Single-step Fermentation Research Articles

Efficient Chitin Extraction from Shrimp Exoskeletons through Single-Step Fermentation by Pseudomonas aeruginosa QF50 and Serratia sp. QCS23

Chitin, an abundant biopolymer with potential applications in agriculture, medicine, and bioremediation, is conventionally extracted using chemical methods that have environmental disadvantages. This study investigates the extraction of chitin from Litopenaeus vannamei shrimp waste by one-step fermentation using the bacterial strains Pseudomonas aeruginosa QF50 and Serratia sp. QCS23. A total of 4 kg of shrimp waste was treated by fermentation with culture media enriched with different concentrations of glucose (1, 5, and 10%) for 7 days at 25 °C, followed by purification and characterization processes using infrared spectroscopy and X-ray diffraction. The results demonstrated an increase in the yield of crude chitin proportional to the glucose concentration, reaching a maximum of 76.81 ± 7.64% for Pseudomonas aeruginosa QF50 and 71.30 ± 1.16% for Serratia sp. QCS23. Both strains showed high efficiencies in deproteinization (80–87%) and demineralization, with significant improvements especially shown at high glucose concentrations. Structural characterization confirmed the presence of the spectral characteristics of α-chitin, with crystallinity indices of 81% and 71% for chitins obtained with Pseudomonas aeruginosa QF50 and Serratia sp. QCS23, respectively. This study concludes that single-step fermentation with Pseudomonas aeruginosa QF50 and Serratia sp. QCS23 is an effective and sustainable method for the extraction of high-quality chitin from shrimp exoskeleton waste, offering a promising alternative to traditional chemical methods.

Screening of protease-producing lactic acid bacteria from Indonesian fermented fish products and its application in chitin extraction from vannamei shrimp (Litopenaeus vannamei) shell

This study aimed to screen lactic acid bacteria (LAB) from traditional Indonesian fermented fish products, namely wadi and jambal, with high protease activity for chitin production from Litopenaeus vannamei shrimp shells. Twenty-eight colonies were successfully isolated and identified through biochemical tests such as catalase and Gram staining. Seven out of the twenty-eight colonies were identified as lactic acid bacteria based on their Gram-positive and catalase-negative results. These strains from wadi samples were named GWB 1.3, GWB 1.7, and GWB 2.5, while those from jambal samples were named GMJB 1.3, GMJB 1.6, GMJB 2.6, and GMJB 2.7. The protease activity of these strains was tested using the well-diffusion method on skim milk agar to assess their deproteinized activity. The diameter of the clear zone formed after 24, 48, and 72 hours of incubation was measured. GWB 1.7 and GMJB 2.6 showed the largest clear zone diameter with 21.3 mm and 22.5 mm, respectively, and were selected for the single step-fermentation of shrimp shells. The fermentation process was conducted in 6 days with different inoculum ranging from 0%, 5%, 10%, 15%, and 20% (v/v) as treatments. All treatments, including moisture, ash, and protein, were evaluated for proximate content in the final days. The results indicate that the 20% inoculum showed the highest deproteinization and demineralization for both bacterial strains. GWB 1.7 exhibited a demineralization of 43.7% and deproteinization of 25.2%, while GMJB 2.6 showed a demineralization of 39% and deproteinization of 16.1%. This study demonstrated that lactic acid bacteria from wadi and jambal samples could produce chitin from shrimp shells, a valuable biopolymer with various applications.

Fermentation of brewer's spent grain liquids to increase shelf life and give an organic acid enhanced ingredient.

Brewer's spent grain (BSG) is an abundant brewing by-product, but a short shelf life limits its use. A rotary drum press can separate BSG into a solid and a liquid fraction (BSG liquid) that contains sugars. Propionic acid producing bacteria (PAB) strains from the family Propionibacteriaceae (DSM 20270, 20272, 20274, 106790) were screened for organic acids formation from BSG liquid in three fermentation setups: A single-step fermentation, a two-step fermentation with Lactiplantibacillus plantarum (NFICC 27) followed by PAB, and a co-culture of PAB with L. plantarum. The two-step fermentation produced 9.5 g/L propionic acid and 5.4 g/L acetic acid. GC-MS analysis of the fermented BSG liquid showed a decrease in off-flavours and an increase in compounds with fruity, chocolate and caramel notes. The fermented BSG liquid also had antimicrobial activity. Fermentation of BSG liquid may therefore be a suitable means of creating a clean label food ingredient with long shelf life.

