Fermentable Carbon Sources Research Articles

Candida albicans Hexokinase 2 Challenges the Saccharomyces cerevisiae Moonlight Protein Model.

Survival of the pathogenic yeast Candida albicans depends upon assimilation of fermentable and non-fermentable carbon sources detected in host microenvironments. Among the various carbon sources encountered in a human body, glucose is the primary source of energy. Its effective detection, metabolism and prioritization via glucose repression are primordial for the metabolic adaptation of the pathogen. In C. albicans, glucose phosphorylation is mainly performed by the hexokinase 2 (CaHxk2). In addition, in the presence of glucose, CaHxK2 migrates in the nucleus and contributes to the glucose repression signaling pathway. Based on the known dual function of the Saccharomyces cerevisiae hexokinase 2 (ScHxk2), we intended to explore the impact of both enzymatic and regulatory functions of CaHxk2 on virulence, using a site-directed mutagenesis approach. We show that the conserved aspartate residue at position 210, implicated in the interaction with glucose, is essential for enzymatic and glucose repression functions but also for filamentation and virulence in macrophages. Point mutations and deletion into the N-terminal region known to specifically affect glucose repression in ScHxk2 proved to be ineffective in CaHxk2. These results clearly show that enzymatic and regulatory functions of the hexokinase 2 cannot be unlinked in C. albicans.

Exploration of processes governing microbial reductive dechlorination in a heterogeneous aquifer flow cell

Although microbial reductive dechlorination (MRD) has proven to be an effective approach for in situ treatment of chlorinated ethenes, field implementation of this technology is complicated by many factors, including subsurface heterogeneity, electron donor availability, and distribution of microbial populations. This work presents a coupled experimental and mathematical modeling study designed to explore the influence of heterogeneity on MRD and to assess the suitability of microcosm-derived rate parameters for modeling complex heterogeneous systems. A Monod-based model is applied to simulate a bioremediation experiment conducted in a laboratory-scale aquifer cell packed with aquifer material from the Commerce Street Superfund site in Williston, VT. Results reveal that (uncalibrated) model application of microcosm-derived dechlorination and microbial growth rates for transformation of trichloroethene (TCE), cis-1,2-dichloroethene (cis-DCE), and vinyl chloride (VC) reproduced observed aquifer cell concentration levels and trends. Mean relative errors between predicted and measured effluent concentrations were quantified as 6.7%, 27.0%, 41.5%, 32.0% and 21.6% over time for TCE, cis-DCE, VC, ethene and total volatile fatty acids (fermentable electron donor substrate and carbon source), respectively. The time-averaged extent of MRD (i.e., ethene formation) was well-predicted (4% underprediction), with modeled MRD exhibiting increased deviation from measured values under electron donor limiting conditions (maximum discrepancy of 14%). In contrast, simulations employing a homogeneous (uniform flow) domain resulted in underprediction of MRD extent by an average of 13%, with a maximum discrepancy of 45%. Model sensitivity analysis suggested that trace amounts of natural dissolved organic carbon served as an important fermentable substrate, providing up to 69% of the reducing equivalents consumed for MRD under donor-limiting conditions. Aquifer cell port concentration data and model simulations revealed that ethene formation varied spatially within the domain and was associated with regions of longer residence times. These results demonstrate the strong influence of subsurface heterogeneity on the accuracy of MRD predictions, and highlight the importance of subsurface characterization and the incorporation of flow field uncertainty in model applications for successful design and assessment of in situ bioremediation.

Isolation and characterization of Lactococcus garvieae from the fish gut for in vitro fermentation with carbohydrates from agro-industrial waste

