Evaporative temperature and moisture control in a rocking reactor for solid substrate fermentation

Summary In the solid substrate fermentation of cooked yellow corn grits with Rhizopus oligosporus in a rocking drum fermenter, temperature was controlled by blowing air through the substrate, forcing water evaporation. The rate of evaporation was controlled by the relative humidity of the air, according to the rate of heat generation during fermentation. Moisture content was maintained constant by spraying cold water on the substrate regulated by the water balance equation of the system. Both controls were operated by computer programs. The rocking motion in the reactor allowed even distribution of air and water in the substrate without disturbing the growing mycelia.     

Pilot production of Clonostachys rosea conidia in a solid‐state fermentor optimized using response surface methodology

The nonpathogenic, saprophytic fungus Clonostachys rosea is one of the most powerful fungal biological control agents (BCAs). However, the production of fungal BCAs is still a major constraint for their large‐scale use and commercialization. Here, we developed a novel solid‐fermentation reactor that is light transparent and ventilated both at the top and the bottom, and optimized C. rosea cultivation conditions in solid‐state fermentation using response surface methodology. The growth area of spores provided by the novel fermentor was two times that of the traditional one. A quadratic polynomial model was developed, which indicated the effects of variables on the conidia yield. The greatest spore production of 3.50 × 10¹⁰ spores/g‐dry‐matter was obtained after 11 days at the initial moisture content of 69.2% w/w, the medium thickness of 3.84 cm, and the porosity of 0.37%. The optimized spore yield was increased by one order of magnitude. The fermentation time was shortened from 15 to 11 days. With the novel solid‐fermentation reactor, increase in C. rosea spores production and decrease in fermentation time were achieved. Current data imply that both the novel solid‐fermentation reactor designed and the optimized fermentation conditions are suitable for industrial‐scale C. rosea spore production.     

纤维素降解菌的分离筛选及对中药废弃物的固相发酵研究

中药在提取炮制后残留大量生物质被简单堆弃,而其中的木质纤维素等生物质能被微生物有效利用降解,筛选到16株对木质纤维素有降解效果的菌株,对其中8株降解效果明显功能菌株进行了深入研究,经16(18)S rDNA鉴定发现5株细菌主要为Bacillus属、Streptomyces属和Enterobacter属,3株真菌为Ascomycete属、Aspergillus属和Trichosporon属;利用8株菌固相发酵中药废弃物,通过监测堆体温度、pH等参数,发现细菌类在发酵初期能迅速提高堆体温度,而真菌类发酵腐熟过程较缓慢,提示利用细菌-真菌联合组成降解菌系是提高中药废弃物固相发酵的重要手段。     

Stimulation by potassium ions of the growth of Rhizopus oligosporus during liquid-and solid-substrate fermentations

Soya beans and several other beans and cereals have been used as substrates for tempe fermentation with the fungus Rhizopus oligosporus Saito. Except for the presence of alkaloids, the chemical composition of lupins (Lupinus mutabilis Sweet) is similar to that of soya beans. Therefore the potential of lupins for tempe production in regions with a long tradition of lupin consumption is promising. The preparation of the fermentation substrate when using bitter lupins (which contain significan quantities of alkaloids) as starting material includes a debittering stage to remove the alkaloids. However, we found that the debittering process yielded lupins that did not support the mycelial growth required in the tempe fermentation. We discovered that potassium is preferentially leached out during the debittering process. The effect of potassium on fungal biomass formation was monitored using a computerized system that determines biomass accretion by measurement of the electrical capacitance at radio frequencies. The importance of potassium for the growth of R. oligosporus was confirmed in liquid cultures. A linear relationship was found between biomass yield and K⁺ concentration in the range of 1 to 10 mg/l. The present report represents one of the few demonstrations of a mineral deficiency during the growth of a fungus on a natural, solid substrate.     

