Efficient Bioconversion Research Articles

Up-flow anaerobic sludge blanket bioreactor for the production of carboxylates: effect of inocula on process performance and microbial communities

This research investigated the acidogenic fermentation (AF) of sugar cane molasses in an up-flow anaerobic sludge blanket (UASB) reactor for the production of carboxylates. The first step was to assess the optimum process temperature (25, 35 or 55 ºC) using two different granular inocula, one from a brewery company (BGS) and other from a paper plant company (PGS). These experiments determined that the most suitable temperature for carboxylates production was 25 ºC, obtaining higher bioconversions (27.3 ± 0.3% using PGS and 39.2 ± 0.2% using BGS), despite the low pH value recorded (4.0-4.2). Then, both inocula were tested in UASB reactors. As a consequence of the operational conditions (25 ºC, pH = 5.5-6, organic loading rate (OLR) = 3 gCOD·L-1·d-1 and hydraulic retention time (HRT) = 10 d), the microbial communities changed from those typical for biogas production to those specialised in the production of volatile fatty acids (VFAs). Indeed, the highest bioconversion efficiency (70.1%) was obtained with BGS, where uncultured Eubacteriaceae family microorganisms (56.0%) prevailed, enhancing the production of butyric acid (59.5 ± 2.4%w/w). Consequently, this inoculum was used to further identify the OLR threshold that should not be exceeded to attain optimal carboxylates production. OLR of 6 gCOD·L-1·d-1 resulted in a decrease in bioconversion efficiency (59.5%). The VFAs pool was dominated by butyric acid (63.0 ± 1.4%w/w at an OLR of 4.5 gCOD·L-1·d-1 and 52.8 ± 2.2%w/w at 6 gCOD·L-1·d-1). The microbial community became even more specialised, increasing the presence of Firmicutes and Actinobacteriota phyla, proving that the imposed conditions favoured the production of VFAs when operating semicontinuously fed UASB reactors.

Hermetia illucens in the Process of Kitchen Waste Biodegradation: The Effect of Different Approaches to Waste Storage on the Microbiological Profile and Nutritional Parameters of the Larvae.

This study assessed the bioconversion efficiency of Hermetia illucens larvae (BSFL) fed on food waste stored under different conditions, focusing on the nutritional and microbial quality of the resulting larval biomass. Food waste was prepared as a fresh diet (FD) or naturally contaminated and stored at 20-22 °C (OS-T, opened storage-tempered) or under refrigeration, at 5-8 °C (CS-C, closed storage-cooled). Refrigerated, closed storage (CS-C) led to the highest rates of waste reduction (91.0%) and bioconversion efficiency (30.2%), with larvae exhibiting the highest protein content (36.83%) compared to the FD (35.5%) and OS-T (34.71%) groups. Microbiome analysis revealed that the CS-C condition promoted beneficial yeasts like Pichia and Diutia, which correlated positively with improved protein content and microbial safety. In contrast, OS-T storage supported spoilage fungi (Mucor, Rhizopus) and elevated total aerobic counts (7.28 log CFU/g), indicating higher microbial risks. The observed trends in waste reduction and protein content most probably relate to differences in microbial profiles, as controlled cooling affected microbial dynamics, preserving substrate quality and supporting larval growth. These findings emphasize the importance of refrigerated, closed storage to optimize bioconversion, improve larval nutritional value, and minimize microbiological hazards.

Macronutrient-Based Predictive Modelling of Bioconversion Efficiency in Black Soldier Fly Larvae (Hermetia illucens) Through Artificial Substrates.