This study focused on agro-industrial waste such as fruit peels by extracting prebiotics as a carbon source for lactic acid bacteria (LAB). Four strains of LAB were selected from Oreochromis niloticus (B2 and B3) and Nemipterus japonicas (R4 and R5), and identified as Lactococcus garvieae through 16S rRNA gene sequencing. The analysis of probiotic characteristics revealed that all four strains were able to tolerate sodium chloride (up to 7 %), bile salt (up to 3 %), and broad range of pH (2–9). Further, analysis of polysaccharide contents in the agro-industrial waste materials such as peels of pineapple, orange, lemon, sugarcane, pomegranate, and sweet lemon revealed that the concentration ranged from 3.91–163.85 mg/g. It was observed that orange peels (20.38–140.99 mg/g), sweet lemon peels (22.03–161.93 mg/g), and pomegranate peels (38.19–163.85 mg/g) yielded maximum indigestible polysaccharide. Evaluation of synbiotic combination of probiotic and prebiotic revealed that L. garvieae strains had better fermentation efficiency with orange, sweet lemon, and pineapple compared to lemon, sugarcane, and pomegranate. In nutshell, different types of agro-industrial waste evaluated in this research were found to be a cheap and fermentable carbon sources for LAB. Further study should be conducted to analyze this symbiotic combination as feed supplements for fish in aquaculture as well as various fermentation industries.

Value-added soluble metabolite production from sugarcane vinasse within the carboxylate platform: An application of the anaerobic biorefinery beyond biogas production

Currently, wastewater processing through anaerobic digestion can generate not only biofuels such as biohydrogen and methane, but also value-added soluble-phase intermediates (e.g. carboxylic acids), which have numerous applications. This study investigates the potential of using sugarcane vinasse, the main byproduct from ethanol production, as substrate in dark fermentation to recover soluble metabolites through the biorefinery approach. The impacts of temperature (30–60 °C) and initial pH (5.0–10.0) were initially assessed in batch tests using microbial consortia obtained from the natural fermentation of vinasse. The yield (401 mg-CODorganic acids g−1CODtinitial) and productivity (653 mg-CODorganic acids L−1 d−1) of organic acids were maximized at alkaline/mesophilic (pH = 8.8–10.0; 40 °C) conditions. Acetic-type fermentation prevailed at 30–40 °C, whilst butyrate was the primary metabolite at a higher temperature (60 °C). Further chain elongation-based experiments were conducted by adding ethanol and lactate as exogenous carbon sources in vinasse fermentation, also using vinasse-derived microbial consortia as the inoculum. Lactate was added as both chemical reactant and fermented cassava flour wastewater (fCFW). Ethanol addition was irrelevant to the acidogenic activity. Conversely, lactate addition directly increased the production of propionic, butyric, (iso)valeric, and caproic acids, with a predominance of butyrate. Chain elongation was particularly favored when adding fCFW with pH adjusted to 7.0, boosting the generation of caproic acid from lactate and butyrate and (iso)valeric acid from acetate and propionate. These results highlight the potential for producing organic acids from vinasse as an alternative to gaseous fuels, expanding the suitability of dark fermentation targeting bioresource recovery from sugarcane.

Production of Rhamnolipids from Soybean Soapstock: Characterization and Comparation with Synthetics Surfactants

In this study, waste from the soybean industry was used as the main carbon source to produce rhamnolipids. This biosurfactant have a great value and can be used in several applications. The biosurfactant produced in this study was characterized through to HPLC–UV, and the emulsification index was determined. The work also made a biodegradation test of lubricating oil residue, using the biosurfactant produced and other synthetics surfactants to make a comparison. After 120 h the total production concentration was 1.434 g L−1 of rhamnolipids. This value was close to order works that used more noble carbon source for fermentation. Also was possible to identify two different homologous on the fermentation production (Rha2C10C10 and Rha2C10). The rhamnolipids synthesized also showed good results in the emulsification of lubrication oil residue, being very close to the synthetic alternative Tween80. Thus, it is possible to conclude that the biosurfactant produced has potential for application in bioremediation of oils residues, and can be an efficient substitute for synthetic surfactants.

Human apolipoprotein L1 interferes with mitochondrial function in Saccharomycescerevisiae.

To the best of our knowledge, the vertebrate apolipoprotein L (APOL) family has not previously been ascribed to any definite pathophysiological function, although the conserved BH3 protein domain suggests a role in programmed cell death or an interference with mitochondrial processes. In the present study, the human APOL1 was expressed in the yeast Saccharomyces cerevisiae in order to determine the molecular action of APOL1. APOL1 inhibited cell proliferation in a non-fermentable carbon source, such as glycerol, while it had no effect on proliferation in fermentable carbon sources, such as galactose. APOL1, expressed in yeast, is localized in the mitochondrial fraction, as determined via western blotting. APOL1 induced a loss of mitochondrial function, demonstrated by a loss of respiratory index, and mitochondrial membrane potential. Green fluorescent protein tagging of mitochondrial protein revealed that APOL1 was associated with abnormal mitochondrial and lysosomal morphologies, observed by a loss of the normal mitochondrial tubular network. Thus, the results of the present study suggest that APOL1 could be a physiological regulator of mitochondrial function.