Hyperactive α-amylase production by Aspergillus oryzae IFO 30103 in a new bioreactor

Aims: To improve the α‐amylase production in solid‐state fermentation (SSF) condition utilizing a new bioreactor (NB) system. Methods and Results: In NB system, 20 g of wheat bran moistened with liquid medium in 1 : 1 ratio (w/v) was taken on the tray present inside the upper vessel and an additional 80 ml medium was supplemented into the lower vessel. Oxygen uptake rate was improved by supplying compressed air that lifted the liquid medium into the upper vessel and touched the substrate bed. This condition probably facilitated the heat transfer to liquid medium, reduce water loss and catabolite repression. With 1% glucose supplementation, maximum α‐amylase activity of 22 317 Ugds^?1 was produced by Aspergillus oryzae IFO 30103 within a very short incubation period (48 h) at 2‐cm bed height with air flow rate of 0·1 l min^?1 g^?1 wheat bran at 32°C and initial medium pH of 6. Conclusions: Within a short incubation period, significantly high α‐amylase activity was obtained and it is higher than those reported to date at bioreactor scale operating with a fungal strain. Significance and Impact of the Study: The reactor is novel and can overcome some of the major problems associated with SSF process. A. oryzae IFO 30103 is reported as the best fungal source for α‐amylase production.     

Microbial production of methane from municipal refuse

A heat and caustic pretreatment process has been tested to determine the increase in gas yield that can be obtained from fermentation of organic municipal refuse. A treatment temperature of 130°C and a NaOH concentration of 3 g/100 g of dry solids resulted in the highest gas yield for the conditions tested. The probable increase in gas yield was 20% for the best conditions tested. The treatment procedure also substantially increased the rates of gas production. A high conversion efficiency is possible at much shorter reactor retention times with pretreatment than without pretreatment.     

Process optimization of an auger pyrolyzer with heat carrier using response surface methodology

A 1 kg/h auger reactor utilizing mechanical mixing of steel shot heat carrier was used to pyrolyze red oak wood biomass. Response surface methodology was employed using a circumscribed central composite design of experiments to optimize the system. Factors investigated were: heat carrier inlet temperature and mass flow rate, rotational speed of screws in the reactor, and volumetric flow rate of sweep gas. Conditions for maximum bio-oil and minimum char yields were high flow rate of sweep gas (3.5 standard L/min), high heat carrier temperature (∼600 °C), high auger speeds (63 RPM) and high heat carrier mass flow rates (18 kg/h). Regression models for bio-oil and char yields are described including identification of a novel interaction effect between heat carrier mass flow rate and auger speed. Results suggest that auger reactors, which are rarely described in literature, are well suited for bio-oil production. The reactor achieved liquid yields greater than 73 wt.%.     

Treatment of coffee waste by slurry-state anaerobic digestion

Slurries containing 20% (w/v) coffee waste solids were treated anaerobically in one- and two-phase thermophilic methane fermentation systems (53°C) with or without pH control. In one-phase methane fermentation using a roller bottle reactor, the maximum gas evolution rate of 0.87 l/l·d was achieved during treatment for 91 d. However, this one-phase methane fermentation did not yield reproducible data. In a two-phase methane fermentation system consisting of a completely stirred tank reactor type (CSTR-type) liquefaction reactor without pH control and an anaerobic fluidized bed type gasification reactor, three-repetitions of treatment were conducted. Each treatment was very stable and the average gas evolution rate per volume of the gasification reactor was about 2.4 l/l·d. Two-repetitions of treatment were then done while controlling pH in the liquefaction at more than 6. The average gas evolution rate per volume of gasification reactor was found to have increased to 10.2 l/l·d, a value which corresponded to 0.84 l/l·d per total volume, including the liquefaction reactor. It was observed that treatment in a two-phase methane fermentation could be repeated in a stable fashion even in the closed system without discharging anything but the coffee waste residues.     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Microbioreactor (micro-Matrix) potential in aerobic and anaerobic conditions with different industrially relevant microbial strains

