Substrate Consumption Research Articles

Bioconversion performances of black soldier fly larvae (<em>Hermetia illucens</em> L. Diptera: Stratiomyidae) on selected organic substrates

Black Soldier Fly Larvae (BSFL; Hermetia illucens L.) is a bioconversion agent of organic substrates and its proven ability to degrade and biotransform of waste strengthens organic waste management and waste recycling strategies. BSFL have the advantages of rapid development and very efficient conversion of various organic wastes into body biomass. This process reduces the disposal costs of organic waste as well as the use of energy providing a sustainable solution to the treatment of organic waste. In addition, it provides valuable products and fertilizers for agriculture, potentially reducing crop production costs for farmers. The current study investigated the potential live biomass production of BSFL using different organic substrates available locally while understanding their decomposition ability. Seven treatments were compared (T1- equal proportion of poultry manure and rice bran, T2- equal proportion of goat manure and rice bran, T3- rabbit droppings, T4- soybean meal, T5- coconut scrapings, T6- equal proportion of vegetable and fruit waste and T7- cow dung) and the experiment was set up as completely randomized design with three replicates. The study results examined the ability of BSFL to reduce the weight of the end-substrate and promote the degradation of organic waste materials. The live biomass production of larvae, changes in live biomass production, substrate consumption rate, the weight of the end-substrate and substrate conversion efficiency were significantly different among treatments. The greatest live biomass production of BSFL was recorded in coconut scrapings and vegetables & fruit waste mixture while the lowest value was observed in cow dung. Coconut scrapings reduced most of their weight at the end of the study indicating the highest substrate consumption rate and the total weight of the end-substrate consisted of < 2mm particle in size. However, the highest substrate conversion rate was observed in cow dung. Aqueous solutions prepared from the end-substrates varied widely in pH, electrical conductivity, total dissolved solids, and salinity. In addition, significant differences were found among treatments for the colour and texture of the end-substrate. However, the appearance and odour did not differ significantly. According to the results of the present study, it can be concluded that the most efficient media for BSFL was coconut scraping though; it cannot be used as organic fertilizer as it exceeded the pH value of the SLS recommendation for compost. Overall, BSFL can be recognized as an effective and environmentally friendly tool to use in the biotransformation process of locally available organic waste materials.

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only). IMPORTANCE A newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Rhamnolipid production by Pseudomonas aeruginosa (SSL-4) on waste engine oil (WEO): Taguchi optimization, soil remediation, and phytotoxicity investigation

ABSTRACT Environmental concerns and rising biosurfactant demand emphasize the need for this study. The objective is to maximize rhamnolipid-biosurfactant production by Pseudomonas aeruginosa (SSL-4) utilizing waste engine oil (WEO) as the sole substrate for use in soil bioremediation and commercial production. Using an L16 Taguchi orthogonal array, a signal-to-noise ratio, and an analysis of variance (ANOVA), the effects of environmental (pH, incubation temperature) and dietary parameters (carbon source concentration, carbon/nitrogen (C/N) and carbon/phosphorus (C/P) ratio) are examined. Variations of the following parameters were made within a carefully selected range: incubation temperature of 25–40℃, pH range of 5–11, WEO concentration of 1–7% (v/v), and C/N and C/P ratios of 10–40. Response variables in this batch study include surface tension reduction (mN/m), dry cell biomass (DCBM) (g/L), and rhamnolipids yield based on substrate consumption, YP/S (g/g). Rhamnolipid was synthesized under optimal conditions, providing a yield of 21.42 g/g. The oil recovery of 74.05 ± 1.481% was achieved from oil-contaminated soil at a CMC of ∼70 mg/L. FTIR, 1H NMR, and UPLC-MS techniques were utilized for the characterization of rhamnolipids, and AAS for determining heavy metals concentration in WEO and residual waste engine oil (RWEO). The Germination Index (GI) of ∼82.55% indicated no phytotoxicity associated with synthesized rhamnolipid.