Purified crude glycerol by acid treatment allows to improve lipid productivity by Yarrowia lipolytica SKY7

In this study, crude glycerol with high potassium concentration was purified using acid treatment and used as carbon source for lipid production using Yarrowia lipolytica SKY7. The crude glycerol was purified using phosphoric acid (pH 2) followed by centrifugation. When purified glycerol was used as carbon source for fermentation, higher biomass productivity (0.54 g/L/h) and lipid productivity (0.2 g/L/h) was observed at 96 h compared to crude glycerol. Results indicated that 6.32 g/L potassium in crude glycerol medium was inhibitory for cell growth and lipid production by Y. lipolytica. Yield coefficients, productivities and specific growth rates were calculated for each glycerol medium. The process performance with purified glycerol medium was comparable to that of pure glycerol medium. A higher lipid yield was obtained in purified glycerol medium (0.21 g/g glycerol) than crude glycerol medium (0.124 g/g glycerol). During purification of crude glycerol, KH2PO4 was also produced as by-product. This study provides a way for valorization of crude glycerol with high potassium concentration for microbial lipid production.

Biosíntesis del agente fibrinolítico uroquinasa por Enterococcus gallinarum aislado de sardine

Fibrinogen degradation by thrombin leads to the formation of fibrin clots in the blood which results in cardiovascular diseases due to which certain fibrinolytic agents are used to treat cardiovascular complications. Urokinase is being favored as a fibrinolytic agent due to its high fibrin specificity and fewer side effects. Therefore, the main objective of this research was the isolation of potent urokinase producing bacteria from soil, seawater, and fermented food samples. Thirty-five isolates that were capable of producing urokinase were isolated using sixty samples and subjected to secondary screening for urokinase estimation. Bacterial isolate QGA-20 that showed maximum enzyme activity 52.6±0.03 FU/ml/min was identified by 18s RNA sequencing as Enterococcus gallinarum which has never been reported before for urokinase production. Different physical and cultural parameters such as fermentation medium, incubation time, temperature, pH, carbon source, nitrogen source, inoculum size, and trace elements were optimized for submerged fermentation to synthesize urokinase. Maximum production of urokinase 81.3±0.02 FU/ml/min was obtained with M-5 fermentation media at 96 hrs of incubation, 37oC temperature, 7 pH, sucrose as carbon, and soya flour as a nitrogen source, 3% inoculum size and CaCl2 as a trace element. In in vitro studies, urokinase enzyme was also applied for the disintegration of clot successfully. This study revealed a potent urokinase producer, reported first time, with appreciable clot lysis ability.

The Antifungal Test: An Efficient Screening Tool for the Discovery of Microbial Metabolites with Respiratory Inhibitory Activity

Valuable natural compounds produced by a variety of microorganisms can be used as lead molecules for development of new agrochemicals. Furthermore, high-throughput in vitro screening systems with specific modes of action can increase the probability of discovery of new fungicides. In the current study, a rapid assay tested with various microbes was developed to determine the degree of respiratory inhibition of Saccharomyces cerevisiae in two different liquid media, YG (containing a fermentable carbon source) and NFYG (containing a non-fermentable carbon source). Based on this system, we screened 100 fungal isolates that were classified into basidiomycetes, to find microbial secondary metabolites that act as respiratory inhibitors. Consequently, of the 100 fungal species tested, the culture broth of an IUM04881 isolate inhibited growth of S. cerevisiae in NFYG medium, but not in YG medium. The result is comparable to that from treatment with kresoxim-methyl used as a control, suggesting that the culture broth of IUM04881 isolate might contain active compounds showing the inhibition activity for respiratory chain. Based on the assay developed in this study and spectroscopic analysis, we isolated and identified an antifungal compound (-)-oudemansin A from culture broth of IUM04881 that is identified as Oudemansiella venosolamellata. This is the first report that (-)-oudemansin A is identified from O. venosolamellata in Korea. Taken together, the development of this assay will accelerate efforts to find and identify natural respiratory inhibitors from various microbes.

