 % on day 1 of SSF. UsP and MWP boosted the availability of bound phenolic compounds like vanillic acid and dihydroxybenzoic acid. Both pretreatments enhanced antioxidant activity compared to untreated fermented WB, with peak activity on day 5 of fermentation at 1411 ± 5.156 μM Trolox/100 g DW for UsP WB and 291.6 ± 1.092 μM Trolox/100 g DW for MWP WB. This integrated approach improved the extraction efficiency of fermentable monosaccharides, particularly glucose and xylose, offering a sustainable bioprocessing strategy for WB valorization and supporting the circular bioeconomy.

Efficient Enzymatic Hydrolysis and Polyhydroxybutyrate Production from Non-Recyclable Fiber Rejects from Paper Mills by Recombinant Escherichia coli

Non-recyclable fiber rejects from paper mills, particularly those from recycled linerboard mills, contain high levels of structural carbohydrates but are currently landfilled, causing financial and environmental burdens. The aim of this study was to develop efficient and sustainable bioprocess to upcycle these rejects into polyhydroxybutyrate (PHB), a biodegradable alternative to degradation-resistant petroleum-based plastics. To achieve high yields of PHB per unit biomass, the specific objective of the study was to investigate various approaches to enhance the hydrolysis yields of fiber rejects to maximize sugar recovery and evaluate the fermentation performance of these sugars using Escherichia coli LSBJ. The investigated approaches included size reduction, surfactant addition, and a chemical-free hydrothermal pretreatment process. A two-step hydrothermal pretreatment, involving a hot water pretreatment (150 °C and 15% solid loading for 10 min) followed by three cycles of disk refining, was found to be highly effective and resulted in an 83% cellulose conversion during hydrolysis. The hydrolysate obtained from pretreated biomass normally requires a detoxification step to enhance fermentation efficiency. However, the hydrolysate obtained from the pretreated biomass contained minimal to no inhibitory compounds, as indicated by the efficient sugar fermentation and high PHB yields, which were comparable to those from fermenting raw biomass hydrolysate. The structural and thermal properties of the extracted PHB were analyzed using various techniques and consistent with standard PHB.

A sustainable bioprocess technology for producing food-flavour (+)-γ-decalactone from castor oil-derived ricinoleic acid using enzymatic activity of Candida parapsilosis: Scale-up optimization and purification using novel composite

Ricinoleic acid (RA) from castor oil was employed in biotransformation of peach-flavoured γ-decalactone (GDL), using a Candida parapsilosis strain (MTCC13027) which was isolated from waste of pineapple crown base. Using four variables—pH, cell density, amount of RA, and temperature—the biotransformation parameters were optimized using RSM and BBD. Under optimized conditions (pH 6, 10 % of microbial cells, 10 g/L RA at 28°C), the conversion was maximum and resulted to 80 % (+)-GDL (4.4 g/L/120 h) yield in shake flask (500 mL). Furthermore, optimization was achieved by adjusting the aeration and agitation parameters in a 3 L bioreactor, which were then replicated in a 10 L bioreactor to accurately determine the amount of (+)-GDL. In bioreactor condition, 4.7 g/L (>85 %) of (+)-GDL is produced with 20 % and 40 % dissolved oxygen (1.0 vvm) at 150 rpm in 72 h and 66 h, respectively. Further, a new Al-Mg-Ca-Si composite column-chromatography method is developed to purify enantiospecific (+)-GDL (99.9 %). This (+)-GDL is 100 % nature-identical as validated through 14C-radio-carbon dating. Thorough chemical investigation of enantiospecific (+)-GDL is authenticated for its use as flavour. This bioflavour has been developed through a cost-effective biotechnological process in response to the demand from the food industry on commercial scale.

The Undeniable Potential of Thermophiles in Industrial Processes.

Extremophilic microorganisms play a key role in understanding how life on Earth originated and evolved over centuries. Their ability to thrive in harsh environments relies on a plethora of mechanisms developed to survive at extreme temperatures, pressures, salinity, and pH values. From a biotechnological point of view, thermophiles are considered a robust tool for synthetic biology as well as a reliable starting material for the development of sustainable bioprocesses. This review discusses the current progress in the biomanufacturing of high-added bioproducts from thermophilic microorganisms and their industrial applications.

Special Issue on “Advances in Bioprocess Technology”

This Special Issue, “Advances in Bioprocess Technology”, focuses on the latest advancements in sustainable bioprocess technologies [...]