From shake flask to bioreactor: Production of Salmonella Typhi porins as a typhoid vaccine candidate

Worldwide, Salmonella Typhi infections persist as an important health problem, accounting for about 9.4 million cases annually. Porins are one of the targets in the protective response against infection with Salmonella Typhi, a vaccine designed with these porins was shown to be safe in a phase 1 clinical trial eliciting a long-lasting response. In this work, an airlift bioreactor was designed and used to produce Salmonella Typhi biomass and purify porins. A comparison between the airlift bioreactor and shaken flasks biomass production rates was carried out considering a constant oxygen mass transfer coefficient. The analysis showed that the change in culture system does not affect the growth kinetic parameters, nor the maximum biomass production. The growth and substrate consumption kinetic parameters were estimated through mathematical models. Porins produced in the bioreactor induced equivalent humoral responses and protective capacity in mice compared to porins produced in shaken flasks. These results suggest that airlift bioreactors provide a promising platform for industrial production of porins.

Regulation of Embden-Meyerhof-Parnas (EMP) Pathway and Tricarboxylic Acid (TCA) Cycle Concerning Aberrant Chilling Injury Behavior in Postharvest Papaya (Carica papaya L.).

Postharvest abnormal chilling injury (CI) behavior in papaya (Carica papaya L.) fruit is a rare phenomenon that may be associated with respiratory metabolism. This study thus aimed to investigate the impacts of storage temperatures (1 and 6 °C) on the respiratory metabolism of postharvest papaya and its impact on CI development. Results demonstrated that 1 °C storage reduced the activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), citrate synthase (CS), and α-ketoglutarate dehydrogenase (α-KGDH) and regulated the expression of corresponding enzymes in the Embden-Meyerhof-Parnas (EMP) pathway and tricarboxylic acid (TCA) cycle compared with 6 °C storage, resulting in a lower respiration rate of the EMP-TCA pathway and mitigating the development of CI. Meanwhile, lower contents of nicotinamide adenine dinucleotide (hydrogen) (NAD(H)) were observed in papaya fruit stored at 1 °C. Notably, papaya fruit stored at 1 °C maintained higher activity and transcriptional levels of SDH and IDH during the whole storage period. These findings suggest that 1 °C storage reduced the respiration rate of the EMP-TCA pathway by reducing the expression level and activity of related enzymes, which is conducive to the reduction of respiration substrate consumption and finally alleviating the occurrence of CI.

Obtaining lactic acid through microbial fermentation from corn residues

The production and kinetics of lactic acid (LA) by batch fermentation were studied, using corn residues as substrate with Lactobacillus delbrueckii spp. Bulgaricus. A factorial design was developed for the pretreatment stage of the residue and lactic fermentation to determine the best conditions for obtaining total reducing sugars and lactic acid. In the pretreatment, the best conditions were obtained by working with HCl at 3% (v/v), 75 °C, and 30 minutes of reaction. Fermentation was carried out, keeping the medium at a pH between 5.5-6, and the best working conditions were 100 µl of the microorganism, 37ºC, and 24 hours of fermentation, reaching a maximum concentration of 19.9 g/L. Kinetic models for LA production, bacterial growth, and substrate consumption were proposed. Likewise, to verify the observed data, different statistical indices were calculated to evaluate the accuracy of the models. Finally the Luedeking-Piret and logistic models, were selected as they presented the best fit to the experimental data for LA production, substrate consumption, and bacterial growth, respectively.

Regulated bacterial interaction networks: A mathematical framework to describe competitive growth under inclusion of metabolite cross-feeding.