Gluten-free flour fermented with autochthonous starters for sourdough production: Effect of the fermentation process

Refined rice flour and wholegrain quinoa and buckwheat flours were used to prepare gluten-free sourdoughs. Two autochthonous LAB strains were isolated, one from quinoa (Limosilactobacillus fermentum Q3) and the other from buckwheat (Lim. fermentum T5). Lactiplantibacillus plantarum ATCC8014 was used as a reference starter. Two fermentation processes were evaluated, a single-step process (30 °C, 24 h) named SD1, and a daily refreshment process (backslopping for 10 days) named SD2. Sourdoughs were freeze-dried prior to analyses. Pasting properties, pentosan and polyphenol contents, and antioxidant activity were analysed. Flour properties were significantly modified by fermentation. Polyphenol extractability increased during fermentation, which resulted in a higher antioxidant capacity, mainly through a reducing mechanism. Q3 and T5 strains produced the most remarkable modifications of flour properties. Differences were found between both fermentation processes, mainly as a result of the development of different microbiota. The single-step fermentation (SD1) showed to be a simple and appropriate method for improving flour properties.

Anaerobic fermentation of rice bran with rumen liquor for reducing their fiber components to use as chicken feed

Rice bran is a very cheap and abundant agricultural by-product in rice producing countries. Additionally, many developing countries used these by products as poultry feed. Rice bran (RB) contains high fiber and chicken is not capable to digest those fibrous feed effectively, resulting in low production performance. The main objectives of this study were to decrease fiber components of RB through fermentation by adding rumen liquor to increase their utilization efficacy in chicken. A single-step fermentation of RB was conducted for 12 h (n = 6) under anaerobic conditions, maintaining proper temperature and ideal pH. Rice bran, buffer substances, and rumen liquor were mixed at the ratio of 1:2:3, respectively. The fresh and fermented rice brans were examined for the content of acid detergent fiber (ADF), crude fiber (CF), dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent lignin (ADL). Other fiber components were determined by substracting the value of ADL from ADF (celluloses) and ADF from NDF (hemicelluloses), respectively. One-way analysis of variance was done to compare the mean nutrient components followed by Tukey's multiple comparison tests at P < 0.05. The pH of fermented brans was decreased with growing fermentation period but appropriate pH was maintained due to the developed protocol. After fermentation, the fiber components of RB were reduced significantly (P < 0.05). However, CP component was not altered significantly after the fermentation of brans. The NDF, ADF, cellulose, hemicellulose contents were reduced by 16.2 ± 0.52, 7.2 ± 0.32, 20.0 ± 0.38 and 23.6 ± 0.54%, respectively compared to the fresh brans. As the fiber content reduced significantly after fermentation that clearly, increases the usability of brans as chicken feed.

METABOLIC PATHWAY MODIFICATION FOR PRODUCTION OF XYLITOL FROM GLUCOSE IN ESCHERICHIA COLI

Glucose is a cheap and readily available substrate for production of large-scale chemicals. Synthesis of xylitol, a high demand chemical in global market is currently done by using xylose, which contributes to its high operational cost. Studies on production of xylitol from glucose have explored several approaches, from sequential fermentation to multiple and single gene expression. Xylitol-5-phosphate dehydrogenase (XPDH), is an enzyme that enables conversion of glucose to xylitol in a single step fermentation. This study explores conversion of xylitol from glucose in E. coli by the expression of xpdh from Clostridium difficile with modifications in metabolic pathways to enhance xylitol production. The xpdh gene was carried by pACYC-Duet-1 expression vector and induced by the addition of IPTG. Initial screening of E. coli expressing xpdh (NA116) was done by shake-flask fermentation for 24 hours and its metabolites were analyzed by HPLC. NA116 was able to produce 0.273 g/L xylitol from 4.33 g/L consumed glucose in 24 hours. Further metabolic pathway modification to eliminate competing pathways yielded four mutants, NA207 (∆rpiA), NA208 (∆rpiB), NA209 (∆pgi) and NA211 (∆rpi∆Apgi). Screening of mutants for xylitol production showed that highest xylitol production from glucose was achieved by NA211 with almost double the amount of the original strain, 0.585 g/L. This showed successful xylitol conversion from glucose in a single fermentation in E. coli with improved yield through metabolic pathway modification.