A newly isolated Klebsiella variicola JYP01 strain with iron-interaction capability for energy-efficient production of 1,3-propanediol

BackgroundGlycerol, a by-product of the biodiesel industry, has garnered attention as a substrate for sustainable bioprocessing. This study investigates the metabolic adaptability of a newly isolated strain, Klebsiella variicola JYP01, focusing on glycerol utilization under both aerobic and anaerobic conditions, as well as the potential of this strain as a proficient producer of 1,3-propanediol (PDO) in sustainable bioprocessing. The research explores the potential of K. variicola JYP01 as the efficient PDO producers with the emphasis on the integration of zero-valent iron (ZVI) to enhance energy production and PDO yield. MethodsIsolation and characterization of the JYP01 strain, assessing its growth and PDO production under both aerobic and anaerobic conditions, and incorporating ZVI (as an electron donor) to identify the metabolic fluxes. Several analyses, including 16S rRNA analysis, high-performance liquid chromatography, and bio-electrochemical system reactors, were employed to elucidate metabolic strategies and the bioconversion efficiency of glycerol to PDO of JYP01 particularly with ZVI. Significant findingsThe integration of ZVI into the bioconversion process significantly enhanced PDO production, leading to 171 % increase from 44.0 mM to 75.2 mM. This demonstrates the distinct ability of the K. variicola JYP01 strain to utilize ZVI as an extra reducing energy in an anaerobic environment. The findings underscore this strain's metabolic capacity, its suitability for glycerol conversion on a large scale, and the novel strategy of using ZVI to elevate PDO production, advancing sustainable bioprocessing techniques.

Unveiling the potential of specific growth rate control in fed-batch fermentation: bridging the gap between product quantity and quality.

During the epoch of sustainable development, leveraging cellular systems for production of diverse chemicals via fermentation has garnered attention. Industrial fermentation, extending beyond strain efficiency and optimal conditions, necessitates a profound understanding of microorganism growth characteristics. Specific growth rate (SGR) is designated as a key variable due to its influence on cellular physiology, product synthesis rates and end-product quality. Despite its significance, the lack of real-time measurements and robust control systems hampers SGR control strategy implementation. The narrative in this contribution delves into the challenges associated with the SGR control and presents perspectives on various control strategies, integration of soft-sensors for real-time measurement and control of SGR. The discussion highlights practical and simple SGR control schemes, suggesting their seamless integration into industrial fermenters. Recommendations provided aim to propose new algorithms accommodating mechanistic and data-driven modelling for enhanced progress in industrial fermentation in the context of sustainable bioprocessing.

Salt stimulates sulfide−driven autotrophic denitrification: Microbial network and metagenomics analyses

Sulfur autotrophic denitrification (SADN) is a promising biological wastewater treatment technology for nitrogen removal, and its performance highly relies on the collective activities of the microbial community. However, the effect of salt (a prevailing characteristic of some nitrogen−containing industrial wastewaters) on the microbial community of SADN is still unclear. In this study, the response of the sulfide−SADN process to different salinities (i.e., 1.5 % salinity, 0.5 % salinity, and without salinity) as well as the involved microbial mechanisms were investigated by molecular ecological network and metagenomics analyses. Results showed that the satisfactory nitrogen removal efficiency (>97 %) was achieved in the sulfide−SADN process (S/N molar ratio of 0.88) with 1.5 % salinity. In salinity scenarios, the genus Thiobacillus significantly proliferated and was detected as the dominant sulfur−oxidizing bacteria in the sulfide−SADN system, occupying a relative abundance of 29.4 %. Network analysis further elucidated that 1.5 % salinity had enabled the microbial community to form a more densely clustered network, which intensified the interactions between microorganisms and effectively improved the nitrogen removal performance of the sulfide−SADN. Metagenomics sequencing revealed that the abundance of functional genes encoding for key enzymes involved in SADN, dissimilatory nitrate reduction to ammonium, and nitrification was up−regulated in the 1.5 % salinity scenario compared to that without salinity, stimulating the occurrence of multiple nitrogen transformation pathways. These multi−paths contributed to a robust SADN process (i.e., nitrogen removal efficiency >97 %, effluent nitrogen <2.5 mg N/L). This study deepens our understanding of the effect of salt on the SADN system at the community and functional level, and favors to advance the application of this sustainable bioprocess in saline wastewater treatment.

A sustainable bioprocessing system leveraging gas fermentation and bipolar membrane electrodialysis system for direct recovery of acetic acid

This study examined the integration of gas fermentation with bipolar membrane electrodialysis (BPMED) for sustainable production of acetic acid and bases, focusing on medium recycling and ion-exchange membrane optimisation. Gas fermentation was performed using an acetogenic microbe, Eubacterium callanderi KIST612, in a custom-designed gas bioreactor. The BPMED system was operated under specific voltage conditions to evaluate the proton and hydroxyl ion generation, ion concentration, and residual ratios. Gas fermentation consistently generated acetate with a stable CO2 consumption rate (0.167 mmol gcell−1) and an increased rate of conversion of carbon into biochemicals via methanol. The BPMED system efficiently produced acetic acid (acetate extraction rate of 99.72 %–99.82 %) and hydroxide ions (from 8 to 18 V), with operational challenges identified at higher voltage levels (≥20 V). The BPMED process, which includes additional membranes and exhibits a lower acetate flux than the ED process, had remarkable cost-effectiveness, surpassing the ED integration process by 17.6 %. This economic advantage was paralleled by a significant environmental benefit, where the BPMED process reduced carbon dioxide emissions by 7.4 % compared with the ED process. These results suggest that integrating gas fermentation with BPMED is a viable and sustainable approach for acetic acid production.

