Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates

Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.

Molecular characterization of xanthan gum producing Xanthomonas Campestris isolated from dark rot spotted leaves in Keffi, Nasarawa State, Nigeria

Background: Despite the wide application of Xanthan gum, its commercial production remains a global challenge. In recent years, considerable research has been carried out using agro-industrial wastes, which are renewable and abundantly available to produce value-added products. The present study was set out for molecular identification of Xanthomonas campestris from leaves of four different plants with indications of dark rot spots and evaluation of their xanthan gum production capacity. Methods: Twenty-five (25) samples of leaves from four different plants with indications of dark rot spots were collected from the study area and isolated for Xanthomonas campestris following standard microbiological methods. Cultural, morphological and biochemical tests were conducted to confirm the organism. Results: The results revealed that of the total 100 samples taken, 6 leaves (24%) were infected with Xanthomonas species in mint, 3(12%) were infected in mango, 1(4%) were infected in rice and 2(8%) were infected in pepper. Further molecular identification of the isolates was carried out to reveal Xanthomonas campestris pv. vesicatoria strain 85-10 and Xanthomonas perforans strain 91-118. These were further used for the production of xanthan gum using sugar cane molasses substrates extracted from sugar cane, which was used as fermentation medium for the production. Isolates from plants varying ability in Xanthan gum production, with the mint plant having the highest Xanthan gum production (0.10 ± 0.02 to 0.9 ± 0.00 g/l). Conclusion: The present study confirmed the high xanthan gum production capacity of Xanthomonas campestris from dark rot spots containing mint leaves and should be considered during local and industrial production of the xanthan gum

Improved Neomycin Sulfate Potency in Streptomyces fradiae Using Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Fermentation Medium Optimization

To improve the screening efficiency of high-yield neomycin sulfate (NM) Streptomyces fradiae strains after mutagenesis, a high-throughput screening method using streptomycin resistance prescreening (8 μg/mL) and a 24-deep well plates/microplate reader (trypan blue spectrophotometry) rescreening strategy was developed. Using this approach, we identified a high-producing NM mutant strain, Sf6-2, via six rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and screening. The mutant displayed a NM potency of 7780 ± 110 U/mL and remarkably stable genetic properties over six generations. Furthermore, the key components (soluble starch, peptone, and (NH4)2SO4) affecting NM potency in fermentation medium were selected using Plackett-Burman and optimized by Box-Behnken designs. Finally, the NM potency of Sf6-2 was increased to 10,849 ± 141 U/mL at the optimal concentration of each factor (73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively), and it exhibited about a 40% and 100% enhancement when compared with before optimization conditions and the wild-type strain, respectively. In this study, we provide a new S. fradiae NM production strategy and generate valuable insights for the breeding and screening of other microorganisms.

Efficacy of Aspergillus sp in the Production of Protease Enzyme with Different Substrates

In the present study, the soil samples were collected from marine environment of Arichalmunai, Dhanushkodi, Ramnad District, Tamilnadu ,India. Fungal species were isolated by plating method, in 50% sea water containing potato dextrose agar medium .Totally 16 fungal species were isolated and identified from the soil sample. The production of protease from Aspergillus niger , A.flavus and A. terreus by liquid state fermentation. The production of protease enzyme was optimized by using fermentation medium containing different substrates. The maximum protease production was observed on wheat bran, containing medium. The protease production was maximum in temperature 35ºC were recorded. Wheat bran produced the maximum level protease. The optimization work also carried out. This study revealed that coastal environment provides impressive density of fungi in the East Coast of India and are unexplored for microbial resources can be useful in industry.

In vitro Modeling of Chicken Cecal Microbiota Ecology and Metabolism Using the PolyFermS Platform

Continuous in vitro fermentation models provide a useful tool for a fast, reproducible, and direct assessment of treatment-related changes in microbiota metabolism and composition independent of the host. In this study, we used the PolyFermS model to mimic the conditions of the chicken cecum and evaluated three nutritive media for in vitro modeling of the chicken cecal microbiota ecology and metabolism. We observed that our model inoculated with immobilized cecal microbiota and fed with a modified Viande Levure medium (mVL-3) reached a high bacterial cell density of up to approximately 10.5 log cells per mL and stable microbiota composition, akin to the host, during 82 days of continuous operation. Relevant bacterial functional groups containing primary fibrolytic (Bacteroides, Bifidobacteriaceae, Ruminococcaceae), glycolytic (Enterococcus), mucolytic (Bacteroides), proteolytic (Bacteroides), and secondary acetate-utilizing butyrate-producing and propionate-producing (Lachnospiraceae) taxa were preserved in vitro. Besides, conserved metabolic and functional Kyoto Encyclopedia of Genes and Genomes pathways were observed between in vitro microbiota and cecal inoculum microbiota as predicted by functional metagenomics analysis. Furthermore, we demonstrated that the continuous inoculation provided by the inoculum reactor generated reproducible metabolic profiles in second-stage reactors comparable to the chicken cecum, allowing for the simultaneous investigation and direct comparison of different treatments with a control. In conclusion, we showed that PolyFermS is a suitable model for mimicking chicken cecal microbiota fermentation allowing ethical and ex vivo screening of environmental factors, such as dietary additives, on chicken cecal fermentation. We report here for the first time a fermentation medium (mVL-3) that closely mimics the substrate conditions in the chicken cecum and supports the growth and metabolic activity of the cecal bacterial akin to the host. Our PolyFermS chicken cecum model is a useful tool to study microbiota functionality and structure ex vivo.

