Two-stage Fermentation System Research Articles

Bioprocessing of corn rind using continuous multiphase-multistage bioreactor for downstream applications

Corn rind is a major agricultural residue which is often problematic for disposal. However, it is a rich lignocellulose source useful for many downstream industrial applications. The technique commonly used in the bioconversion of organic waste is a static two-stage fermentation system; in the first stage, corn rind is converted to simple sugars, and at the second stage, reduced sugars are used to produce commercial products. The stage one is a resource-consuming process as the product removal is not dynamic, and hence, the bioconversion process comes to an equilibrium due to product accumulation. Therefore, a novel concept of continuous multiphase-multistage bioreactor was introduced, in which the sugars formed are continuously removed to enhance the bioconversion efficiency. A lab-scale model with three digestion modules, each with 2000-ml working capacity, is developed for degradation of corn rind to partially optimize the process parameters that eventually improve corn rind degradation. The multiphase in each stage contributes cumulatively in achieving better degradation of corn rind. In the first stage, out of 150-g feed-stock introduced, a total of 2353 mg of free reducing sugar was released on completion of five phases (1st phase; 431 mg, 2nd phase; 690 mg, 3rd phase; 523 mg, 4th phase; 374 mg, 5th phase; 335 mg) which accounts for 44% of the yield potential. The lab-scale prototype showed promising trend of progressive degradation of corn rind that requires further process optimization to achieve maximum productivity. The reducing sugars thus obtained could be used for subsequent bioconversion processes by using suitable microorganisms.

Biohythane production from organic waste: Recent advancements, technical bottlenecks and prospects

The availability of fossil fuels is a major factor that determines the economy of a country. However, possible exhaustion of fossil fuel deposits as well as increased pollution, and other adverse effects on the environment has prompted us to search for alternative fuels. This resulted in the development of hythane, a blend of hydrogen with methane, at concentrations of 10%–30%. The breakdown of organic substrates using sequential dark fermentation (DF) and anaerobic digestion (AD) leads to biohythane production. The quality and quantity of biohythane can be improved by altering the following aspects: selection, development, and/or genetic engineering of suitable microbial consortium; the use of cheap, appropriate substrates; improved design of bioreactors; and the implementation of two-stage fermentation system. This review focusses on the mechanism of biohythane production and the different aspects involved in increasing both its production rate and quality. A comparative study has also been done to demonstrate the superiority of biohythane over other biofuels.

Value-added lipid production from brown seaweed biomass by two-stage fermentation using acetic acid bacterium and thraustochytrid.

Thraustochytrid production of polyunsaturated fatty acids and xanthophylls have been generally sourced from crop-derived substrates, making the exploration of alternative feedstocks attractive since they promise increased sustainability and lower production costs. In this study, a distinct two-stage fermentation system was conceptualized for the first time, using the brown seaweed sugar mannitol as substrate for the intermediary biocatalyst Gluconobacter oxydans, an acetic acid bacterium, along with the marine thraustochytrid Aurantiochytrium sp. to produce the value-added lipids and xanthophylls. Jar fermenter culture resulted in seaweed mannitol conversion to fructose with an efficiency of 83 % by G. oxydans and, after bacteriostasis with sea salts, production of astaxanthin and docosahexaenoic acid by Aurantiochytrium sp. KH105. Astaxanthin productivity was high at 3.60 mg/L/day. This new system, therefore, widens possibilities of obtaining more varieties of industrially valuable products including foods, cosmetics, pharmaceuticals, and biofuel precursor lipids from seaweed fermentation upon the use of suitable thraustochytrid strains.

Conversion of organic solid waste to hydrogen and methane by two-stage fermentation system with reuse of methane fermenter effluent as diluting water in hydrogen fermentation

In this study, a two-stage system converting organic solid waste (food waste+sewage sludge) to H2 and CH4 was operated. In the first stage of dark fermentative hydrogen production (DFHP), a recently proposed method that does not require external inoculum, was applied. In the second stage, anaerobic sequencing batch reactor (ASBR) and an up-flow anaerobic sludge blanket reactor (UASBr) were followed to treat H2 fermenter effluent. (H2+CH4-ASBR) system showed better performance in terms of total biogas conversion (78.6%), while higher biogas production rate (2.03 L H2/Lsystem/d, 1.96 L CH4/Lsystem/d) was achieved in (H2+CH4-UASBr) system. To reduce the alkali addition requirement in DFHP process, CH4 fermenter effluent was tested as a diluting water. Both the ASBR and UASBr effluent was effective to keep the pH above 6 without CH4 production. In case of using ASBR effluent, H2 production dropped by 15%, but alkali addition requirement was reduced by 50%.