Evaluation of gamma-aminobutyric acid (GABA) production by Lactobacillus plantarum using two-step fermentation.

Lactic acid bacteria (Lactobacillus plantarum KCTC 3103) were fermented to produce gamma-aminobutyric acid (GABA). The conditions of the modified synthetic medium were optimized as 5g/L glucose, 10g/L yeast extract, 100g/L rice bran extract, and 1.0g/L ascorbic acid for GABA production. Single-step fermentation of cell growth and GABA production with a modified synthetic medium was higher than those with an MRS medium. Two-step fermentation was evaluated by separating the cell growth and GABA production under a modified synthetic medium. The cell concentration of 1.65g dcw/L produced by the modified synthetic medium was higher than that of 1.0g dcw/L produced by the MRS medium at 36h from the first step of two-step fermentation. The highest GABA production of L. plantarum KCTC 3103 was 0.67g/L with monosodium glutamate addition at 60h in the second step of fermentation. Two-step fermentation with the modified synthetic medium is suitable for GABA production because of its high GABA productivity and favorable cell growth.

Coproduction of hydrogen and volatile fatty acids via integrated two-step fermentation of sweet sorghum stalks by alkaline and enzymatic treatment

Coproduction of H2 and volatile fatty acids from sweet sorghum stalks was successfully developed via two-step fermentation process involving alkaline and enzymatic treatment of the residual slurry obtained from the first step fermentation of the raw material. The optimum treatment conditions to obtain the highest yield of the end products included 2% (w/v) alkali at 120 °C for the residual slurry and 32 FPU cellulase per gram of the alkali-treated materials. The two-step fermentation using Clostridium thermosaccharolyticum increased the end product yields of H2 (6.37 mmol/g-substrate) by 95%, acetic acid (2.33 g/L) by 97% and butyric acid (2.36 g/L) by 143% compared with those obtained in the single step fermentation without any treatment. As a result, the H2 energy recovery efficiency of the two-step fermentation process with commercially attractive butyric acid as a coproduct reached 10.54%. These results provide a promising approach for sweet sorghum biorefining.

Chitin extraction from shrimp wastes by single step fermentation with Lactobacillus acidophilus FTDC3871 using response surface methodology

Chitin extraction from shrimp wastes by single step fermentation with <i>Lactobacillus acidophilus</i> FTDC3871 using response surface methodology

Production of high-purity galacto-oligosaccharides by depleting glucose and lactose from galacto-oligosaccharide syrup with yeasts.

Galacto-oligosaccharides (GOS) are prebiotic compounds, widely used as ingredients in various food, nutraceutical and pharmaceutical products. Enzymatic synthesis of GOS results in low-purity products that contain high amounts of glucose and lactose beside the valuable GOS. In this study, a systematic approach was used to develop yeast-based fermentation strategies to purify crude GOS. Potentially applicable yeast strains were identified based on an extensive search in literature databases followed by a series of laboratory-scale fermentation tests. Single- and two-step fermentation processes were designed for the removal of glucose alone or together with lactose from crude GOS syrup. Single-step fermentation trials with two strains of previously unreported species, Cyberlindnera jadinii NCAIM Y.00499 and Kluyveromyces nonfermentans NCAIM Y.01443, resulted in purified products free of both glucose and ethanol from a crude GOS syrup diluted to 15 and 10 w/v%, respectively. Simultaneous removal of glucose and lactose was achieved by Kluyveromyces marxianus DMB Km-RK in a single-step fermentation process with a yield of 97.5% and final purity of 100%. A two-step fermentation approach was designed to allow conversion of a glucose-free product into a high-purity GOS by removing glucose with C. jadinii Y.00499 in the first step, and lactose by Kluyveromyces lactis DMB Kl-RK in the second step, resulting in a final product with a yield of 100% and a final purity of 92.1%. These results indicate that the selected nonconventional yeasts are promising candidates for the removal of non-GOS components from commercial crude GOS products by selective fermentation.