When bacterial species with the same resource preferences share the same growth environment, it is commonly believed that direct competition will arise. A large variety of competition and more general 'interaction' models have been formulated, but what is currently lacking are models that link monoculture growth kinetics and community growth under inclusion of emerging biological interactions, such as metabolite cross-feeding. In order to understand and mathematically describe the nature of potential cross-feeding interactions, we design experiments where two bacterial species Pseudomonas putida and Pseudomonas veronii grow in liquid medium either in mono- or as co-culture in a resource-limited environment. We measure population growth under single substrate competition or with double species-specific substrates (substrate 'indifference'), and starting from varying cell ratios of either species. Using experimental data as input, we first consider a mean-field model of resource-based competition, which captures well the empirically observed growth rates for monocultures, but fails to correctly predict growth rates in co-culture mixtures, in particular for skewed starting species ratios. Based on this, we extend the model by cross-feeding interactions where the consumption of substrate by one consumer produces metabolites that in turn are resources for the other consumer, thus leading to positive feedback in the species system. Two different cross-feeding options were considered, which either lead to constant metabolite cross-feeding, or to a regulated form, where metabolite utilization is activated with rates according to either a threshold or a Hill function, dependent on metabolite concentration. Both mathematical proof and experimental data indicate regulated cross-feeding to be the preferred model to constant metabolite utilization, with best co-culture growth predictions in case of high Hill coefficients, close to binary (on/off) activation states. This suggests that species use the appearing metabolite concentrations only when they are becoming high enough; possibly as a consequence of their lower energetic content than the primary substrate. Metabolite sharing was particularly relevant at unbalanced starting cell ratios, causing the minority partner to proliferate more than expected from the competitive substrate because of metabolite release from the majority partner. This effect thus likely quells immediate substrate competition and may be important in natural communities with typical very skewed relative taxa abundances and slower-growing taxa. In conclusion, the regulated bacterial interaction network correctly describes species substrate growth reactions in mixtures with few kinetic parameters that can be obtained from monoculture growth experiments.

ATP yield of plant respiration: potential, actual and unknown.

The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c-ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase - a typically observed fraction - ATP yield falls 15 % below its potential. Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36-38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c-ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs.

Enforcing energy consumption promotes microbial extracellular respiration for xenobiotic bioconversion.

Extracellular electron transfer (EET) empowers electrogens to catalyse the bioconversion of a wide range of xenobiotics in the environment. Synthetic bioengineering has proven effective in promoting EET output. However, conventional strategies mainly focus on modifications of EET-related genes or pathways, which leads to a bottleneck due to the intricate nature of electrogenic metabolic properties and intricate pathway regulation that remain unelucidated. Herein, we propose a novel EET pathway-independent approach, from an energy manipulation perspective, to enhance microbial EET output. The Controlled Hydrolyzation of ATP to Enhance Extracellular Respiration (CHEER) strategy promotes energy utilization and persistently reduces the intracellular ATP level in Shewanella oneidensis, a representative electrogenic microbe. This approach leads to the accelerated consumption of carbon substrate, increased biomass accumulation and an expanded intracellular NADH pool. Both microbial electrolysis cell and microbial fuel cell tests exhibit that the CHEER strain substantially enhances EET capability. Analysis of transcriptome profiles reveals that the CHEER strain considerably bolsters biomass synthesis and metabolic activity. When applied to the bioconversion of model xenobiotics including methyl orange, Cr(VI) and U(VI), the CHEER strain consistently exhibits enhanced removal efficiencies. This work provides a new perspective and a feasible strategy to enhance microbial EET for efficient xenobiotic conversion.

Selective Removal of Various Resilient Ionic and Halides-Based Surface Contaminants by Wet Cleaning

In this paper, we investigate the use of a wet cleaner developed to dissolve residues left after plasma etching selectively to aluminum in new applications such as the removal of various resilient ionic and halides-based surface contaminants. The compromise between efficiency and selectivity of this fluorinated acid cleaner makes it an interesting chemistry for many cleaning steps during wafer manufacturing. Two new applications were investigated: the selective removal of fluoride-based defects on aluminum pads and surface decontamination of various ionic metals with low substrate consumption. These studies showed very encouraging results in beakers level tests, but also on partly industrial equipment, opening new possibilities for this cleaner. Indeed, “water lily” defects removal seems to be possible with a low aluminum consumption in TechniClean IK73. Decontamination study carried out in static bath and in single wafer tool showed rather similar results, enabling static bath protocol and ionic metals removal to be validated and VPD-ICPMS results, after a short process time in a single wafer tool were very promising with metal levels close to the lower limit of detection of all elements tested.