A sustainable bioprocess to produce bacterial cellulose (BC) using waste streams from wine distilleries and the biodiesel industry: evaluation of BC for adsorption of phenolic compounds, dyes and metals

BackgroundThe main challenge for large-scale production of bacterial cellulose (BC) includes high production costs interlinked with raw materials, and low production rates. The valorization of renewable nutrient sources could improve the economic effectiveness of BC fermentation while their direct bioconversion into sustainable biopolymers addresses environmental pollution and/or resource depletion challenges. Herein a green bioprocess was developed to produce BC in high amounts with the rather unexplored bacterial strain Komagataeibacter rhaeticus, using waste streams such as wine distillery effluents (WDE) and biodiesel-derived glycerol. Also, BC was evaluated as a bio-adsorbent for phenolics, dyes and metals removal to enlarge its market diversification.ResultsBC production was significantly affected by the WDE mixing ratio (0–100%), glycerol concentration (20–45 g/L), type of glycerol and media-sterilization method. A maximum BC concentration of 9.0 g/L, with a productivity of 0.90 g/L/day and a water holding capacity of 60.1 g water/g dry BC, was achieved at 100% WDE and ≈30 g/L crude glycerol. BC samples showed typical cellulose vibration bands and average fiber diameters between 37.2 and 89.6 nm. The BC capacity to dephenolize WDE and adsorb phenolics during fermentation reached respectively, up to 50.7% and 26.96 mg gallic acid equivalents/g dry BC (in-situ process). The produced BC was also investigated for dye and metal removal. The highest removal of dye acid yellow 17 (54.3%) was recorded when 5% of BC was applied as the bio-adsorbent. Experiments performed in a multi-metal synthetic wastewater showed that BC could remove up to 96% of Zn and 97% of Cd.ConclusionsThis work demonstrated a low-carbon approach to produce low-cost, green and biodegradable BC-based bio-adsorbents, without any chemical modification. Their potential in wastewater-treatment-applications was highlighted, promoting closed-loop systems within the circular economy era. This study may serve as an orientation for future research towards competitive or targeted adsorption technologies for wastewater treatment or resources recovery.

Shared and more specific genetic determinants and pathways underlying yeast tolerance to acetic, butyric, and octanoic acids

BackgroundThe improvement of yeast tolerance to acetic, butyric, and octanoic acids is an important step for the implementation of economically and technologically sustainable bioprocesses for the bioconversion of renewable biomass resources and wastes. To guide genome engineering of promising yeast cell factories toward highly robust superior strains, it is instrumental to identify molecular targets and understand the mechanisms underlying tolerance to those monocarboxylic fatty acids. A chemogenomic analysis was performed, complemented with physiological studies, to unveil genetic tolerance determinants in the model yeast and cell factory Saccharomyces cerevisiae exposed to equivalent moderate inhibitory concentrations of acetic, butyric, or octanoic acids.ResultsResults indicate the existence of multiple shared genetic determinants and pathways underlying tolerance to these short- and medium-chain fatty acids, such as vacuolar acidification, intracellular trafficking, autophagy, and protein synthesis. The number of tolerance genes identified increased with the linear chain length and the datasets for butyric and octanoic acids include the highest number of genes in common suggesting the existence of more similar toxicity and tolerance mechanisms. Results of this analysis, at the systems level, point to a more marked deleterious effect of an equivalent inhibitory concentration of the more lipophilic octanoic acid, followed by butyric acid, on the cell envelope and on cellular membranes function and lipid remodeling. The importance of mitochondrial genome maintenance and functional mitochondria to obtain ATP for energy-dependent detoxification processes also emerged from this chemogenomic analysis, especially for octanoic acid.ConclusionsThis study provides new biological knowledge of interest to gain further mechanistic insights into toxicity and tolerance to linear-chain monocarboxylic acids of increasing liposolubility and reports the first lists of tolerance genes, at the genome scale, for butyric and octanoic acids. These genes and biological functions are potential targets for synthetic biology approaches applied to promising yeast cell factories, toward more robust superior strains, a highly desirable phenotype to increase the economic viability of bioprocesses based on mixtures of volatiles/medium-chain fatty acids derived from low-cost biodegradable substrates or lignocellulose hydrolysates.