Use of real time gas production data for more accurate comparison of continuous single-stage and two-stage fermentation

Changes in fermenter gas composition within a given 24h period can cause severe bias in calculations of biogas or energy yields based on just one or two measurements of gas composition per day, as is common in other studies of two-stage fermentation. To overcome this bias, real time recording of gas composition and production were used to undertake a detailed and controlled comparison of single-stage and two-stage fermentation using a real world substrate (wheat feed pellets). When a two-stage fermentation system was used, methane yields increased from 261Lkg−1VS using a 20day HRT, single-stage fermentation, to 359Lkg−1VS using a two-stage fermentation with the same overall retention time – an increase of 37%. Additionally a hydrogen yield of 7Lkg−1VS was obtained when two-stage fermentation was used. The two-stage system could also be operated at a shorter, 12day HRT and still produce higher methane yields (306Lkg−1VS). Both two-stage fermentation systems evaluated exhibited methane yields in excess of that predicted by a biological methane potential test (BMP) performed using the same feedstock (260Lkg−1VS).

Continuous fermentative hydrogen and methane production from Laminaria japonica using a two-stage fermentation system with recycling of methane fermented effluent

In this study, a two-stage fermentation system to produce H2 and CH4 from Laminaria japonica was developed. In the first stage (dark fermentative H2 production, DFHP), response surface methodology (RSM) with a Box-Behnken design (BBD) was applied for optimization of operational parameters, including cycle-frequency, HRT, and substrate concentration, using an intermittent-continuously stirred tank reactor (i-CSTR). Overall performance revealed that the degree of importance of the three variables in terms of H2 yield is as follows: cycle-frequency > substrate concentration > HRT. In the confirmation test, H2 yield of 113.1 mL H2/g dry cell weight (dcw) was recorded, corresponding with 96.3% of the predicted response value under desirable operational conditions (cycle-frequency of 17 hr, HRT of 2.7 days, and substrate concentration of 31.1 g COD/L). In the second stage, an anaerobic sequencing batch reactor (ASBR) and an up-flow anaerobic sludge blanket reactor (UASBr) were employed for CH4 production from H2 fermented solid state (HFSS) and H2 fermented liquid state (HFLS), respectively. The CH4 producing ASBR and UASBr showed a stable CH4 yield and COD removal until a HRT of 12 days and OLR of 3.5 g COD/L/d, respectively. Subsequently, for recycling of CH4 fermented effluent from the UASBr (MFEUASBr) as diluting water in DFHP, the tap water and MFEUASBr mixing ratio (T/M ratio) was optimized (a T/M ratio of 5:5) in a batch test using heat pretreated MFEUASBr at 90 °C for 20 min, resulting in the best performance. Although slight decreases of H2 yield (7.6%) and H2 production rate (3.5%) were recorded, 100% reduction of alkali addition was possible, indicating potential to maximize economic benefits. However, a drastic decrease of H2 productivity and a change of liquid-state metabolites were observed with the use of non-heat pretreated MFEUASBr. These results coincided with those of the microbial analysis, where non-H2 producing bacteria, such as Selenomonas sp., were detected. The results indicate that pretreatment of MFEUASBr may be required in order to recycle it in DFHP.

Feasibility of Hydrogen Production by a Continuous Two-Stage (Dark/Dark) Fermentation System

Anaerobic hydrogen production in a continuous two-stage fermentation system was studied. Two continuously stirred tank reactors (CSTR) were employed to evaluate performances of the system. The first stage was fed with molasses wastewater, and the effluent discharged from the first stage was subsequently fed into the second stage. The hydrogen production rate (HPR) in the second stage achieved a remarkable increase from 1.76 L/d to 6.45 L/d during the operation by re-utilizing the residual substrates from the first reactor effluent. The two stages showed a similar metabolic pathway for biohydrogen fermentation. The hydrogen production yield (HY) and acidification efficiency increased markedly by more than 70% and 50% respectively, which indicated the hydrogen recovery and anaerobic acidification of organic substrates can be improved by the combined continuous two-stage hydrogen production process.