The genome-scale metabolic model for the purple non-sulfur bacterium Rhodopseudomonas palustris Bis A53 accurately predicts phenotypes under chemoheterotrophic, chemoautotrophic, photoheterotrophic, and photoautotrophic growth conditions.

The purple non-sulfur bacterium Rhodopseudomonas palustris is recognized as a critical microorganism in the nitrogen and carbon cycle and one of the most common members in wastewater treatment communities. This bacterium is metabolically extremely versatile. It is capable of heterotrophic growth under aerobic and anaerobic conditions, but also able to grow photoautotrophically as well as mixotrophically. Therefore R. palustris can adapt to multiple environments and establish commensal relationships with other organisms, expressing various enzymes supporting degradation of amino acids, carbohydrates, nucleotides, and complex polymers. Moreover, R. palustris can degrade a wide range of pollutants under anaerobic conditions, e.g., aromatic compounds such as benzoate and caffeate, enabling it to thrive in chemically contaminated environments. However, many metabolic mechanisms employed by R. palustris to breakdown and assimilate different carbon and nitrogen sources under chemoheterotrophic or photoheterotrophic conditions remain unknown. Systems biology approaches, such as metabolic modeling, have been employed extensively to unravel complex mechanisms of metabolism. Previously, metabolic models have been reconstructed to study selected capabilities of R. palustris under limited experimental conditions. Here, we developed a comprehensive metabolic model (M-model) for R. palustris Bis A53 (iDT1294) consisting of 2,721 reactions, 2,123 metabolites, and comprising 1,294 genes. We validated the model using high-throughput phenotypic, physiological, and kinetic data, testing over 350 growth conditions. iDT1294 achieved a prediction accuracy of 90% for growth with various carbon and nitrogen sources and close to 80% for assimilation of aromatic compounds. Moreover, the M-model accurately predicts dynamic changes of growth and substrate consumption rates over time under nine chemoheterotrophic conditions and demonstrated high precision in predicting metabolic changes between photoheterotrophic and photoautotrophic conditions. This comprehensive M-model will help to elucidate metabolic processes associated with the assimilation of multiple carbon and nitrogen sources, anoxygenic photosynthesis, aromatic compound degradation, as well as production of molecular hydrogen and polyhydroxybutyrate.

Six Functions of Respiration: Isn't It Time to Take Control over ROS Production in Mitochondria, and Aging Along with It?

Cellular respiration is associated with at least six distinct but intertwined biological functions. (1) biosynthesis of ATP from ADP and inorganic phosphate, (2) consumption of respiratory substrates, (3) support of membrane transport, (4) conversion of respiratory energy to heat, (5) removal of oxygen to prevent oxidative damage, and (6) generation of reactive oxygen species (ROS) as signaling molecules. Here we focus on function #6, which helps the organism control its mitochondria. The ROS bursts typically occur when the mitochondrial membrane potential (MMP) becomes too high, e.g., due to mitochondrial malfunction, leading to cardiolipin (CL) oxidation. Depending on the intensity of CL damage, specific programs for the elimination of damaged mitochondria (mitophagy), whole cells (apoptosis), or organisms (phenoptosis) can be activated. In particular, we consider those mechanisms that suppress ROS generation by enabling ATP synthesis at low MMP levels. We discuss evidence that the mild depolarization mechanism of direct ATP/ADP exchange across mammalian inner and outer mitochondrial membranes weakens with age. We review recent data showing that by protecting CL from oxidation, mitochondria-targeted antioxidants decrease lethality in response to many potentially deadly shock insults. Thus, targeting ROS- and CL-dependent pathways may prevent acute mortality and, hopefully, slow aging.

Mutagenesis enhances gellan gum production by a novel Sphingomonas spp.: upstream optimization, kinetic modeling, and structural and physico-functional evaluation.