Microscale fermentation systems are important high throughput tools in clone selection, and bioprocess set up and optimization, since they provide several parallel experiments in controlled conditions of pH, temperature, agitation, and gas flow rate. In this work we evaluated the performance of biotechnologically relevant strains with different respiratory requirements in the micro-Matrix microbioreactor. In particular Escherichia coli K4 requires well aerated fermentation conditions to improve its native production of chondroitin-like capsular polysaccharide, a biomedically attractive polymer. Results from batch and fed-batch experiments demonstrated high reproducibility with those obtained on 2 L reactors, although highlighting a pronounced volume loss for longer-term experiments. Basfia succiniciproducens and Actinobacillus succinogenes need CO₂ addition for the production of succinic acid, a building block with several industrial applications. Different CO₂ supply modes were tested for the two strains in 24 h batch experiments and results well compared with those obtained on lab-scale bioreactors. Overall, it was demonstrated that the micro-Matrix is a useful scale-down tool that is suitable for growing metabolically different strains in simple batch process, however, a series of issues should still be addressed in order to fully exploit its potential.     

Monitoring the microbial community succession and diversity of Liangzhou fumigated vinegar during solid-state fermentation with next-generation sequencing

This study reveals the microbial community succession and diversity during the whole solid-fermentation processes of naturally fermented Liangzhou fumigated vinegar (LZFV). Dynamics and diversity of microbial community succession in “Daqu” starter and other fermentation stages (starch saccharification, alcoholic fermentation, and acetic acid fermentation) were monitored using a metagenomic approach involving high-throughput sequencing. Meanwhile, dynamic changes of characteristic flavor compounds of vinegar were determined by gas chromatograph (GC) analysis. The result showed that the microbiota composition exhibited rich diversity. Twenty-five bacterial and 18 fungal genera were found in the whole fermentation process where Lactobacillus, Acetobacter, Aspergillus, Saccharomyces, and Alternaria were the predominant microorganisms. Alpha diversity metrics showed that bacterial diversity in Daqu was greater than that in AF and AAF. By contrast, fungal diversity increased from Daqu to AF and decreased in the initial stage (5–8 days) of AAF then remained relatively steady. Hence, these results could help understand dynamics of microbial community succession in continuous fermentation of traditional Chinese vinegars. The LZFV fermentation is a continuous process with spontaneous growth that affects the dynamics of microbial communities. Continuous changes of micro-environment conditions in substrate affect the diversity and structure of microbiota. Microbial growth and metabolism were closely related to the changes in the physicochemical characteristics of the cultures. The microbial flora composition showed rich diversity, and with the increase in brewing time and the change in micro-ecological environmental conditions; the microbial community showed a complex dynamic changes.

     

Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.     

Model-based control of feed rate and illuminance in a photosynthetic fed-batch reactor for H2S removal

Traditional application of computer to fermentation processes has focused on the measurement and control of parameters such as temperature, pH, vessel pressure, sparge rate, dissolved oxygen, substrate concentration, and product concentration. In a fed-batch reactor with the photosynthetic green sulfur bacterium Chlorobium thiosulfatophilum which converts hydrogen sulfide to elementary sulfur or sulfate, separate measurement of cell mass concentration and sulfur particle concentration turbidimetrically was difficult due to their combined contributions to the total turbidity. Instead of on-line measurement of many process variables, a model-based control of feed rate and illuminance was designed. Optimal operation condition relating feed rate vs. light intensity was obtained to suppress the accumulation of sulfate and sulfide, and to save light energy in a 4-1 photosynthetic fed-batch reactor. This relation was correlated with the inreasing cell mass concentration. A model which describes the cell growth by considering the light attenuation effects due to scattering and absorption, and to crowding effect of the cells, was established beforehand with the results from the experiments. Based on these optimal operating conditions and the cell growth model, automatic controls of feed rate and illuminance were carried out alternatively to the traditional application of computer to fermentation with on-line measurement, realtime response and adjustment of process variables.List of Symbols F ml/min Flow rate of gas mixture - hV lux Average illuminance - Q mmol/(l h) Removal rate of hydrogen sulfide - X mg protein/l Cell mass concentration as protein - X ₀ mg protein/l Initial cell mass concentration - X _m mg protein/l Maximum cell mass concentration - _a h^–1 Apparent specific growth rate     

