Mixed acid fermentation of paper fines and industrial biosludge

This paper uses countercurrent fermentation to anaerobically convert paper fines and industrial biosludge to carboxylate salts using a mixed culture of acid-forming microorganisms. Using the MixAlco process, the carboxylate salts can be thermally converted to ketones and hydrogenated into mixed alcohol fuels. Continuum particle distribution modeling (CPDM) correlated batch fermentation data to countercurrent fermentation data, allowing the prediction of product concentrations and conversions over a wide range of solid loading rates and liquid residence times. For 80% paper/20% biosludge, the predicted product concentrations agreed with the data within 7.7%. The predicted conversion agreed with the actual conversion within 27.8%. By correcting for varying selectivity, the predicted conversion agreed with the actual conversions within 15.2%. For 40% paper/60% biosludge, the predicted product concentrations agreed with the data within 9.6%. The predicted conversion agreed with the actual conversion within 28.3%. By correcting for varying selectivity, the predicted conversion agreed with the actual conversions within 15.4%. For both the 80/20 and 40/60 cases, CPDM predicts that 90% conversion is possible with a 20 g/l product concentration, 300 g/l substrate concentration, 16 day liquid residence time, and 2.5 g/(ld) solids loading rate. Before proceeding to an industrial plant, these predictions must be verified in a pilot plant.     

Fermentation of corn stover to carboxylic acids

This article describes countercurrent fermentation to anaerobically convert corn stover and pig manure to mixed carboxylic acids using a mixed culture of mesophilic microorganisms. Corn stover was pretreated with lime to increase digestibility. The Continuum Particle Distribution Model (CPDM) was used to simulate continuous fermentors based on data collected from batch experiments. This model saves considerable time in determining optimum operating conditions. For 80% corn stover/20% pig manure, the highest total carboxylic acid productivity was 1.81 g/(L of liquid. d) at a concentration of 21.4 g total acid/L. The highest total acid selectivity, yield, and conversion were 0.714 g total acid/g volatile solids (VS) digested, 0.550 g total acid/g VS fed, and 0.770 g VS digested/g VS fed, respectively, at a concentration of 16.0 g total acid/L. CPDM predicted the acid concentration and conversion within 13.4 and 11.6%, respectively.     

Anaerobic mixed‐culture fermentation of aqueous ammonia‐treated sugarcane bagasse in consolidated bioprocessing

The MixAlco process is an example of consolidated bioprocessing (CBP) in which anaerobic mixed‐culture fermentation biochemically converts any biodegradable feedstock into carboxylate salts. Downstream processing thermochemically transforms the resulting salts into mixed alcohol fuels or gasoline. To enhance digestibility, sugarcane bagasse was treated under mild conditions (55°C, 24 h, and 30% aqueous ammonia solution with a loading of 10 mL/g dry biomass). Using NH₄HCO₃ buffer, the feedstock (80% ammonia‐treated sugarcane bagasse/20% chicken manure) was anaerobically fermented by a mixed culture of marine microorganisms at 55°C. Four‐stage countercurrent fermentations were performed at various volatile solids loading rates (VSLRs) and liquid residence times (LRTs). The highest acid productivity (1.14 g/(L day)) occurred at a total acid concentration of 29.8 g/L. The highest conversion (65%) occurred at a total acid concentration of 27.6 g/L. The continuum particle distribution model (CPDM) predicted the experimental total acid concentrations and conversions within 4.98% and 10.41%, respectively. When using NH₄HCO₃ buffer, ammonia pretreatment is an attractive option. The CPDM “map” shows that both high volatile solid conversions (78.8%) and high acid concentrations (32.6 g/L) are possible with 300 g/(L liquid) substrate concentration, 30 days LRT, 2 g/(L day) solid loading rate and NH₄HCO₃ buffer. Biotechnol. Bioeng. 2010;106: 216–227. © 2010 Wiley Periodicals, Inc.     