Gellan gum (GG) has gained tremendous attention owing to its diversified applications. However, its high production and hence market cost are still a bottleneck in its widespread utilization. In the present study, high GG producing mutant of Sphingomonas spp. was developed by random mutagenesis using ethyl methylsulphonate (EMS) for industrial fermentation and identified as Sphingomonas trueperi after 16S rRNA and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. The fermentation conditions such as pH, temperature, and inoculum ratio were optimized by one factor at a time (OFAT) followed by screening of medium components by the Plackett-Burman statistical design. The most critical nutrients were further optimized by response surface methodology for maximizing GG production. The effect of dissolved oxygen tension in bioreactor on cell growth, substrate consumption, GG production, and batch productivity was elucidated. The highest GG titer (23 ± 2.4g/L) was attained in optimized medium at 10% inoculum (6.45 ± 0.5 log cfu/mL) under controlled fermentation conditions of pH (7), temperature (30°C), agitation (300-600rpm), and aeration (0.5-2.0 SLPM) at 22 ± 2% dissolved oxygen tension in a 10-L bioreactor. Kinetic modeling of optimized batch process revealed that logistic growth model could best explain biomass accumulation, while GG formation and substrate consumption were best explained by Luedeking-Piret and exponential decay model, respectively. Structural and physico-functional features of GG produced by mutant Sphingomonas spp. were characterized by HPLC, FTIR, NMR, DSC, TGA, GPC, SEM, and rheological analysis. The higher productivity (0.51g/L/h) under optimized fermentation conditions suggests potential consideration of mutant and process for commercial utilization.

Ethanol production by Kluyveromyces marxianus ATCC 36907: Fermentation features and mathematical modeling

The effects of different temperatures (30 °C, 35 °C, 40 °C, and 45 °C) and initial glucose concentrations (50 g/L, 120 g/L, and 190 g/L) on the fermentative metabolism of Kluyveromyces marxianus ATCC 36907 were evaluated to optimize ethanol production. Mathematical models were developed based on experimental data to describe cell growth, substrate consumption, and ethanol production. Various kinetic models integrated with material balances were examined. The Akaike information criterion (AIC) was employed to identify the most accurate model representing the experimental behavior. Interestingly, the ethanol production rate was independent of the growth rate of K. marxianus. The growth kinetics indicated that the growth rate could be controlled by the oxygen concentration, which was influenced by cell concentration rather than glucose consumption. A desirability function was utilized to optimize ethanol productivity, substrate conversion efficiency and temperature, considering the conditions for future applications in simultaneous saccharification and fermentation (SSF) processes. The optimized experimental conditions obtained were validated, resulting in a new model specific to these conditions. This new model analysis confirmed the achievement of the objective to prioritize ethanol production. The optimized conditions for maximum ethanol production were initial glucose concentration of 91 g/L and temperature of 40 °C. Under these conditions, the ethanol concentration reached 12 g/L, with glucose conversion efficiency into ethanol of 0.13 g/g and productivity of 1.09 g/L/h after 11 h. This approach provided valuable insights into the fermentative behavior of K. marxianus, serving as a foundation for the design, optimization, and process control in biorefineries.

Comparative Metagenomic and Metatranscriptomic Analyses Reveal the Response of Black Soldier Fly (Hermetia illucens) Larvae Intestinal Microbes and Reduction Mechanisms to High Concentrations of Tetracycline.