This study explores the optimisation of rearing substrates for black soldier fly larvae (BSFL). First, the ideal dry matter content of substrates was determined, comparing the standard 30% dry matter (DM) with substrates hydrated to their maximum water holding capacity (WHC). Substrates at maximal WHC yielded significantly higher larval survival rates (p = 0.0006). Consequently, the WHC approach was adopted for further experiments. Using these hydrated artificial substrates, fractional factorial designs based on central composite and Box-Behnken designs were employed to assess the impact of macronutrient composition on bioconversion efficiency. The results demonstrated significant main, interaction, and quadratic effects on bioconversion efficiency. Validation with real-life substrates of varied protein content, including indigestible feather meal, affirmed the predictive model's accuracy after accounting for protein source digestibility. This research underscores the importance of optimal hydration and macronutrient composition in enhancing BSFL growth and bioconversion efficiency.

Microbial synthesis of m-tyrosine via whole-cell biocatalysis.

Meta-tyrosine (m-tyrosine), a nonproteinogenic amino acid, has shown significant potential for applications as an herbicide in agriculture and for various medical uses. However, the natural abundance of m-tyrosine is very low, limiting its widespread use. In this study, we successfully achieved microbial production of m-tyrosine by establishing the in vivo enzyme activity of phenylalanine 3-hydroxylase (PacX from Streptomyces coeruleoribudus) in E. coli, which catalyzes the meta-hydroxylation of phenylalanine to produce m-tyrosine. Remarkably, PacX is capable of utilizing the native E. coli cofactor tetrahydromonapterin (MH4) for its hydroxylation activity. The integration of a non-native MH4 regeneration system significantly improved the bioconversion efficiency, resulting in the accumulation of m-tyrosine at a concentration of up to 368 mg/L. Additionally, we attempted to modify a well-characterized phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris to alter its regioselectivity through protein engineering. Remarkably, a double mutant (F184C/G199T) successfully shifted the enzyme's hydroxylation specificity from the para- to the meta-position, demonstrating the feasibility of altering the regioselectivity of aromatic amino acid hydroxylases (AAAHs). To the best of our knowledge, this is the first report of microbial production of m-tyrosine through whole-cell biocatalysis.

Evaluating Different Supplements on the Growth Performance and Bioconversion Efficiency of Kitchen Waste by Black Soldier Fly Larvae.

Black soldier fly larvae (BSFL) convert kitchen waste into high-quality insect feed. However, the optimal amount of auxiliary materials needed to improve the physical and chemical properties of kitchen waste and enhance BSFL bioconversion efficiency remains unresolved. In this study, maize stover and BSFL frass were added to kitchen waste (in groups G2 and G3, respectively) to explore their effects on the growth performance and bioconversion efficiency of BSFL. The group with only kitchen waste, without the addition of maize stover or BSF frass, was used as the control group and labeled as G1. On the 5th day, the body length of the BSFL in the G2 group was significantly greater than that in G1 and G3 (p < 0.05). The dry matter weight loss rate in the G3 group was significantly lower compared to that of G1 and G2 (p < 0.05), and the feed conversion rate (FCR) of G1 was significantly lower than that of G2 and G3 (p < 0.01). In summary, adding maize stover and BSFL frass increased BSFL feed intake and improved body weight gain. However, these additives did not significantly enhance BSFL bioconversion efficiency. The organic matter in maize stover and BSFL frass was utilized by the BSFL, and the heavy metal levels in each group of BSFL did not exceed standard limits.

Bioconversion efficiency and chemical composition of Hermetia illucens larvae fed spent mushroom substrates