Clostridium thermocellum: A microbial platform for high-value chemical production from lignocellulose.

Second generation biorefining, namely fermentation processes based on lignocellulosic feedstocks, has attracted tremendous interest (owing to the large availability and low cost of this biomass) as a strategy to produce biofuels and commodity chemicals that is an alternative to oil refining. However, the innate recalcitrance of lignocellulose has slowed progress toward economically viable processes. Consolidated bioprocessing (CBP), i.e., single-step fermentation of lignocellulose may dramatically reduce the current costs of 2nd generation biorefining. Metabolic engineering has been used as a tool to develop improved microbial strains supporting CBP. Clostridium thermocellum is among the most efficient cellulose degraders isolated so far and one of the most promising host organisms for application of CBP. The development of efficient and reliable genetic tools has allowed significant progress in metabolic engineering of this strain aimed at expanding the panel of growth substrates and improving the production of a number of commodity chemicals of industrial interest such as ethanol, butanol, isobutanol, isobutyl acetate and lactic acid. The present review aims to summarize recent developments in metabolic engineering of this organism which currently represents a reference model for the development of biocatalysts for 2nd generation biorefining.

BIOETHANOL PRODUCTION FROM RICE STRAW BY SINGLE STEP FERMENTATION USING CO-CULTURE CLOSTRIDIUM THERMOPHILIC: A REVIEW

Bioethanol is a future renewable energy for gasoline substitution. Market need for bioethanol has been increased significantly in a decade. That will lead to unbalance between bioethanol supply and bioethanol demand if bioethanol production only come from starch and sugar source (first generation of bioethanol). Using lignocellulose waste (second generation of bioethanol) which abundand in Indonesia is one of good solution for this problem. Rice Straw production in Indonesia approximately 103.57 million ton in a year which make it is the most abundant lignocellulosa waste in this country. Optimizing bioethanol production using rice straw need two adjustment step. First step is adjustment in pre-treatment which important to breakdown insoluble lignin from the waste. Second step is selecting bioconvertion agent that has ability to carry single step fermentation process. Thermophilic clostridium is one of best bioconvertion agent for this process which has several advantage compare to others bioconvertion agent. This will lead to increase of bioethanol production to balance between bioethanol supplay and bioethanol demand.

Single-step production of arabino-xylooligosaccharides by recombinant Bacillus subtilis 3610 cultivated in brewers’ spent grain

Brewers’ spent grain (BSG) is an inexpensive and abundant brewery by-product that can be used to produce prebiotic arabino-xylooligosaccharides (AXOS). In this study, Bacillus subtilis 3610 was used, for the first time, to produce AXOS through direct fermentation of BSG. Additionally, the microorganism was genetically modified to improve the AXOS production. The xylanase gene xyn2 from Trichoderma reesei coupled with a secretion tag endogenous to B. subtilis was cloned in pDR111 and integrated into its chromosome. After optimization by experimental design, AXOS with a degree of polymerization ranging from 2 to 6 were obtained. The maximum production yield expressed in xylose equivalents per amount of BSG (54.2 ± 1.1 mg/g) represents an increase of 33% comparing to the wild type. When compared with the enzymatic hydrolysis process, single-step fermentation with B. subtilis proved to be a very promising low-cost strategy for the simultaneous production of AXOS and valorization of BSG.

Effect of two-step fermentation by Chrysonilia crassa and Bacillus subtilis on nutritional values and antioxidative properties of agro-industrial by-products as poultry feed ingredients.