Black soldier fly (Hermetia illucens L) larvae (BSFL) possess remarkable antibiotic degradation abilities due to their robust intestinal microbiota. However, the response mechanism of BSFL intestinal microbes to the high concentration of antibiotic stress remains unclear. In this study, we investigated the shift in BSFL gut microbiome and the functional genes that respond to 1250 mg/kg of tetracycline via metagenomic and metatranscriptomic analysis, respectively. The bio-physiological phenotypes showed that the survival rate of BSFL was not affected by tetracycline, while the biomass and substrate consumption of BSFL was slightly reduced. Natural BSFL achieved a 20% higher tetracycline degradation rate than the germ-free BSFL after 8 days of rearing. Metagenomic and metatranscriptomic sequencing results revealed the differences between the entire and active microbiome. Metatranscriptomic analysis indicated that Enterococcus, Vagococcus, Providencia, and Paenalcaligenes were the active genera that responded to tetracycline. Furthermore, based on the active functional genes that responded to tetracycline pressure, the response mechanisms of BSFL intestinal microbes were speculated as follows: the Tet family that mediates the expression of efflux pumps expel tetracycline out of the microbes, while tetM and tetW release it from the ribosome. Eventually, tetracycline was degraded by deacetylases and novel enzymes. Overall, this study provides novel insights about the active intestinal microbes and their functional genes in insects responding to the high concentration of antibiotics.

Substrate type and CO2 addition significantly influence succinic acid production of Basfia succiniciproducens.

Metabolic engineering has shown that optimizing metabolic pathways' fluxes for industrial purposes requires a methodical approach. Accordingly, in this study, in silico metabolic modeling was utilized to characterize the lesser-known strain Basfia succiniciproducens under different environmental conditions, followed by the use of industrially relevant substrates for succinic acid synthesis. Based on RT-qPCR carried out in flask experiments, we discovered a relatively large difference in the expression levels of ldhA gene compared to glucose in both xylose and glycerol cultures. In bioreactor-scale fermentations, the impact of different gas phases (CO2, CO2/AIR) on biomass yield, substrate consumption, and metabolite profiles was also investigated. In the case of glycerol, the addition of CO2 increased biomass as well as target product formation, while using CO2/AIR gas phase resulted in higher target product yield (0.184mM⋅mM-1). In case of xylose, using CO2 alone would result in higher succinic acid production (0.277mM⋅mM-1). The promising rumen bacteria, B. succiniciproducens, has shown to be suitable for succinic acid production from both xylose and glycerol. As a result, our findings present new opportunities for broadening the range of raw materials used in this significant biochemical process. Our study also sheds light on fermentation parameter optimization for this strain, namely that, CO2/AIR supply has a positive effect on target product formation.

Holistic Approach to Process Design and Scale-Up for Itaconic Acid Production from Crude Substrates

Bio-based bulk chemicals such as carboxylic acids continue to struggle to compete with their fossil counterparts on an economic basis. One possibility to improve the economic feasibility is the use of crude substrates in biorefineries. However, impurities in these substrates pose challenges in fermentation and purification, requiring interdisciplinary research. This work demonstrates a holistic approach to biorefinery process development, using itaconic acid production on thick juice based on sugar beets with Ustilago sp. as an example. A conceptual process design with data from artificially prepared solutions and literature data from fermentation on glucose guides the simultaneous development of the upstream and downstream processes up to a 100 L scale. Techno-economic analysis reveals substrate consumption as the main constituent of production costs and therefore, the product yield is the driver of process economics. Aligning pH-adjusting agents in the fermentation and the downstream process is a central lever for product recovery. Experiments show that fermentation can be transferred from glucose to thick juice by changing the feeding profile. In downstream processing, an additional decolorization step is necessary to remove impurities accompanying the crude substrate. Moreover, we observe an increased use of pH-adjusting agents compared to process simulations.

Obesity-induced metabolic imbalance allosterically modulates CtBP2 to inhibit PPAR-alpha transcriptional activity