Spent mushroom substrate (SMS) is a by-product remaining after harvesting mushrooms. We evaluated the effect of substituting chicken feed with 0–100% of Pleurotus eryngii and Lentinula edodes SMS at different stocking densities (200–1000 larvae/box) on development, composition, and substrate reduction of black soldier fly (Hermetia illucens) larvae. Although the survival rate was not significantly different, feeding pure SMS led to a low growth rate. The substitution level of SMS negatively correlated with individual larval weight, total harvested biomass, larval growth rate (LGR), feed conversion ratio (FCR), substrate reduction, and waste reduction index (WRI) except for the 20% substitution. Feeding 40% SMS resulted in the highest number of prepupae. In the density experiment, the heaviest larvae (220–239 mg fresh weight) were obtained at 200 larvae/box in the 0% SMS group. The frass residue and FCR decreased with increased density. Remarkably, when feeding 20% SMS at 250 larvae/box, the harvested biomass, LGR, and FCR did not differ significantly from the 0% SMS control, whereas some of the higher densities led to a deterioration. In fact, the frass residue, substrate reduction, and WRI were even improved at 250 larvae/box in the 20% SMS group. The chemical analyses of larvae reared on 20% SMS at 250 larvae/box showed comparable ash and fat contents and a higher protein content compared to the 0% SMS group. Accordingly, up to 20% of a standard diet such as chicken feed can be replaced by low-cost SMS without disadvantages for breeding.

The Effect of Polyethylene Glycol Addition on Improving the Bioconversion of Cellulose.

In recent years, many studies have focused on improving the bioconversion of cellulose by adding non-ionic surfactants. In our study, the effect of the addition of a polymer, polyethylene glycol (PEG 4000), on the bioconversion of different cellulose materials was evaluated, focusing on the hydrolysis efficiency and structural changes in pure cellulose after the enzymatic hydrolysis process. The obtained results showed that the addition of non-ionic surfactant significantly improved the digestibility of cellulosic materials. The highest hydrolysis efficiency was observed for Sigmacel 101 (Cel-S101) cellulose, which consists mainly of amorphous regions. In the case of Avicel cellulose (Cel-A), PEG had a lesser effect on the bioconversion's efficiency due to limited access to the crystal structure and limited substrate-cellulase interactions. The consistency of the obtained results is confirmed by qualitative and quantitative analyses (XRD, FTIR, and HPLC). Our findings may be helpful in further understanding the mechanism of the action of surfactants and improving the enzymatic hydrolysis process.

High-level production of free fatty acids from lignocellulose hydrolysate by co-utilizing glucose and xylose in yeast.

Lignocellulose bio-refinery via microbial cell factories for chemical production represents a renewable and sustainable route in response to resource starvation and environmental concerns. However, the challenges associated with the co-utilization of xylose and glucose often hinders the efficiency of lignocellulose bioconversion. Here, we engineered yeast Ogataea polymorpha to effectively produce free fatty acids from lignocellulose. The non-oxidative branch of the pentose phosphate pathway, and the adaptive expression levels of xylose metabolic pathway genes XYL1, XYL2 and XYL3, were systematically optimized. In addition, the introduction of xylose transporter and global regulation of transcription factors achieved synchronous co-utilization of glucose and xylose. The engineered strain produced 11.2g/L FFAs from lignocellulose hydrolysates, with a yield of up to 0.054g/g. This study demonstrated that metabolic rewiring of xylose metabolism could support the efficient co-utilization of glucose and xylose from lignocellulosic resources, which may provide theoretical reference for lignocellulose biorefinery.

Effects of biochar-loaded microbial agent in regulating nitrogen transformation and integration into humification for straw composting

Exogenous additives can impact organic matter transformation in composting, but their effects on nitrogen conversion and humification in straw composting require clarification. This study investigated how rice husk biochar-loaded microbial agent (RM) affects nitrogen transformation and humification during straw composting. Results showed that the addition of RM enhanced ammonia oxidation and assimilation during composting, leading to a 10.32%–22.27% increase in total nitrogen content. Furthermore, the RM treatment enriched nitrogen-converting microbes such as Longispora and Coprinopsis, enhancing synergistic relationships among microbes, facilitating the accumulation of pivotal nitrogenous humus precursors (amino acid nitrogen), and promoting humus formation. This research not only guides reducing nitrogen loss during composting and elucidating the relationship between nitrogen transformation and humification but also contributes to enhancing bioconversion efficiency of agricultural waste to explore new ways of straw waste management.