Objective:This current study was subjected to investigate the influence of two-stage fermentation by Chrysonilia crassa and Bacillus subtilis on nutritional values and antioxidative properties of agro-industrial by-products.Materials and methods:Two-stage fermentation with Ch. crassa (inoculated in advance; single-step fermentation) and B. subtilis (inoculated later; two-step fermentation) was conducted on agro-industrial by-products, i.e., banana peel meals, cassava pulp, and rice bran. The pH measurement, microbial enumeration, proximate, and antioxidant analyses were conducted following 4- and 2-days aerobic incubation with Ch. crassa and B. subtilis, respectively.Results:The pH of banana peels and cassava pulp increased with Ch. crassa-fermentation, but then decreased following B. subtilis-fermentation. Chrysonilia crassa-fermentationdid not change, but B. subtilis-fermentation decreased pH of rice bran. The number of lactic acid bacteria was higher in two-stage than in single-stage fermented by-products. Crude protein and fat were higher in fermented than in unfermented banana peels. Crude protein was higher in single- and two-stage fermented, while fat higher in single-stage fermented than in unfermented cassava pulp. Crude fat and ash contents increased with fermentation in rice bran. Single-stage fermentation increased some of amino acids contents in banana peels and cassava pulp. The concentration of polyphenols, tannins, and antioxidant potential of banana peels reduced with fermentation. Total polyphenols and tannins were higher, whereas antioxidant activity was lower in fermented than in unfermented cassava pulp. Total polyphenols, tannins, and antioxidant activity were lower in two-stage than in single-stage fermented and unfermented rice bran.Conclusion:Single-stage fermentation with Ch. crassa improved nutritional characteristics of agro-industrial by-products.

Co-production of polyhydroxyalkanoates and carotenoids through bioconversion of glycerol by Paracoccus sp. strain LL1

Polyhydroxyalkanoates (PHA) have emerged as a potential alternative to synthetic plastics, although its large-scale production is limited by the expenditure incurred in feed materials. Co-production of various valuable bioproducts along with PHA has been proposed to alleviate the overall production cost. Here, high-yield co-production of PHA and carotenoids was achieved in single step fermentation by Paracoccus sp. LL1. The halophilic bacterial strain could metabolize a wide range of substrates including methanol, lactose, galactose, glycerol, fructose, mannitol, etc. Under batch fermentation using mineral media supplemented with 2% glycerol, Paracoccus sp. LL1 synthesized 3.77gL−1 PHA with concomitant production of 3.6mgL−1 of carotenoids after 96h. An enhancement of 2.2-folds in total dry cell weight (DCW) was achieved through cell retention culture of Paracoccus sp. LL1, resulting in a maximum DCW (24.2gL−1) containing PHA (39.3%, ww−1) with concomitant enhancement in the production of total carotenoids. Paracoccus sp. LL1 could convert glycerol into carotenoids and PHA to a level of 7.14mgL−1 and 9.52gL−1, respectively. It is the first report showing value addition in PHA production process through co-production of high value carotenoids by Paracoccus sp. using glycerol as substrate.

Recombinant Lactococcus lactis for efficient conversion of cellodextrins into L‐lactic acid

Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a β-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA.

Single-step production of the simvastatin precursor monacolin J by engineering of an industrial strain of Aspergillus terreus

Monacolin J is a key precursor for the synthesis of simvastatin (Zocor), an important drug for treating hypercholesterolemia. Industrially, monacolin J is manufactured through alkaline hydrolysis of lovastatin, a fungal polyketide produced by Aspergillus terreus. Multistep chemical processes for the conversion of lovastatin to simvastatin are laborious, cost expensive and environmentally unfriendly. A biocatalysis process for monacolin J conversion to simvastatin has been developed. However, direct bioproduction of monacolin J has not yet been achieved. Here, we identified a lovastatin hydrolase from Penicillium chrysogenum, which displays a 232-fold higher catalytic efficiency for the in vitro hydrolysis of lovastatin compared to a previously patented hydrolase, but no activity for simvastatin. Furthermore, we showed that an industrial A. terreus strain heterologously expressing this lovastatin hydrolase can produce monacolin J through single-step fermentation with high efficiency, approximately 95% of the biosynthesized lovastatin was hydrolyzed to monacolin J. Our results demonstrate a simple and green technical route for the production of monacolin J, which makes complete bioproduction of the cholesterol-lowering drug simvastatin feasible and promising.