Continuous acetic acid production by the bioreactor system loading a new ceramic carrier for microbial attachment

Summary We have developed a bioreactor system for aerobic fermentation, using a new ceramic carrier APHROCELL which has a suitable shape for liquid and gas passage. In acetic acid fermentation byAcetobacter cells from ethanol, as a typical example of aerobic fermentation, a productivity of 17.25 g/l h was attained at continuous production of 23 g-acetic acid/l; at an acetic acid concentration around 53 g/l, the productivity was 6.4 g/l h. Thus a marketable vinegar can be obtained continuously by this bioreactor system. Because of the simplicity of the APHROCELL reactor, scale up should be relatively easy.     

Continuous fermentation of soy sauce by immobilized cells of Zygosaccharomyces rouxii in an airlift reactor

Summary The optimum conditions for continuous alcohol fermentation of soy sauce with immobilized Zygosaccharomyces rouxii cells were investigated using an airlift reactor. The optimum pH and temperature of the fermentation were 4.5–5.5 and 25°–27.5° C, respectively. Ethanol content in the fermented liquid was increased with increasing height to diameter ratio of the reactor and the ratio of air to nitrogen in the supplied gas (total supplied gas: 0.08 vvm). A notable decrease in ethanol content was observed when only nitrogen gas was supplied. The products fermented by supplying air (0.02 vvm) had a higher conent of aroma components than that by supplying only nitrogen gas, and the aroma of the former products was similar to that of conventional soy sauce. This alcohol fermentation using an airlift reactor was continued for about 50 days without problems even if conditions such as residence time and aeration were altered.     

Integrated biogas upgrading and hydrogen utilization in an anaerobic reactor containing enriched hydrogenotrophic methanogenic culture

Biogas produced by anaerobic digestion, is mainly used in a gas motor for heat and electricity production. However, after removal of CO₂, biogas can be upgraded to natural gas quality, giving more utilization possibilities, such as utilization as autogas, or distant utilization by using the existing natural gas grid. The current study presents a new biological method for biogas upgrading in a separate biogas reactor, containing enriched hydrogenotrophic methanogens and fed with biogas and hydrogen. Both mesophilic‐ and thermophilic anaerobic cultures were enriched to convert CO₂ to CH₄ by addition of H₂. Enrichment at thermophilic temperature (55°C) resulted in CO₂ and H₂ bioconversion rate of 320 mL CH₄/(gVSS h), which was more than 60% higher than that under mesophilic temperature (37°C). Different dominant species were found at mesophilic‐ and thermophilic‐enriched cultures, as revealed by PCR–DGGE. Nonetheless, they all belonged to the order Methanobacteriales, which can mediate hydrogenotrophic methanogenesis. Biogas upgrading was then tested in a thermophilic anaerobic reactor under various operation conditions. By continuous addition of hydrogen in the biogas reactor, high degree of biogas upgrading was achieved. The produced biogas had a CH₄ content, around 95% at steady‐state, at gas (mixture of biogas and hydrogen) injection rate of 6 L/(L day). The increase of gas injection rate to 12 L/(L day) resulted in the decrease of CH₄ content to around 90%. Further study showed that by decreasing the gas–liquid mass transfer by increasing the stirring speed of the mixture the CH₄ content was increased to around 95%. Finally, the CH₄ content around 90% was achieved in this study with the gas injection rate as high as 24 L/(L day). Biotechnol. Bioeng. 2012; 109: 2729–2736. © 2012 Wiley Periodicals, Inc.     