Genome-wide association studies unveils the genetic basis of cell wall composition and saccharification of cassava pulp

Cassava (Manihot esculenta Crantz) is a key crop for starch and biofuels production. This study focuses on the polysaccharide composition and saccharification efficiency in cassava pulp through genome-wide association studies (GWAS), targeting the improvement of root characteristics for industrial use. We analyzed 135 partially inbred lines population, performing monosaccharide composition and saccharification analyses to reveal substantial variability in storage root biomass. Among 33 traits examined, 128 significant SNPs were associated with 23 biomass traits, highlighting a complex genetic architecture. Saccharification potential varied from 39 to 95 nmol Glu mg−1 h−1, with high broad-sense heritability for saccharification and several monosaccharide traits, indicating a strong genetic control. Our findings revealed that cassava pulp comprises similar proportions of pectin, hemicellulose, and cellulose in all genotypes. Correlation analysis showed significant associations between cellulose content and saccharification, suggesting that enhancing these traits can improve bioconversion efficiency. Negative correlations with glucose and glucuronic acid in hemicellulose and pectin fractions imply these components may inhibit saccharification. We identified 118 candidate genes associated with 21 traits, with many involved in stress responses affecting cell wall composition. This study verified 12 key candidate genes through sequence and expression analysis, including MANES_07G081200, a YTH domain-containing protein associated with saccharification. Several stress-response genes, such as MANES_04G118600 and MANES_09G174600, were linked to monosaccharide traits, suggesting that adaptive stress pathways influence biomass characteristics. This study provides insights into the genetic determinants of cassava pulp's saccharification and polysaccharide composition, aiding breeding efforts to develop cassava varieties optimized for industrial applications.

Optimizing food waste bioconversion with sodium selenite-enhanced Lucilia sericata maggots: a sustainable approach for chicken feed production and heavy metal mitigation.

Recycling food waste by feeding it to insects can result in the continuous production of high-quality animal feed protein and organic fertilizer. However, the bioconversion efficiency and safety of using insects as feed protein for animal breeding are important factors limiting the development of this technology. Therefore, we aimed to optimize the efficiency of bioconversion of food waste using Lucilia sericata maggot (LSM). Sodium selenite (SS) was used to improve the quality and safety of each trophic-level organism. The results showed that an SS concentration of 15mgkg-1 w.w. in the food waste culture substrate (SS15), the yield and quality of the obtained LSMs were optimal. The total selenium (Se) content of LSMs was 82.4 ± 1.16mgkg-1 d.w., and non-inorganic Se accounted for 96.4% ± 2.01% of the total Se content. Additionally, the conversion efficiency of food waste was 18.7% higher than that in the control group (p < 0.05). When SS15 was used to raise maggots as a protein substitute for fish meal (commercial feed), the weight of the chickens and the crude protein content were 1.09-1.26times and 1.09-1.13times, respectively (p < 0.05), in comparison with the corresponding findings obtained with the use of ordinary maggots and commercial feed. In this group, glutathione peroxidase, superoxide dismutase, catalase, and immunoglobulin A and G activities were significantly higher than those obtained with the other feeds (p < 0.05). During this cyclic utilization process, the total Se content in chickens (0.31 ± 0.05mgkg-1 w.w. in the breast, 0.19 ± 0.01mgkg-1 w.w. in the leg, and 0.57 ± 0.01mgkg-1 w.w. in the liver) significantly increased (p < 0.05). Meanwhile, the Cu and Zn contents in the LSMs and chickens increased, whereas cadmium, lead, chromium, and nickel absorption was inhibited (p < 0.05). Health risk assessment based on the levels of Se and heavy metals showed that Se-enriched chickens produced using this method can be safely consumed.