Biogasification of Garbage and Waste-paper by Hydrogen-methane Two-stage Fermentation System

Biogasification of Garbage and Waste-paper by Hydrogen-methane Two-stage Fermentation System

Feasibility of hydrogen production from ripened fruits by a combined two-stage (dark/dark) fermentation system

Anaerobic fermentation for hydrogen (H2) production was studied in a two-stage fermentation system fed with different ripened fruit feedstocks (apple, pear, and grape). Among the feedstocks, ripened apple was the most efficient substrate for cumulative H2 production (4463.7 mL-H2 L(-1)-culture) with a maximum H2 yield (2.2 mol H2 mol(-1) glucose) in the first stage at a hydraulic retention time (HRT) of 18 h. The additional cumulative biohydrogen (3337.4 mL-H2 L(-1)-culture) was produced in the second stage with the reused residual substrate from the first stage. The major byproducts in this study were butyrate, acetate, and ethanol, and butyrate was dominant among them in all test runs. During the two-stage system, the energy efficiency (H(2) conversion) obtained from mixed ripened fruits (RF) increased from 4.6% (in the first stage) to 15.5% (in the second stage), which indicated the energy efficiency can be improved by combined hydrogen production process. The RF could be used as substrates for biohydrogen fermentation in a two-stage (dark/dark) fermentation system.

Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

BackgroundThermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis.ResultsAn about 10.5 kb long genomic region was isolated and sequenced from Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a formate dehydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, membrane-bound hydrogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase.ConclusionThe formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase (formate dehydrogenase coupled hydrogenase, FDH-MHY) complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation.

Utilization of keratin-containing biowaste to produce biohydrogen

A two-stage fermentation system was constructed to test and demonstrate the feasibility of biohydrogen generation from keratin-rich biowaste. We isolated a novel aerobic Bacillus strain (Bacillus licheniformis KK1) that displays outstanding keratinolytic activity. The isolated strain was employed to convert keratin-containing biowaste into a fermentation product that is rich in amino acids and peptides. The process was optimized for the second fermentation step, in which the product of keratin fermentation--supplemented with essential minerals--was metabolized by Thermococcus litoralis, an anaerobic hyperthermophilic archaeon. T. litoralis grew on the keratin hydrolysate and produced hydrogen gas as a physiological fermentation byproduct. Hyperthermophilic cells utilized the keratin hydrolysate in a similar way as their standard nutrient, i.e., bacto-peptone. The generalization of the findings to protein-rich waste treatment and production of biohydrogen is discussed and possible means of further improvements are listed.

Chlamydospore Production, Inoculation Methods and Pathogenicity of Fusarium oxysporum M12-4A, a Biocontrol for Striga hermonthica

Fusarium oxysporum isolate M12-4A is currently being evaluated for the biological control of Striga hermonthica . Inoculum production, inoculum delivery to the target, chlamydospore germination, and weed growth suppression of this weed-pathogen system were investigated. Liquid fermentation systems using organic material were evaluated for the production of large numbers of chlamydospores. A 1% sorghum straw powder (< 1 mm) substrate, exposed to black light at 21°C for 21 days, yielded 3.23 X 10 8 colony forming units (CFU) l -1 medium. A two-stage fermentation system using 5% w/v straw substrate under black light at 30°C for 14 days yielded 3.5 X 10 8 CFU l -1 medium. In vitro variations in chlamydospore germination were governed by the presence of exogenous carbon, nitrogen, and sorghum root exudates. Ammonium-nitrogen compounds and urea, in combination with glucose had a stronger stimulatory effect on chlamydospore germ tube growth than did potassium nitrate. Maximal germ tube elongation occurred when chlamydospores were exposed to urea at a C/N ratio of 10. Some mineral solutions and sorghum root exudates inhibited chlamydospore germ tube elongation; however, arabic gum, a complex polysaccharide, stimulated chlamydospore germ tube elongation and the production of secondary chlamydospores. In field trials, chlamydospore powder harvested from small-scale fermenters reduced S. hermonthica emergence by 92%. Complete inhibition of S. hermonthica emergence occurred when the chlamydospore powder was added to the soil at sowing and when sorghum seeds coated with chlamydospores were sown. Effective biological control of S. hermonthica was achieved using a simple fermentation system with sorghum straw as the inoculum growth substrate. For inoculum delivery to the farmers' fields, sorghum seeds were coated with the inoculum using arabic gum as the adhesive. This simple delivery system permits a uniform inoculation of the field as well as the proper positioning of the inoculum in the immediate environment of sorghum roots, where S. hermonthica attaches to its host. To facilitate a broad usage of F. oxysporum M12-4A for the biocontrol of S. hermonthica , we propose an inoculum production strategy based on a cottage industry model that utilizes a liquid fermentation process and inexpensive locally-available substrates including sorghum straw and arabic gum.