Biotransformation of agro-industrial waste to produce lignocellulolytic enzymes and bioethanol with a zero waste

The use of lignocellulosic wastes reduces dependence on fossil fuel resources, contributes to sustainable waste management, and reinforces the circular economy model of continual use of resources. Typically, the second generation of bioethanol production involves several steps to transform lignocellulosic material into bioethanol. The more complicated step of the overall process is to define a tailor-made to each lignocellulosic material available with a wide variety of complex structures of the lignin, hemicellulose, and cellulose. However, the thermochemicals are the most frequently reported pretreatments, and they have the bottleneck of producing an additional waste stream with a high charge of pollution owing to chemical products implicated. This problem harms on the zero waste policy that we must include in our technological process to be considered sustainable and ecological processes. Consolidated bioprocessing (CBP) is a viable alternative to produce bioethanol from lignocellulosic materials, using a single microorganism, in one-step, and no chemical products are implied. Three agro-industrial wastes with lignocellulosic characteristics were evaluated as a substrate for bioethanol production with a Mexican native white-rot fungus Trametes hirsuta CS5 in a one-step process. Qualitative and quantitative analyses of lignocellulolytic enzymes produced by native fungus were carried out. Instead, Trametes hirsuta is a lignin degrader white-rot fungus; it was capable to produce until 500 U/L of cellulase titers and a maximum xylanase activity of 45 U/mL when it was cultivated in orange peel substrate. Substantial ethanol yields were achieved using lignocellulosic materials like brewer’s spent grain (BSG), orange peel (OP), and wheat bran (WB) as a carbon source in fermentation with no chemicals, which represents a zero waste environment-friendly ethanol production system. Ethanol yield on wheat bran was the highest of all evaluated substrates, reaching value of 34.9% at 7 days, being T. hirsuta able to degrade other hexoses and pentoses present in the structural polymers of cellulose and hemicellulose.

Transcriptional Activity and Protein Levels of Horizontally Acquired Genes in Yeast Reveal Hallmarks of Adaptation to Fermentative Environments.

In the past decade, the sequencing of large cohorts of Saccharomyces cerevisiae strains has revealed a landscape of genomic regions acquired by Horizontal Gene Transfer (HGT). The genes acquired by HGT play important roles in yeast adaptation to the fermentation process, improving nitrogen and carbon source utilization. However, the functional characterization of these genes at the molecular level has been poorly attended. In this work, we carried out a systematic analysis of the promoter activity and protein level of 30 genes contained in three horizontally acquired regions commonly known as regions A, B, and C. In three strains (one for each region), we used the luciferase reporter gene and the mCherry fluorescent protein to quantify the transcriptional and translational activity of these genes, respectively. We assayed the strains generated in four different culture conditions; all showed low levels of transcriptional and translational activity across these environments. However, we observed an increase in protein levels under low nitrogen culture conditions, suggesting a possible role of the horizontally acquired genes in the adaptation to nitrogen-limited environments. Furthermore, since the strains carrying the luciferase reporter gene are null mutants for the horizontally acquired genes, we assayed growth parameters (latency time, growth rate, and efficiency) and the fermentation kinetics in this set of deletion strains. The results showed that single deletion of 20 horizontally acquired genes modified the growth parameters, whereas the deletion of five of them altered the maximal CO2 production rate (Vmax). Interestingly, we observed a correlation between growth parameters and Vmax for an ORF within region A, encoding an ortholog to a thiamine (vitamin B1) transporter whose deletion decreased the growth rate, growth efficiency, and CO2 production. Altogether, our results provided molecular and phenotypic evidence highlighting the importance of horizontally acquired genes in yeast adaptation to fermentative environments.

Extraction and biological activity of exopolysaccharide produced by Leuconostoc mesenteroides SN-8

An exopolysaccharide (EPS)-producing strain SN-8 isolated from Dajiang was identified as Leuconostoc mesenteroides. When sucrose was used as the carbon source for fermentation, the output of EPS was 2.42 g/L. High performance liquid chromatography analysis confirmed the presence of monomers such as glucan and mannose. The molecular weight detection value is 2.0 × 105 Da. Fourier transform infrared spectroscopy displayed the EPS had the basic skeleton and functional groups of a typical polysaccharide structure. Scanning electron microscopy showed smooth surfaces and compact structure. Thermal performance analysis showed that the highest heat resistance temperature of the EPS was 80 °C. Compared with vitamin C, its hydroxyl radical scavenging rate was as high as 32% and 1,1-diphenyl-2-picrylhydrazyl scavenging rate was as high as 40% under the same concentration. The peanut oil was the most emulsifiable at a concentration of 1.5 mg/mL, and the emulsification index was 0.55. These results might show that the EPS had high application value.