Dose-response effects of starch in sterilised and unsterilised chicken excreta on Musca domestica larval growth and bioconversion efficiency

Abstract Housefly (Musca domestica L.) larvae can convert animal manure into valuable protein and fat but little is known about their nutritional requirements. We assessed the effects of differential starch content in unsterilised (UE) and heat sterilised (SE) chicken excreta on larval performance and bioconversion. Gelatinized corn starch was added to excreta resulting in 11 diets with 0 to 50% starch of dry mass. Individual larval mass increased from (mean ± SD) 12 ± 0.2 (UE) and 13 ± 0.4 (SE) mg at 0% starch up to 18 ± 0.4 (UE) and 18 ± 0.5 (SE) mg at 15% starch and decreased to 5 ± 0.4 (UE and SE) mg in 50% starch. Higher starch concentrations resulted in non-normal distributions of individual larva mass. Larval wet yield showed a curvilinear relation with starch content, with maxima of 39 ± 2.3 g in UE at 10% starch, and 43 ± 2.8 g in SE at 15% starch. The decrease in yield after the maxima was faster in UE compared to SE. Larval survival was highest in 0% starch, 80 ± 8% (UE) and 72 ± 13 % (SE), linearly decreasing to 14 ± 7% (UE) and 18 ± 3% (SE) in 50% starch. Dry matter bioconversion increased from 4 ± 0.1% (UE) and 4 ± 0.3% (SE) in 0% starch to 6 ± 0.2 in UE with 10% starch and 7 ± 0.3 in SE with 15% starch, decreasing to close to 0 in 50% starch. Highest nitrogen bioconversion was 15 ± 0.5% in UE with 10% starch and 15 ± 0.5% in SE with 15% starch. Optimal starch inclusions increased nitrogen bioconversion with 40% (UE) and 100% (SE) compared to pure excreta. Finally, around 20 to 25% starch uric acid and ammonia contents, pH, and diet residue C/N decreased, indicating a tipping point. In conclusion, adding starch to chicken excreta can substantially increase larval yield and bioconversion while reducing ammonia content in the substrate residue.

Feed-shifting strategy for increasing biodiesel production from black soldier fly larvae

The aim of this study was to increase the bioconversion efficiency (lipid accumulation) of black soldier fly larvae while simultaneously increasing biodiesel production through a feed-shifting strategy. Feeding with low-lipid feed promoted an increase in larval weight, while high-lipid feed resulted in greater lipid accumulation. Based on this result, a feed-shifting strategy was introduced, which consisted of two stages: first, increasing larval body weight using low-lipid feed, followed by lipid induction for biodiesel production using high-lipid feed. The use of this strategy resulted in an increase in the dry weight of larvae by ≥16 % compared to single feeding systems. This led to a 20 % increase in biodiesel productivity. The waste reduction ratio was enhanced due to the higher bioconversion rate in the feed-shifting strategy compared to that in the single feeding systems. The feed-shifting strategy would contribute to the enhancement of waste-to-energy efficiency.

Improvement of cellobiose 2-epimerase expression in Bacillus subtilis for efficient bioconversion of lactose to epilactose

Epilactose, a lactose derivative known for its prebiotic properties and potential health benefits, has garnered significant interest. Cellulose 2-epimerase (CEase) is responsible for catalyzing the conversion of lactose to epilactose. In this study, the enhancement of food-grade CEase expression in Bacillus subtilis WB600 was systematically investigated. Among seven selected epilactose-producing CEases, Rhodothermus marinus CEase (RmCE) exhibited the highest epimerization activity when expressed in B. subtilis. Translational and transcriptional regulations were employed to enhance CEase expression by screening effective N-terminal coding sequences (NCSs) and promoters. The final strain demonstrated efficient production of CEase, with epimerization activity reaching 273.6 ± 6.5 U/mL and 1255 ± 26.4 U/mL in shake-flask and fed-batch cultivation, respectively. Utilizing only 0.25 % (V/V) of the fed-batch cultivation broth for lactose biotransformation, epilactose was efficiently produced from 300 g/L of lactose within 4 h, achieving a yield of 29.5 %. These findings provide significant support for the potential industrialization of enzymatic epilactose production.