Fixed-bed fermentation of rice straw and chicken manure using a mixed culture of marine mesophilic microorganisms

A mixture of rice straw (80%) and chicken manure (20%) was pretreated and fermented to carboxylic acids by using a mixed culture of marine mesophilic microorganisms. Two sets of four fermentors, built from PVC pipes, were used for both biomass pretreatment and fermentation. Four 1L fermentors (F1-F4) were arranged in series, where liquid fermentation products were transferred from one fermentor to the other, to form a train. A liquid volume of 10mL and 15mL were transferred every four days for Trains A and B, respectively. The maximum total acid concentration for F1 in Train A was 34.2g/L and the maximum acid concentration in F2-F4 was approximately 44g/L. The maximum total acid concentration in F1 in Train B was 30.5g/L and the maximum acid concentration in F2-F4 was approximately 48g/L. The conversion in each of the fermentors in Train A varied from 0.821 to 0.879g VS digested/g VS fed and the yield was in the range 0.489-0.609g total acids/g VS fed. The conversion and yield in Train B were 0.741-0.914g VS digested/g VS fed and 0.563-0.669g total acids/g VS fed, respectively. The continuum particle distribution model (CPDM) predicted acid concentrations and retention times in the fixed-bed fermentation system with R(2) of 0.67-0.84 in Trains A and B.     

Anaerobic thermophilic fermentation for carboxylic acid production from in-storage air-lime-treated sugarcane bagasse

Wet storage and in situ lime pretreatment (50 °C, 1-atm air, 56 days, excess lime loading of 0.3 g Ca(OH)₂/g dry biomass) of sugarcane bagasse (4,000 g dry weight) was performed in a bench-scale pile pretreatment system. Under thermophilic conditions (55 °C, NH₄HCO₃ buffer, methane inhibitors), air-lime-treated bagasse (80 wt.%) and chicken manure (20 wt.%) were anaerobically co-digested in 1-L rotary fermentors by a mixed culture of marine microorganisms (Galveston, TX). During four-stage countercurrent fermentation, the resulting carboxylic acids consisted of primarily acetate (average 87.7 wt.%) and butyrate (average 9.0 wt.%). The experimental fermentation trains had the highest yield (0.47 g total acids/g volatile solids (VS) fed) and highest selectivity (0.79 g total acids/g VS digested) at a total acid concentration of 28.3 g/L, which is equivalent to an ethanol yield of 105.2 gal/(tonne VS fed). Both high total acid concentrations (>44.7 g/L) and high substrate conversions (>77.5%) are predicted for countercurrent fermentations of bagasse at commercial scale, allowing for an efficient conversion of air-lime-treated biomass to liquid transportation fuels and chemicals via the carboxylate platform.     

Biosurfactant yields and nutrient consumption of Pseudomonas fluorescens 378 studied in a microcomputer controlled multifermentation system

Production of biosurfactant AP-6 and consumption of carbon (succinic acid) and nitrogen (ammonium ions) by Pseudomonas fluorescens 378 were studied under different growth conditions. The study was performed in a microcomputer controlled multibatch fermentation system which enabled simultaneous running of 10 fermentors. The fermentors were mantled glass vessels, temperature controlled by circulated water, and mixing was arranged by magnetic stirrers. They were connected to the computer system (pH measurement and control) via signal conditioning cards. The microcomputer had a 128 kbytes RAM, two 800-kbyte floppy disc drives, a graphic terminal, and expansion cards. Biosurfactant production was independent of the carbon-to-nitrogen ratio and the phosphorus content in the medium. Omitting the Fe(III) supplement to the medium increased the product yield by 120%. Changes in oxygen transfer rate and pH in the iron deficient cultures did not have any effect on the product yield. Iron deficiency increased the cell consumption of carbon source. Consumption of carbon source in relation to nitrogen uptake (carbon/nitrogen quotient) increased with increasing quotient in the growth medium. The uptake of carbon and nitrogen changed in the intervals of 1.2-1.5 g/g biomass and 0.09-0.16 g/g biomass, respectively. The consumption of carbon increased from 1.5 g/g biomass to 2.0 g/g biomass when the medium concentration of phosphorus was decreased from 0.18 to 0.027 g/L.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