The Study of Sphingolipids Metabolism and Its Effect on Brain Disorders

Sphingolipids are molecules involved in important processes like cell growth, signaling, cell structure and apoptosis. It is known that sphingolipids metabolism is involved in the proper development of the brain and deficiency in sphingolipids homeostasis lead to psychiatric disorders Sphingolipids are also known to play a role in other disorders, such: necrosis. In this research, sphingolipids metabolism is studied using Saccharomyces cerevisiae as a model. A CSG2 deletion mutant, accumulates inositolphosphoryl ceramide (IPC), the first complex sphingolipid. When it is exposed to nitrogen starvation conditions with a fermentable carbon source present, the cells do not adapt properly to this stressor because the mitochondria cannot increase the MMP (mitochondria membrane potential), which leads to an increase in reactive oxygen species (ROS) and cell death. Although, when the same mutant is exposed to nitrogen starvation conditions with a nonfermentable carbon source present, cells grow normally. Cells are rescued by the ISC1 gene activity. ISC1human’s homologue gene codes for the protein sphingomyelinase. This protein breaks down complex sphingolipids into ceramide. It is well known that ISC1 is involved in process related to nutrient availability and it is activated under nonfermentable carbon sources. Currently, Isc1p is the only protein known that can break down complex sphingolipids in yeast. This organism has two main types of complex sphingolipids, mannosylated and non‐mannosylated. In this research, we do not only study IPC (non‐mannosylated complex sphingolipid) but also the other two more complex mannosylated sphingolipids: mannose‐inositol‐phosphoceramide (MIPC) and mannose‐(inositol‐P)2‐ceramide (M(IP)2C). It has been shown that Ipt1D, mutant that accumulates MIPC since, the gene, IPT1, that codes for the protein that turns MIPC into M(IP)2C has been deleted, survive under nitrogen starvation when a fermentable carbon source is present. Our results suggest that Isc1p cannot bind to complex sphingolipids when a mannose group is attached to them and another protein, not known yet, that carries out the function of breaking down mannosylated complex sphingolipids when a fermentable carbon source is present. Therefore, this research focuses on the study of sphingolipids metabolism and homeostasis, studying genes that are involved in the anabolism as well as the catabolism pathways. This research uses data collected form hands‐on experiment, for instance: TLC, fluorescence microscope, cell viability assay; and from computational methods. The results will lead to a better understanding of sphingolipids homeostasis and can help to target the treatment of disorders caused by these molecules.Support or Funding InformationPresbyterian College, Chemistry and Biochemistry Department

Capacity Analysis of Photo-Fermentation Bio-Hydrogen Production from Different Food Wastes

Food waste is rich in starch or cellulose, which can be utilized as carbon source for fermentation. Hence, in this paper, different food wastes (vegetable, rice, corn, potato) were taken as substrate to evaluate their hydrogen yield potential. The characteristics of fermentation broth, cumulative hydrogen yield, and hydrogen production rate were investigated in the photo-fermentation bio-hydrogen production process. Modified Gompertz Model was utilized to deal with experiment data. Results showed that food waste can be effectively utilized by photosynthetic bacteria. Waste rice was determined to have the best hydrogen production capacity with hydrogen yield of 696 mL, and the maximum hydrogen production rate of 17.71 mL/h, the average hydrogen concentration was 55.78%.