Influence of ambient air temperature on the cooling/heating load of a single cell protein jacketed fermenter operating on cheese whey under continuous conditions

The heat generated by mixing and lactose metabolism, during the continuous production of single cell protein from cheese whey lactose using a jacketed fermenter with running cooling water, was calculated using a heat balance equation. The technique quantified the heat produced in and lost from the fermentation unit. Most of the heat generated by mixing in the cell-free system (97.47%) was lost with exhaust gas, while a very small amount (2.53%) was lost through the fermenter lid, wall, and bottom. The heat generated by mixing was significant (26.31% of the total heat generated in the fermentation system with an active yeast population present) and, therefore, cannot be ignored in heat balance calculations. About 19.71% of the total heat generated in the reactor was lost through the coolant at an ambient temperature of 22 +/- 0.5 degrees C, showing the need for a cooling system. A yeast population size of 986 million cells/mL and a lactose removal efficiency of 95.6% were observed. About 72.5% and 27.5% of the lactose consumed were used for growth and respiration, respectively. A yield of 0.66 g of cells/g of lactose was achieved. The heat released by unit biomass was 7.05 kJ/g of cells. The results showed the significant impact of ambient air temperature on the cooling load. The heat to be removed from the medium by the cooling system varied from 3.46 to 281.56 kJ/h when the temperature increased from 16 to 30 degrees C. A heating system is needed to maintain the medium temperature at 34 degrees C when the ambient air temperature is below 16 degrees C.     

A rapid column technique for trapping and collecting of volatile fungal hydrocarbons and hydrocarbon derivatives

A custom-made stainless steel column was designed to contain various materials that would trap the hydrocarbons and hydrocarbon derivatives during the processes of fungal fermentation ultimately yielding preparative amounts of volatile organic substances (VOCs). Trapping materials tested in the column were Carbotrap materials A and B (Supelco) as well as bentonite-shale from the oil bearing areas of Eastern Montana, the former allowed for the effective and efficient trapping of VOCs from purged cultures of Hypoxylon sp. Trapping efficiencies of various materials were measured by both gravimetric as well as proton transfer reaction mass spectroscopy with the Carbotraps A and B being 99% efficient when tested with known amounts of 1,8-cineole. Trapped fungal VOCs could effectively be removed and recovered via controlled heating of the stainless steel column followed by passage of the gases through a liquid nitrogen trap at a recovery rate of ca 65–70%. This method provides for the recovery of mg quantities of compounds normally present in the gas phase that may be needed for spectroscopy, bioassays and further separation and analysis and may have wide applicability for many other biological systems involving VOCs. Other available Carbotraps could be used for other applications.     

Temperature constraints on the growth and functioning of root organ cultures with arbuscular mycorrhizal fungi

In this study we investigated the effects of temperature on fungal growth and tested whether the differences in fungal growth were related to the effects of temperature on carbon movement to, or within, the fungus. Growth curves and C uptake-transfer-translocation measurements were obtained for three arbuscular mycorrhizal fungi (AMF) isolates cultured within a 6-30 degrees C temperature range. A series of experiments with a model fungal isolate, Glomus intraradices, was used to examine the effects of temperature on lipid body and 33P movement, and to investigate the role of acclimation and incubation time. Temperature effects on AMF growth were both direct and indirect because, despite clear independent root and AMF growth responses in some cases, the uptake and translocation of 13C was also affected within the temperature range tested. Root C uptake and, to a lesser extent, C translocation in the fungus, were reduced by low temperatures (< 18 degrees C). Uptake and translocation of 33P by fungal hyphae were, by contrast, similar between 10 and 25 degrees C. We conclude that temperature, between 6 and 18 degrees C, reduces AMF growth, and that C movement to the fungus is involved in this response.