An increase of curdlan productivity by integration of carbon/nitrogen sources control and sequencing dual fed-batch fermentors operation

Curdlan is produced by Agrobacterium sp. ATCC 31749 under nitrogen-limited conditions not associated with cell growth. A novel curdlan production process was developed based on the different nutrient requirements for microbial cell growth and its efficiency was increased by integrating carbon/nitrogen sources control and sequencing dual fed-batch fermentors operation. By feeding ammonium solution to supply abundant nitrogen source and controlling pH in Fermentor I, cell growth was accelerated. High cell density of 29 g/L was attained. The culture broth in Fermentor I was then inoculated into sequencing Fermentor II which alleviated the high requirement for dissolved oxygen and accumulation of inhibitory metabolic by-products during curdlan production. Fermentor I promoted cell growth. Curdlan production started instantaneously in Fermentor II. By feeding nutrient solution with high carbon/nitrogen ratio and NaOH solution for pH adjustment, a feasible and optimal curdlan production process was formulated. The productivity, conversion efficiency and curdlan yield were achieved of 0.98 g/(L h), 57% (w) and 67 g/L, respectively. Such novel process can be scaled up for significant cost reduction at the industrial level.     

Performance characteristics of continuous yeast-alcohol fermentors with no mechanical stirring

Continuous fermentation of grape juice was studied in three basic kinds of nonmechanically stirred fermentors, classified according to the direction in which flow is applied against settling: upward-flow, slant-flow, and horizontal-flow. Fermentation kinetics were found to be zero-order, thus rate was directly proportional to cell density. Completeness of fermentation depends upon yeast characteristics (growth rate flocculation), fermentor geometry (settling depth, flow path length), and upon flow velocity. Fermentor analysis demonstrated that to achieve complete fermentation in the minimum juice holding time, efficient cell retention as well as rapid yeast growth and fermentation rates are required. Fermentor design and operation are discussed in the light of this analysis.     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

Production of Fumaric Acid in 20-Liter Fermentors

The conditions necessary for the production of fumaric acid in 20-liter fermentors by fermentation of glucose with Rhizopus arrhizus strain NRRL 2582 were determined. Continuous neutralization of fumaric acid was necessary for optimal yields. Yields of the calcium salt were in excess of 65 g of fumaric acid from 100 g of sugar consumed during fermentation of sugar concentrations of 10 to 16%. Conditions established for calcium fumarate production include a simple mineral salts medium, 0.5 v:v:min aeration rate, 300 rev/min agitation rate in a baffled tank, 33 C incubation temperature, CaCO(3) to neutralize the acid formed, and a 4 to 5% (v/v) vegetative inoculum. A suitable procedure and medium for the preparation of a vigorous vegetative inoculum were established. The tendency for calcium fumarate fermentations to foam excessively was controlled with a proper antifoam agent added prior to sterilization of the medium and again at daily intervals during fermentation. The production of soluble sodium or potassium fumarates was inhibited when the concentration of fumarates reached 3.5 to 4.0%. No means of overcoming this inhibition was found. Starches and certain other grain-derived carbohydrates were fermented to form calcium fumarate in flask experiments with approximately the same efficiency as was glucose.     

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.     