Production of D -tagatose, bioethanol, and microbial protein from the dairy industry by-product whey powder using an integrated bioprocess.

We designed and constructed a green and sustainable bioprocess to efficiently coproduce D -tagatose, bioethanol, and microbial protein from whey powder. First, a one-pot biosynthesis process involving lactose hydrolysis and D -galactose redox reactions for D -tagatose production was established in vitro via a three-enzyme cascade. Second, a nicotinamide adenine dinucleotide phosphate-dependent galactitol dehydrogenase mutant, D36A/I37R, based on the nicotinamide adenine dinucleotide-dependent polyol dehydrogenase from Paracoccus denitrificans was created through rational design and screening. Moreover, an NADPH recycling module was created in the oxidoreductive pathway, and the tagatose yield increased by 3.35-fold compared with that achieved through the pathway without the cofactor cycle. The reaction process was accelerated using an enzyme assembly with a glycine-serine linker, and the tagatose production rate was 9.28-fold higher than the initial yield. Finally, Saccharomyces cerevisiae was introduced into the reaction solution, and 266.5g of D -tagatose, 162.6g of bioethanol, and 215.4g of dry yeast (including 38% protein) were obtained from 1kg of whey powder (including 810g lactose). This study provides a promising sustainable process for functional food (D -tagatose) production. Moreover, this process fully utilized whey powder, demonstrating good atom economy.

Exploring The Potential of Microalgae-Fungi Co-Cultivation for Sustainable Bioprocessing in Microalgae Biorefinery

Developing co-cultivation systems involving microalgae and fungi has shown promising potential for microalgae harvesting technology. As discussed in this review, the co-cultivation of microalgae and fungi has emerged as a novel approach for enhancing biomass and lipid production, wastewater treatment, biofuel production, and high-value products. However, despite being used for a few years, this technique is still in its early stages of development and has yet to be widely applied in the industry. The main challenges associated with co-cultivation include designing effective cultivation systems, managing nutrient requirements, selecting compatible strains, and implementing contamination control measures. In this study, bibliometric analysis was conducted (using the Web of Science database) to examine global trends and developments in microalgae-fungi co-cultivation research between 2014 and 2023, which aimed to identify the research progression, prominent contributors, and leading countries in the research field. The dataset comprised 682 articles, 242 reviews, 31 book chapters, and 22 conference papers. The results showed a rapid increment of publications with China as an active nation in this research area, followed by India, the USA, Italy, Spain, etc. As demonstrated in this study, the immense potential of co-cultivation techniques suggests further exploration, particularly in employing different microalgae species with exceptional characteristics in conjunction with non-pathogenic and edible fungi for profitable industrialization.

Sustainable Bioprocess Combining Fed-Batch Pretreatment Followed by Semi-Continuous Anaerobic Digestion of Brewer’s Spent Grains for Biomethane Production

Sustainable Bioprocess Combining Fed-Batch Pretreatment Followed by Semi-Continuous Anaerobic Digestion of Brewer’s Spent Grains for Biomethane Production

Complementation of reducing power for 5-hydroxyvaleric acid and 1,5-pentanediol production via glucose dehydrogenase in Escherichia coli whole-cell system

One of the key intermediates, 5-hydroxyvaleric acid (5-HV), is used in the synthesis of polyhydroxyalkanoate monomer, δ-valerolactone, 1,5-pentanediol (1,5-PDO), and many other substances. Due to global environmental problems, eco-friendly bio-based synthesis of various platform chemicals and key intermediates are socially required, but few previous studies on 5-HV biosynthesis have been conducted. To establish a sustainable bioprocess for 5-HV production, we introduced gabT encoding 4-aminobutyrate aminotransferase and yqhD encoding alcohol dehydrogenase to produce 5-HV from 5-aminovaleric acid (5-AVA), through glutarate semialdehyde in Escherichia coli whole-cell reaction. As, high reducing power is required to produce high concentrations of 5-HV, we newly introduced glucose dehydrogenase (GDH) for NADPH regeneration system from Bacillus subtilis 168. By applying GDH with D-glucose and optimizing the parameters, 5-HV conversion rate from 5-AVA increased from 47% (w/o GDH) to 82% when using 200 mM (23.4 g/L) of 5-AVA. Also, it reached 56% conversion in 2 h, showing 56 mM/h (6.547 g/L/h) productivity from 200 mM 5-AVA, finally reaching 350 mM (41 g/L) and 14.6 mM/h (1.708 g/L/h) productivity at 24 h when 1 M (117.15 g/L) 5-AVA was used. When the whole-cell system with GDH was expanded to produce 1,5-PDO, its production was also increased 5-fold. Considering that 5-HV and 1,5-PDO production depends heavily on the reducing power of the cells, we successfully achieved a significant increase in 5-HV and 1,5-PDO production using GDH.