Conditions for a continuous production of transient microbial products in a two-stage fermentation system

The optimal residence time in the inducing reactor of a continuous two-stage system has been studied in order to maximize the yield in such a process. The attention has been focused on the case in which the product (or one of its forms) appears in the culture only transiently after the induction. Whereas in some cases the two-stage system is able to improve the yield and to allow a continuous product concentration in the outgoing stream, in others, and depending on how the product is accumulated in the induced culture, no optimal residence time can be found, and a higher productivity will be achieved by using the one-stage continuous strategy or a batch fermentation.

Production of idoheptulosan from sedoheptulosan by microorganisms

Microbial production of idoheptulosan has been established using a two-stage fermentation system. Sedoheptulosan was first converted to 5-ketosedoheptulosan by a strain of Agrobacterium, 449-1, in a medium containing sorbitol as the energy source. After 4 d of cultivation, sedoheptulosan (100 g/ l was almost completely converted to its 5-keto form. Many strains of Gluconobacter were also found to have the same oxidizing activity. Asymmetric reduction of 5-ketosedoheptulosan to idoheptulosan was performed with strains of Aureobacterium, Pseudomonas, Paracoccus, Micrococcus or soil isolates of coryneform bacteria. By incubating these bacteria with 5-ketosedoheptulosan in medium containing d-glucose as the hydrogen donor for reduction, 5-ketosedoheptulosan was converted specifically to idoheptulosan after 5 d. The overall yield of idoheptulosan was about 60% when soil isolate CF-987 was used. Other stereoisomers of idoheptulosan and other metabolites were not detected in the final broth of the strain.

Optimization of a two‐stage recombinant fermentation process: The dilution rate effect

The effects of dilution rates on the performance of a two-stage fermentation system for a recombinant Escherichia coli culture were studied. Dilution rate determines the apparent or averaged specific growth rate of a heterogeneous population of cells in the recombinant culture. The specific growth rate affects the genetic parameters involved in product formation in the second stage, such as plasmid stability, plasmid content, and specific gene expression rate. Kinetic models and correlations were developed for these parameters based on experimental data. Simulations of plasmid stability in the first stage showed that for longer fermentation periods, plasmid stability is better at higher dilution rates. However, the plasmid content is lower at these dilution rates. The optimal apparent specific growth rate for maximum productivity in the second stage was determined using two methods: (1) direct search for a constant specific growth rate, and (2) dynamic optimization using the maximum principle for a time-dependent specific growth rate profile. The results of the calculations showed that the optimum constant apparent specific growth rate for maximum over-all productivity is 0.40 h(-1). This coincides with the optimal specific growth rate for maximum plasmid content in the expressed stage. A 3.5% increase in overall productivity can be obtained by using a linear time dependent apparent specific growth rate control, micro(2)(t) = 0.0007t, in the course of the fermentation time.

Optimal temperature control policy for a two-stage recombinant fermentation process.

The optimal temperature control policy to be followed in the operation of a two-stage fermentation system in which gene expression is induced by a temperature-sensitive gene switching system was studied. A genetically structured model was used to describe product formation, and kinetic equations based on experimental data were used to quantify the specific gene expression rate and parameters that affect plasmid instability. A constant temperature control policy and temperature profiling control policy including temperature cycling were studied and compared. Maximum average production rate was obtained from a temperature control policy in which the second stage was operated initially at about 40.5 degrees C and the temperature decreased slightly to a constant value at 40.0 degrees C. The maximum average production rate, which corresponds to the optimal temperature control policy, for an operation of 180 h was 29.7 units of protein (mg of cells)-1 h-1.

Digestion of cheese whey with anaerobic rotating biological contact reactors

A laboratory-scale anaerobic rotating biological contact reactor receiving full strength cheese whey was studied over a range of hydraulic retention times from 11 to 5 days at 35°C. Methane production rates ranging from 1·68 to 3·26 litres CH 4 litre −1 and a 76 to 93% reduction in chemical oxygen demand were achieved. At hydraulic retention times shorter than 5 days, steady-state operation could not be maintained for reactors receiving either full strength or diluted whey. A two-stage fermentation system was also studied; the results indicated that stable operation and treatment efficiency (89·5% COD removal) could be achieved.