Enhanced Biosynthesis of D-arabitol by Metschnikowia Reukaufii Through Optimizing Medium Composition and Fermentation Conditions

D-arabitol is an important functional sugar alcohol, which can be used in the preparation of foods, chemicals, and medicines. Despite biological production of D-arabitol from low-cost substrates has recently been the focus of research, low yield of this technology has limited its large-scale exploitation. Optimization of this bioprocess could be a promising option to improve the yield of D-arabitol. In this study, one-factor-at-a-time (OFAT) strategy and Box-Behnken design (BBD) were used to increase D-arabitol production by Metschnikowia reukaufii CICC 31858 through optimizing the fermentation conditions and medium composition. The OFAT optimization provided the optimal conditions for temperature, agitation speed, and fermentation time of 30℃, 220 rpm, and 144 h, respectively. Likewise, the optimum concentrations of peptone, ammonium sulfate, KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were (g/L) 7.5, 1, 2 , 0.5 and 7.5, respectively. Under these optimum conditions, 80.43 g/L of D-arabitol was produced from 200 g/L of glucose, with a productivity of 0.56 g/L/h. The BBD optimization with three important components of fermentation medium (KH2PO4, MgSO4·7H2O and fumaric acid) showed that the predicted titer of D-arabitol varied from 47.21 g/L to 89.27 g/L, and the actual titer of D-arabitol ranged from 47.36 to 89.83 g/L. The optimum concentrations (g/L) of KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were found to be 1.0, 0.5, and 4.7g/L, respectively. Under the optimum conditions, 92.45 g/L of D-arabitol was finally produced with the yield and productivity of 0.46 g/g and 0.64 g/L/h, respectively.

An Overview of Bioprocesses Employing Specifically Selected Microbial Catalysts for γ-Aminobutyric Acid Production

Gamma-aminobutyric acid (GABA) is an important chemical compound in the human brain. GABA acts as an inhibitory neurotransmitter by inducing hyperpolarization of cellular membranes. Usually, this pharmaceutically important compound is synthesized using a chemical process, but in this short overview we have only analysed microbial processes, which have been studied for the biosynthesis of this commercially important compound. The content of this article includes the following summarised information: the search for biological processes showed a number of lactic acid bacteria and certain species of fungi, which could be effectively used for the production of GABA. Strains found to possess GABA-producing pathways include Lactobacillus brevis CRL 1942, L. plantarum FNCC 260, Streptococcus salivarius subsp. thermophilus Y2, Bifidobacterium strains, Monascus spp., and Rhizopus spp. Each of these strains required specific growth conditions. However, several factors were common among these strains, such as the use of two main supplements in their fermentation medium—monosodium glutamate and pyridoxal phosphate—and maintaining an acidic pH. Optimization studies of GABA production were comprised of altering the media constituents, modifying growth conditions, types of cultivation system, and genetic manipulation. Some strains increased the production of GABA under anaerobic conditions. Genetic manipulation focused on silencing some genes or overexpression of gadB and gadC. The conclusion, based on the review of information available in published research, is that the targeted manipulation of selected microorganisms, as well as the culture conditions for an optimised bioprocess, should be adopted for an increased production of GABA to meet its increasing demand for food and pharmaceutical applications.

Bacterial Nanocellulose Derived from Banana Leaf Extract: Yield and Variation Factors

Bananas are one of the most important crops worldwide. However, a large amount of residual lignocellulosic biomass is generated during its production and is currently undervalued. These residues have the potential to be used as feedstock in bio-based processes with a biorefinery approach. This work is based on the valorization of banana leaf and has the following objectives (i) to determine the effect of certain physical and environmental factors on the concentration of glucose present in banana leaf extract (BLE), using a statistical regression model; (ii) to obtain Bacterial Nanocellulose (BNC), using BLE (70% v/v) and kombucha tea as fermentation medium. In addition, the physicochemical properties of BNC were evaluated by X-ray diffraction (XRD), Fourier transform infrared (FTIR), and thermogravimetric analysis (TGA). The results indicate that storage time, location, leaf color, and petiole type are factors related to BLE concentration, which is reduced by approximately 28.82% and 64.32% during storage times of five days. Regarding BNC biosynthesis, the results indicate that the highest yield, 0.031 g/g, was obtained at 21 days. Furthermore, it was determined that the highest production rate was 0.11 gL−1h−1 at 11 days of fermentation. By FTIR, it was determined that the purification step with NaOH (3M) should be carried out for approximately two hours. This research supports the development of a circular bioeconomy around the banana value chain, as it presents a way of bioprocessing residual biomass that can be used to produce bioproducts.