Efficient Bioconversion of Mango Waste into Ethanol Employing Plackett-Burman and Central Composite Models.

The current study focuses on the idea of "Energy from Waste" that intends to address energy crises and manage waste. Fruit waste is one of the most common forms of organic waste due to its inedible portion and perishable nature. In Pakistani regions, an extensive amount of mango pulp (MP)/juice waste is produced due to excessive consumption during summers, which poses huge environmental challenges. The study aims at effective valorization of perishable waste and elimination of deteriorating waste that causes a polluting environment. Experimental work has been conducted to evaluate the sucrolytic potential of Bacillus cereus FA3 for the bioconversion of sucrose from mango waste into reducing sugars for ethanologenesis. The Plackett-Burman model was designed to analyze enzymatic hydrolytic parameters for sugar conversion. The model was significant for reducing sugars with F and p values of 43.99 and 0.0013 correspondingly. 11.43 ± 0.068 g/L maximum reducing sugars were analyzed in MP after hydrolysis with 12.58 IU of crude enzyme dosage of B. cereus FA3 at 30 °C within 5 days with a 22% enzyme conversion rate. Additionally, the ethanologenic potentials of experimental Metschnikowia cibodasensis Y34 and standard Saccharomyces cerevisiae K7 yeasts were investigated from mango hydrolyzate when subjected to central composite design as a statistical optimization tool. These findings exhibited significantly higher response outcomes and good development for waste management.

Bioconversion of sugar beet molasses into functional exopolysaccharides by beet juice-derived lactic acid bacteria

Sugar beet molasses, a sugar-rich byproduct of sugar beet industry, have great potential as a biorefinery feedstock for the production of value-added bio-products by microbial fermentation. The production of functional exopolysaccharides (EPS) by lactic acid bacteria (LAB) represents a promising strategy for valorizing sugar processing waste. This study screened and identified high-efficiency EPS-producing LAB isolates from beet juice. Among the 32 isolates, six LAB strains including Limosilactobacillus fermentum YL7, Lactiplantibacillus plantarum YL4, TC10, and CJ10, Leuconostoc mesenteroides TCT3 and TCT4, exhibited efficient bio-conversion of beet molasses to LAB-EPS, with yields varying from 13.96 to 22.26 g/L. Antioxidant activity analysis revealed that EPS from these strains displayed significant scavenging activities against ABTS+, DPPH, and -OH radicals. Notably, EPS produced by L. plantarum TC10 and L. fermentum YL7 exhibited superior ABTS + scavenging capacity, with rates up to 99.48% and 95.89% at 4 mg/mL, respectively. Additionally, EPS from strains YL4, YL7, TCT3 and TCT4 showed significant antibiofilm activity against S. aureus and E. coli, with EPS-YL7 inhibiting S. aureus biofilm formation by up to 90.49% at 8.0 mg/mL. Furthermore, EPS from strains TCT4 demonstrated great tolerance to digestive fluid compared to EPS from other strains, with a retention rate of 72% at the end of intestinal digestion. These findings suggest that EPS-producing LAB strains from beet juice can efficiently convert beet molasses into valuable EPS utilized as functional food ingredients.

Bioconversion of L-Tyrosine into p-Coumaric Acid by Tyrosine Ammonia-Lyase Heterologue of Rhodobacter sphaeroides Produced in Pseudomonas putida KT2440.

p-Coumaric acid (p-CA) is a valuable compound with applications in food additives, cosmetics, and pharmaceuticals. However, traditional production methods are often inefficient and unsustainable. This study focuses on enhancing p-CA production efficiency through the heterologous expression of tyrosine ammonia-lyase (TAL) from Rhodobacter sphaeroides in Pseudomonas putida KT2440. TAL catalyzes the conversion of L-tyrosine into p-CA and ammonia. We engineered P. putida KT2440 to express TAL in a fed-batch fermentation system. Our results demonstrate the following: (i) successful integration of the TAL gene into P. putida KT2440 and (ii) efficient bioconversion of L-tyrosine into p-CA (1381 mg/L) by implementing a pH shift from 7.0 to 8.5 during fed-batch fermentation. This approach highlights the viability of P. putida KT2440 as a host for TAL expression and the successful coupling of fermentation with the pH-shift-mediated bioconversion of L-tyrosine. Our findings underscore the potential of genetically modified P. putida for sustainable p-CA production and encourage further research to optimize bioconversion steps and fermentation conditions.