Microstructural and carbohydrate compositional changes induced by enzymatic saccharification of green seaweed from West Africa

The use of marine macroalgae as carbon source for fermentation is gaining increasing attention due to their high carbohydrate content and availability. Three green seaweed species Chaetomorpha linum, Ulva fasciata and Caulerpa taxifolia were investigated for their amenability to enzymatic saccharification. Microstructural changes were studied in order to understand the physical changes occurring in the seaweeds during saccarification and to identify structural barriers. C. linum had highest glucan content (20%), compared to 16% in U. fasciata and 6% in C. taxifolia indicating large differences in composition. Glucose yields obtained after 24 h of enzymatic saccharification were 59, 38 and 60% for C. taxifolia, U. fasciata and C. linum, respectively, based on the glucan content. Pre-autoclaving increased the saccharification yield to 81, 99 and 71%, respectively. Morphologically, C. linum displayed unbranched filaments, U. fasciata two-cell layer large sheets and C. taxifolia featured leaf like structures. Enzymatic saccharification resulted in cell wall degradation and release of the chlorophyll content in C. linum, delamination of sheets in U. fasciata and surface erosion of leaves in C. taxifolia. C. taxifolia deviated in being very rich in β-1,3 linked xylan (46%), which was only hydrolysed at 1% xylose yield due to lack of β-1,3-xylanase. Based on the high cellulose content and no need for pre-treatment C. linum was the best source of glucose among the three types of green seaweed species. Further studies are warranted to assess fermentation. We also conclude that for C. taxifolia enzymatic saccharification with β-1,3-xylanase might enable further xylose release than obtained here with the enzymatic cellulase treatment.

High-Protein Solid-State Symbiotic Fermentation of Sauce Residue for Probiotic Feed

The abuse of antibiotics in animal feed has caused a series of problems in livestock breeding, meat products and the natural environment. In this study, sauce residue was used as the main raw material that includes 25.98% of dry-based crude protein, 3.91% of NaCl, 16.28% of crude fat, 11.06% of the ash and the multi-strain solid-state symbiotic fermentation technology was used to increase the amount of probiotics and the crude protein content of feed. Meanwhile, the addition of functional probiotics improved the feed palatability, while reducing the use of antibiotics further improved the cycle utilization of soy sauce residue. This study systematically optimized the symbiotic conditions of two probiotics, Lactobacillus bulgaricus and Saccharomyces cerevisiae. This study defined viable cell count and crude protein content as the core targets for analysis and evaluation. Firstly, the optimal carbon source concentration, the temperature and the inoculation process of symbiotic fermentation were determined by single factors; then the symbiotic fermentation conditions (inoculation volume, material-liquid ratio, fermentation time) were optimized by orthogonal test. The results showed that 40 g/L glucose/lactose (50/50%) was the best carbon source for sauce residue fermentation. The optimum fermentation conditions were a ratio of material to water of 1:0.8 and an inoculation volume of 15% (v/w) and a fermentation duration of 48 h. Meanwhile, the study found that multi-step temperature control could effectively improve the corresponding targets. The inoculation with Saccharomyces cerevisiae first and then with Lactobacillus bulgaricus after 24 h, boosted the probiotic quantity and protein content. In optimized conditions, probiotic viable count reached 7.2×108 CFU·mL-1 and the crude protein content increased from 25.98% (dry content) to 38.86%, which is 49.6% increase compared with the raw material. This research could provide a reference for the development of high-protein probiotic feed using sauce residue.

Production of Lactic Acid from Seaweed Hydrolysates via Lactic Acid Bacteria Fermentation

Biodegradable polylactic acid material is manufactured from lactic acid, mainly produced by microbial fermentation. The high production cost of lactic acid still remains the major limitation for its application, indicating that the cost of carbon sources for the production of lactic acid has to be minimized. In addition, a lack of source availability of food crop and lignocellulosic biomass has encouraged researchers and industries to explore new feedstocks for microbial lactic acid fermentation. Seaweeds have attracted considerable attention as a carbon source for microbial fermentation owing to their non-terrestrial origin, fast growth, and photoautotrophic nature. The proximate compositions study of red, brown, and green seaweeds indicated that Gracilaria sp. has the highest carbohydrate content. The conditions were optimized for the saccharification of the seaweeds, and the results indicated that Gracilaria sp. yielded the highest reducing sugar content. Optimal lactic acid fermentation parameters, such as cell inoculum, agitation, and temperature, were determined to be 6% (v/v), 0 rpm, and 30 °C, respectively. Gracilaria sp. hydrolysates fermented by lactic acid bacteria at optimal conditions yielded a final lactic acid concentration of 19.32 g/L.