The relationship between hydrogen gas and butanol production by Clostridium saccharoperbutylacetonicum

Two simultaneous fermentations were performed at 26 degrees C with simultaneous inocula using Clostridium saccharoperbutylacetonicum. Fermentation 1 prevented the gas formed by the biomass from escaping the fermentor while 2 allowed the gas formed to escape. Fermentor 1 provided for the production of butanol, acetone, and ethanol, while when the H(2) formed was allowed to escape with fermentor 2, neither butanol nor acetone were produced. Ethanol was also formed in both fermentors and began along with the initial growth of biomass and continued until the fermentations were complete. Butanol and acetone production began after biomass growth had reached a maximum and began to subside. The butanol-acetone-ethanol millimolar yields and ratios were 38:1:14 respectively. The fermentor 2 results show that a yield of 2.1 L H(2), 93 or 370 mmol H(2)/mol glucose, was formed only during the growing stage of growth; neither butanol nor acetone were produced; ethanol was formed throughout the fermentation, reaching a yield of 15.2 mmolar. It appears that hydrogen gas is required for butanol production during the resting stage of growth.     

Anaerobic digestion of processed municipal solid waste using a novel high solids reactor: Maximum solids levels and mixing requirements

Summary Novel, laboratory-scale, high solids reactors operated under mesophilic conditions were used to study the anaerobic fermentation of processed municipal solid waste (MSW) to methane. The anaerobic digestion consortium was introduced to high solids levels through gradual adaptation. The maximum sludge solids level for stable anaerobic fermentation performance was identified as approximately 36% wt/wt. Recovery of the anaerobic consortium, following dilution of inhibitory high solids levels, was swift. Reactor mixing requirements were also studied. No significant difference in fermentation performance was observed between agitator speeds of 1 and 25 rpm. Preliminary fermentation performance tests showed that solids loading rates as high as 9.5 g VS (volatile solids) feed/L sludge^.d, at 32% solids within the reactor, were possible. Under these conditions, operation was stable with an average pH of 7.8–8.0, total volatile fatty acid pools of <20 mM, and a biogas composition of 55%–60% methane.     

Production of carotenoids by phaffia rhodozyma growing on media made from hemicellulosic hydrolysates of eucalyptus globulus wood

Phaffia rhodozyma NRRL Y-17268 cells were proliferated in xylose-containing media made from Eucalyptus wood. Wood samples were subjected to acid hydrolysis under mild operational conditions, and hydrolysates were neutralized with lime. Neutralized hydrolysates were treated with charcoal for removing inhibitors and then supplemented with nutrients to obtain culture media useful for proliferation of the red yeast P. rhodozyma. A set of experiments carried out in orbital shakers proved that hydrolysates containing 16.6 g xylose/L supplemented only with 3 g peptone/L performed well as fermentation media. At the end of experiments, xylose was depleted and 10.5 g cells/L were obtained. Biomass was highly pigmented and volumetric carotenoid concentrations up to 5.8 mg carotenoids/L (with 4.6 mg astaxanthin/L) were reached. Further experiments in batch fermentors using concentrated hydrolysates (initial xylose concentrations within 16.6 and 40.8 g/L) led to good biomass concentrations (up to 23.2 g cells/L) with increased pigment concentration (up to 12.9 mg total carotenoids/L, with 10.4 mg astaxanthin/L) and high volumetric rates of carotenoid production (up to 0.079 mg/L.h). Copyright 1998 John Wiley & Sons, Inc.     

Biomass production and alcoholic fermentation performance of Saccharomyces cerevisiae as a function of nitrogen source

Nitrogen limitation is one of the most common causes for stuck or sluggish fermentation. A broad range of values have been reported as the minimum nitrogen concentration necessary for the completion of alcoholic fermentation. We have analyzed the minimum nitrogen concentration required to yield the maximum biomass (nitrogen reference value) using a microwell plate reader to monitor fermentation with different nitrogen sources and sugar concentrations. The biomass yield was dependent on the amount of available nitrogen, the nature of nitrogen source, and the sugar concentration in the medium. Nevertheless, achieving the maximum biomass was not sufficient to ensure the completion of the alcoholic fermentation, because the fermentation of 280?g?sugar?L(-1) stuck, regardless of the nature and concentration of nitrogen source. However, a mixture of five amino acids (Leu, Ile, Val, Phe and Thr) as the nitrogen source allowed for maximum sugar consumption. Analysis of cell vitality by impedance showed a significant improvement in the vitality for cells fermenting using this amino acid combination.     