Oriented regulation of earthworm production and vermicompost quality by carbon bioavailability management

Vermicomposting is an efficient bioconversion technology for recycling nutrients from organic waste materials. The biodegradability of raw materials has a significant impact on the earthworm transformation product. However, the management of carbon bioavailability is often overlooked during the vermicomposting process due to the varying degradability of C-rich source in different organic waste. This research aims to investigate the impact of different bioavailable carbon compositions on vermicomposting and to develop a strategy for efficient carbon management. The study involved systematic vermicomposting using four different biodegradable carbon sources (pineapple peels, rice straw, tomato straw, and sawdust) with varying carbon‑nitrogen ratios (ranging from 24 to 42). The earthworm production and vermicompost quality were comprehensively evaluated, along with the influence of carbon components on microbial community structure. The results indicated that the optimal vermicomposting treatments were achieved at PCM24, RCM30, TCM30, and MCM30 treatments. Maintaining an approximate ratio of 1:(0.5–1.3) between available and recalcitrant carbon components based on the optimal carbon‑nitrogen ratio was found to be optimal for regulating vermicomposting products. Increasing the proportion of available carbon enhanced the quality of vermicompost fertilizer, while a higher proportion of recalcitrant carbon could improve earthworm biomass production efficiency. Labile carbon proportion I (LCP1) and available carbon component (ACC) were identified as key indicators in influencing the formation of microbial community structure. Different carbon compositions led to the specific development and formation of microbial communities, further resulting in significant variations in vermicompost quality under the mediation of microbes. This study, for the first time, clarifies the impact of vermicomposting performance and microbial community from the perspective of carbon bioavailability, which is of great significance for the oriented regulation the vermicomposting efficiency and product in practice.

Systems biology of electrogenic Pseudomonas putida - multi-omics insights and metabolic engineering for enhanced 2-ketogluconate production

BackgroundPseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production.ResultsWhen incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol).ConclusionsBy integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida.

Fatty Acid Bioconversion and Scaling-Up Effects of Swine Manure Treatment with Black Soldier Fly Larvae

Black soldier fly larvae (BSFL) treatment offers a promising avenue for manure valorization. However, there is a lack of larval density studies and ton-scale exploration in swine manure bioconversion. This study delves into the efficiency of larval fatty acid (FA) bioconversion, examining the impact of larval density on a kilogram scale and extending the analysis to a ton scale. Across a range of 50 to 600 larvae/kg, the larval FA content decreased from 15.3% to 7.85%. The peak larval FA yield, at 3.04% (based on manure dry matter), occurred at a density of 200 larvae/kg. Both low (50 larvae/kg) and high (600 larvae/kg) densities adversely affected BSFL bioconversion performance. Dominant larval FAs included C12:0 (39.7%), C16:1 (24.2%), C18:1 (17.5%), and C16:0 (8.3%). The scaling-up process maintained a consistent larval FA content and composition but resulted in decreased larvae FA yield due to increased larval mortality. Ultimately, each ton of swine manure yielded 12.4 kg of fresh larvae or 0.71 kg of larval FA components, corresponding to a 1.14% larval FA yield. This study underscores the feasibility of upscaling swine manure treatment using BSFL for FA bioconversion and emphasizes the necessity for large-scale studies to enhance larval survivorship and bioconversion efficiency.