Pilot-scale operation and economic assessment of a two-stage, straw—manure fermentation system

A two-stage fermentation system was evaluated. The system consisted of a high-rate fermenter (to produce methane from beef-cattle manure) and a low-rate fermenter (to produce methane from straw and slowly degrading material in the effluent from the high-rate fermenter). Three trials were completed. Results from these trials showed that methane yields of 0.25 m 3 CH 4/kg volatile solids (VS) straw can be achieved in less than 100 days of fermentation at 35°C, and that injection of anhydrous ammonia does not significantly increase the methane yield. Using only the effluent from the straw fermenter as make-up liquid in the system inhibited methane production. This inhibition was attributed to salt accumulation. Based on the assumptions used to design the system (40 Mg total solids (TS) per day) and in the economic analysis, a straw cost of slightly over $15/Mg TS was the break-even cost below which it was economically advantageous to add straw to manure for fermentation to methane. It was economically advantageous to compact the straw to densities between 150 and 300 kg/m 3 to reduce the straw fermenter volume and cost. Economies of scale were apparent, especially at the smaller plant sizes. The discounted cash flow rate of return (DCFRR) increased rapidly up to a plant size of about 20 Mg TS/day, then increased linearly as plant size continued to increase. The plant sizes necessary to achieve a DCFRR of 15% were 6.4, 13, and 43 Mg/day for straw costs of $0, $15, and $30/Mg, respectively. Income tax bracket had only minimal effect on the DCFRR.

Semi-continuous and Continuous Production of Aspergillus niger Spores in Submerged Liquid Culture

SUMMARY: Sporulation of Aspergillus niger was induced in continuous tower fermenters by restricting growth with nitrate limitation. Semi-continuous production of spores was achieved in a single-stage fermentation by alternating full-nutrient and nitrogen-deficient media to the culture. Continuous spore production was obtained in a two-stage fermentation system using the first stage for the growth of vegetative mycelium under optimum conditions and the second, larger, stage for sporulation induction in a constant nitrogen-deficient environment. Spores were produced from much simplified sporulation structures. Using a new sporulation index (Ω), which relates spore numbers produced to the quantity of substrate utilized, the average production efficiency of the two-stage continuous system was shown to be more than twice that of the semi-continuous production system. The tower fermenter was shown to be ideal for controlling organism morphology, thus providing conditions which allowed the development of continuous fungal sporulation.

Production of 2-Keto- l -Gulonic Acid from d -Glucose by Two-Stage Fermentation

A practical method for the production of calcium 2-keto-l-gulonate (an intermediate in the Reichstein synthesis of l-ascorbic acid) from d-glucose has been established by using a two-stage fermentation system. d-Glucose was first converted to calcium 2,5-diketo-d-gluconate by a mutant strain of Erwinia sp. in a medium containing d-glucose, corn steep liquor, (NH(4))(2)HPO(4), and CaCO(3). After a 26-h cultivation, 328.6 mg of calcium 2,5-diketo-d-gluconate per ml was obtained, with a 94.5% yield from d-glucose. This broth was used directly for the next conversion without removal of cells by treatment with sodium dodecyl sulfate. The stereospecific reduction of calcium 2,5-diketo-d-gluconate to calcium 2-keto-l-gulonate was performed with a mutant strain of Corynebacterium sp. When the cell growth reached a maximum (about 16 h) in a medium containing d-glucose, corn steep liquor, NaNO(3), KH(2)PO(4), and trace elements, NaNO(3) was added to the culture, and then the calcium 2,5-diketo-d-gluconate broth was fed over a period of about 50 h. Since the mutant strain requires a hydrogen donor for reduction, the calcium 2,5-diketo-d-gluconate broth was mixed with d-glucose before being fed. The results of four two-stage fermentations in 10-m conventional fermentors showed that an average of 106.3 mg of calcium 2-keto-l-gulonate per ml was obtained, with a 84.6% yield from d-glucose, the starting material of calcium 2,5-diketo-d-gluconate production. Calcium 2-keto-l-gulonate was stable in the broth. Neither 2-keto-d-gluconic acid nor 5-keto-d-gluconic acid was detected in the final broth.