Effect of stabilization on biomass activity

The study aimed to compare aerobic and aerobic/anoxic stabilization processes in terms of organic matter and the biomass removal efficiencies using a municipal sludge sample. The efficiency of stabilization process was assessed monitoring suspended solids (SS), volatile suspended solids (VSS), total and dissolved organic carbon (TOC, DOC), nitrate, nitrite, and phosphate parameters. The oxygen uptake rate (OUR) measurements were conducted to determine active biomass concentration. On the 30th day of the aerobic stabilization, the SS, VSS and TOC removal efficiencies were 22%, 28% and 55%, respectively. Under aerobic/anoxic conditions, removal efficiencies for SS, VSS and TOC were 25%, 27% and 67%. On the 17th day of the stabilization, SS and VSS removal rates were 60 mg SS/L day and 47 mg VSS/L day for aerobic and 102 mg SS/L day and 63 mg VSS/L day for aerobic/anoxic conditions, respectively. These findings reflected the higher stabilization performance of the aerobic/anoxic conditions. Based on respirometric results, the ratios of the active biomass were decreased to 30% and 24% for the 17th and 30th day of the aerobic stabilization, respectively. Such results have significant implications relative to the activity decrease quantification of the biomass as well as its further application potentials after aerobic or aerobic/anoxic sludge stabilization.     

The metabolism of lactose byClostridium acetobutylicum

Summary Acetone and butanol biosynthesis byClostridium acetobutylicum ATCC 824 was affected by lactose concentration and by agitation during the fermentation. At 1% and 3% lactose concentrations acid production predominated, while butanol production predominated at 5% lactose concentration. Higher solvent production was observed in fermentors without agitation than in fermentors with agitation.     

Effect of Dietary Monensin or Chlortetracycline on Methane Production from Cattle Waste

Wastes from feedlot cattle fed finishing diets containing either monensin, chlortetracycline, or no antibiotic were investigated as substrates for methane production. We used continuously mixed anaerobic fermentors with 3-liter working volumes at 35 and 55°C; these fermentors were fed once per day. Within a few days after waste from animals fed monensin was added, the volume of methane produced began to decrease in the 55°C fermentors. After 9 days of daily feeding, methane production was severely inhibited, the pH dropped from 7.6 to 5.9, and the concentration of volatile acids increased from 543 to 6,300 mg/liter (as acetate). Although additions of waste from cattle fed monensin were discontinued after 9 days, the fermentors did not resume gas production within 8 weeks. The addition of waste from cattle which had been fed chlortetracycline reduced the methane production rate approximately 20%; however, pH and volatile acid values were comparable to control fermentor values after 40 days. Similar effects were observed with the 35°C fermentors. In a batch fermentation experiment in which 50-g portions of volatile solids from waste of animals fed monensin, chlortetracycline, or no antibiotics were added to fermentors, monensin delayed the onset of methane production for about 40 days, but then these fermentors began to produce methane at a rate comparable to the control rate. The ultimate methane yields from the three types of waste after 180 days were not significantly different. These studies indicate that monensin has a detrimental effect on the conversion of feedlot wastes to methane, unless microorganisms can be adapted to the levels that are present in these wastes.     

Efficient acetic acid production by repeated fed-batch fermentation using two fermentors

Summary In acetic acid fermentation, the number of viable cells decrease as the acetic acid concentration increases to more than about 40 g/l, which means that the productivity attainable by conventional fed-batch and repeated fed-batch operations using one fermentor is limited. In this paper, based on a fed-batch experiment using Acetobacter aceti 2096, a mathematical model was developed. The optimization carried out showed the superiority of repeated fed-batch operation using two fermentors. The performance evaluation was made with respect to productivity and product concentration. It was shown to be attractive in practice to use multiple fermentors, in particular for high product concentrations. Experiments were then conducted to ascertain the simulation results. Offprint requests to: T. Kobayashi