Optimization of Citric Acid Production by Immobilized Cells of Novel Yeast Isolates

Citric acid is a commercially valuable organic acid widely used in food, pharmaceutical, and beverage industries. In this study, 260 yeast strains were isolated from soil, bread, juices, and fruits wastes and preliminarily screened using bromocresol green agar plates for their ability to produce organic acids. Overall, 251 yeast isolates showed positive results, with yellow halos surrounding the colonies. Citric acid production by 20 promising isolates was evaluated using both free and immobilized cell techniques. Results showed that citric acid production by immobilized cells (30–40 g/L) was greater than that of freely suspended cells (8–19 g/L). Of the 20 isolates, two (KKU-L42 and KKU-L53) were selected for further analysis based on their citric acid production levels. Immobilized KKU-L42 cells had a higher citric acid production rate (62.5%), while immobilized KKU-L53 cells showed an ∼52.2% increase in citric acid production compared with free cells. The two isolates were accurately identified by amplification and sequence analysis of the 26S rRNA gene D1/D2 domain, with GenBank-based sequence comparison confirming that isolates KKU-L42 and KKU-L53 were Candida tropicalis and Pichia kluyveri, respectively. Several factors, including fermentation period, pH, temperature, and carbon and nitrogen source, were optimized for enhanced production of citric acid by both isolates. Maximum production was achieved at fermentation period of 5 days at pH 5.0 with glucose as a carbon source by both isolates. The optimum incubation temperature for citric acid production by C. tropicalis was 32 °C, with NH4Cl the best nitrogen source, while maximum citric acid by P. kluyveri was observed at 27 °C with (NH4)2 SO4 as the nitrogen source. Citric acid production was maintained for about four repeated batches over a period of 20 days. Our results suggest that apple and banana wastes are potential sources of novel yeast strains; C. tropicalis and P. kluyveri which could be used for commercial citric acid production.

Potential assessment and kinetic modeling of carboxylic acids production using dairy wastewater as substrate

Carboxylic acids (CA) are high added-value compounds that can be produced via anaerobic fermentation by using agroindustrial residues as substrates. However, different compounds in wastewater impose uncontrolled metabolic pathways, in which the acidogenic fermentation kinetics need to be elucidated. This work aimed to assess the potential production of CA from dairy wastewater (DW) and to perform the kinetic modeling of the process. The experiments were conducted in quadruplicate batch reactors (250 mL of working volume) with inoculum from a brewery UASB at 0.61 ± 0.04 gCOD gVSS−1. To inhibit methanogenesis, 0.05 % (v/v) chloroform was added to the reactors. The tests showed that DW is a readily fermentable substrate to acidogenic microorganisms because it presents high rates of short-chain CA formation in the first two days of the experiment. The low concentrations of medium-chain CA found indicate that fats and proteins did not function as the main carbon source for DW fermentation. The yield obtained was 0.66 mgCA mgCODA−1, which corresponds to 0.83 mgCODCA mgCODA−1. Kinetic modeling studies have shown that mathematical models that can describe an exponential phase, such as First-order and Fitzhugh models, are suitable for simulating the production of carboxylic acids. Finally, DW seems to be a promising substrate to be investigated in the carboxylic platform.

Improved succinate production from galactose-rich feedstocks by engineered Escherichia coli under anaerobic conditions.

It is of great economic interest to produce succinate from low-grade carbon sources, which can make it more economically competitive against petrochemical-based succinate. Galactose sugars constitute a significant fraction of the soluble carbohydrate in a meal from agricultural sources which is considered a low value or waste byproduct of oilseed processing. To improve the galactose utilization, the effect of galR and glk on sugars uptake was investigated by deactivation of each gene in three previously engineered host strains. As expected, glk plays an important role in glucose uptake, while, the effect of deactivation of galR is highly dependent on the strength of the downstream module (succinate production module). A new succinate producer FZ661T was constructed by enhancement of the succinate producing module and manipulation of the gal operon. The succinate productivity reached 4.57 g/L/hr when a mixed sugar feedstock was used as a carbon source in shake-flask fermentation, up to 812 mM succinate was accumulated in 80 hr in fed-batch fermentation. When SoyMolaGal hydrolysate was used as a carbon source, 628 mM (74 g/L) succinate was produced within 72 hr. In this study, we demonstrate that FZ661T can produce succinate quickly with relatively high yield, giving it the potential for industrial application.