Saccharification and Single Step Fermentation of Cassava Peel by Mixed Culture of Saccharomycopsis fibuligera NCIM 3161 and Zymomonas mobilis MTCC 92

The main objective of this study was to utilize the cassava peel by saccharification and single step fermentation (SSF) by mixed culture of Saccharomycopsis fibuligera NCIM 3161 and Zymomonas mobilis MTCC 92. Single step fermentation was performed in conical flasks with various concentrations of cassava peel (30, 50, 70 and 90 g/L), pH (3.5, 4.5 and 5.5), temperature (37, 47 and 57° C), reaction time (72, 720 and 168 h), inoculum size (5, 10 and 15 % v/v) and agitation speed (50, 100 and 150 rpm). Cell growth was identified by measuring optical density at 660 nm. The total reducing sugars were determined by centrifuging at 5000 rpm for 10 min by 3,5-dinitrosalicylic acid method. Starch concentration was estimated by anthrone method. Ethanol concentration was determined by acid dichromate method. The optimum process conditions were substrate concentration of 70 g/L, pH of 4.5, temperature of 37° C, reaction time of 120 h, inoculum size of 10 % (v/v) and agitation speed of 100 rpm. Under these conditions, the highest ethanol concentration of 26.46 g/L was obtained for cassava peel (93.75 % of theoretical yield).

4-Hydroxyisoleucine production of recombinant Corynebacterium glutamicum ssp. lactofermentum under optimal corn steep liquor limitation.

4-Hydroxyisoleucine (4-HIL) is a nonproteinogenic amino acid that exhibits insulinotropic biological activity. Here, L-isoleucine dioxygenase gene (ido) derived from Bacillus thuringiensis YBT-1520 was cloned and expressed in an L-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, in order to directly convert its endogenous L-isoleucine (Ile) into 4-HIL through single-step fermentation. The effects of corn steep liquor limitation as well as ido and truncated idoΔ6 overexpression on 4-HIL production were researched. 4-HIL production by ido-overexpressing strain was improved to 65.44 ± 2.27 mM after fermented for 144 h under corn steep liquor-subsufficient condition, obviously higher than that under corn steep liquor-rich and insufficient conditions. The conversion ratio of Ile to 4-HIL increased to 0.85 mol/mol. In addition, 4-HIL production by ido-overexpressing strain was higher than that by idoΔ6-overexpressing strain, in accord with the relatively higher affinity of Ido as compared to IdoΔ6. This research generated a novel system for 4-HIL de novo biosynthesis and demonstrated corn steep liquor limitation as a useful strategy for improving 4-HIL production in recombinant C. glutamicum ssp. lactofermentum.

Single-step fermentation of agricultural hemp residues for hydrogen and ethanol production

We have investigated a one-step fermentation process using purified hemp cellulose (PHC), hemp fibres (HF), and hemp hurds (HH), compared to reagent-grade α-cellullose (AC) for ethanol and hydrogen production by Clostridium thermocellum. Exponential phase growth and production rates on PHC were comparable to those observed with AC. Net production of ethanol for AC (8.47 mM) was only slightly higher than PHC (6.56 mM), but significantly higher than HF (5.48 mM) and HH (3.52 mM), while the final hydrogen yield was comparable for AC (12.70 mM), PHC (11.01 mM), HF (10.91 mM), and HH (4.72 mM). End-product yields were dependent on the intrinsic cellulosic content as well as the presence of other polymers present in substrate biomass. Rates of ethanol and hydrogen production were similar in early log phase but varied in mid-log, and accessibility to cellulose was shown to determine yield and metabolic flux distribution. Our data suggest that improving the accessibility of the cellulolytic bacterium, C. thermocellum, to cellulose fibres has a greater effect on increasing the yield of fermentation products than simply increasing the concentration of cellulose in the substrate.