Maintenance of metabolic homeostasis is secured by metabolite-sensing systems, which can be overwhelmed by constant macronutrient surplus in obesity. Not only the uptake processes but also the consumption of energy substrates determine the cellular metabolic burden. We herein describe a novel transcriptional system in this context comprised of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator for fatty acid oxidation, and C-terminal binding protein 2 (CtBP2), a metabolite-sensing transcriptional co-repressor. CtBP2 interacts with PPARα to repress its activity, and the interaction is enhanced upon binding to malonyl-CoA, a metabolic intermediate increased in tissues in obesity and reported to suppress fatty acid oxidation through inhibition of carnitine palmitoyltransferase 1 (CPT1). In line with our preceding observations that CtBP2 adopts a monomeric configuration upon binding to acyl-CoAs, we determined that mutations in CtBP2 that shift the conformational equilibrium toward monomers increase the interaction between CtBP2 and PPARα. In contrast, metabolic manipulations that reduce malonyl-CoA decreased the formation of the CtBP2/PPARα complex. Consistent with these in vitro findings, we found that the CtBP2/PPARα interaction is accelerated in obese livers while genetic deletion of CtBP2 in the liver causes derepression of PPARα target genes. These findings support our model where CtBP2 exists primarily as a monomer in the metabolic milieu of obesity to repress PPARα, representing a liability in metabolic diseases that can be exploited to develop therapeutic approaches.

Melomel Production by Saccharomyces bayanus Using Hydrolyzed Jackfruit (Artocarpus heterophyllus) Pulp, Pollen, and Rice (Oryza sativa) Bran Extract as Unconventional Supplements

This work evaluated the effect of hydrolyzed jackfruit pulp, rice (Oryza sativa) bran extract, and pollen concentration as unconventional supplements on mead production by Saccharomyces bayanus. Fermentations were carried out in 500-mL Erlenmeyer flasks containing 250 mL of honey wort (30°Brix). The flasks were supplemented with hydrolyzed jackfruit pulp (10, 20, and 30%), rice bran extract (5, 32.5, and 60 g∙L−1), and pollen (0, 20, and 30 g∙L−1), inoculated with 0.2 g∙L−1 cells and incubated at 30°C for 288 h. Higher cell growth (11.5 g∙L−1 cells) and ethanol production (18.2 %v/v) were observed in the wort supplemented with 32.5 g∙L−1 rice bran extract, 20 g∙L−1 pollen, and 20% hydrolyzed jackfruit pulp. The highest substrate consumption (59%) was verified in the wort containing the highest concentration of hydrolyzed jackfruit pulp (30%). The S. bayanus Premier Blanc yeast had the best ethanol volumetric yield (1.982 g∙(L∙h)−1), product yield coefficient (0.427 g∙g−1), and fermentation efficiency (83.52%) after 48 h of fermentation.

Investigation of exopolysaccharide formation and its impact on anaerobic succinate production with Vibrio natriegens.

Vibrio natriegens is an emerging host for biotechnology due to its high growth and substrate consumption rates. In industrial processes typically fed-batch processes are applied to obtain high space-time yields. In this study, we established an aerobic glucose-limited fed-batch fermentation with the wild type (wt) of V. natriegens which yielded biomass concentrations of up to 28.4 gX L-1 . However, we observed that the viscosity of the culture broth increased by a factor of 800 at the end of the cultivation due to the formation of 157 ± 20 mg exopolysaccharides (EPS) L-1 . Analysis of the genomic repertoire revealed several genes and gene clusters associated with EPS formation. Deletion of the transcriptional regulator cpsR in V. natriegens wt did not reduce EPS formation, however, it resulted in a constantly low viscosity of the culture broth and altered the carbohydrate content of the EPS. A mutant lacking the cps cluster secreted two-fold less EPS compared to the wt accompanied by an overall low viscosity and a changed EPS composition. When we cultivated the succinate producer V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) under anaerobic conditions on glucose, we also observed an increased viscosity at the end of the cultivation. Deletion of cpsR and the cps cluster in V. natriegens Succ1 reduced the viscosity five- to six-fold which remained at the same level observed at the start of the cultivation. V. natriegens Succ1 ΔcpsR and V. natriegens Succ1 Δcps achieved final succinate concentrations of 51 and 46 g L-1 with a volumetric productivity of 8.5 and 7.7 gSuc L-1 h-1 , respectively. Both strains showed a product yield of about 1.4 molSuc molGlc -1 , which is 27% higher compared with that of V. natriegens Succ1 and corresponds to 81% of the theoretical maximum.