Conversion of municipal solid waste into carboxylic acids by anaerobic countercurrent fermentation: effect of using intermediate lime treatment

Municipal solid waste (MSW) and sewage sludge (SS) were combined and anaerobically converted into carboxylate salts by using a mixed culture of acid-forming microorganisms. MSW is an energy source and SS is a source of nutrients. In this study, MSW and SS were combined, so they complemented each other. Four fermentors were arranged in series for a countercurrent fermentation process. In this process, the solids and liquid were transferred in opposite directions, with the addition of fresh biomass to fermentor 1 and fresh liquid media to fermentor 4. An intermediate lime treatment of solids exiting fermentor 3 before entering fermentor 4 was applied to improve the product acid concentration from the untreated MSW/SS fermentations. All fermentations were performed under anaerobic conditions at 40 degrees C. Calcium carbonate was added to neutralize the carboxylic acids and to control the pH. Iodoform was used as a methanogen inhibitor. Carboxylic acid concentration and gas composition were determined by gas chromatography. Substrate conversion was measured by volatile solids loss, and carboxylic acid productivity was calculated as the function of the total carboxylic acids produced, the amount of liquid in all fermentors, and time. The addition of intermediate lime treatment increased product concentration and conversion by approx 30 and 15%, respectively. The highest carboxylic acid concentrations for untreated MSW/SS fermentations with and without intermediate lime treatment were 22.2 and 17.7 g of carboxylic acid/L of liquid, respectively. These results confirm that adding a treatment step between fermentor 3 and fermentor 4 will increase the digestibility and acid productivity of the fermentation.     

Conversion of sugarcane bagasse to carboxylic acids using a mixed culture of mesophilic microorganisms

Using the MixAlco process, biomass can be converted into carboxylic acids, which can be chemically converted into mixed alcohol fuels. This study focused on the use of countercurrent fermentation to anaerobically convert sugarcane bagasse and chicken manure to mixed carboxylic acids using a mixed culture of mesophilic microorganisms from terrestrial and marine sources. Bagasse was pretreated with lime to increase digestibility. The continuum particle distribution model (CPDM) simulated continuous fermentors based on data collected from batch experiments. This model saves considerable time in determining optimum operating conditions. For an 80% bagasse/20% chicken manure fermentation with terrestrial inoculum at a volatile solids loading rate (VSLR) of 7.36 g/(L of liquid·d) and a liquid residence time (LRT) of 8.88 d, total carboxylic acid productivity, total acid selectivity, and yield were 2.49 g/(L of liquid·d), 0.581 g of total acid/g of VS digested, and 0.338 g of total acid/g of VS fed, respectively, at a concentration of 18.7 g of total acid/L. At the same VSLR and LRT, fermentation with marine inoculum gave higher total acid productivity, total acid selectivity, and yield than fermentation with terrestrial inoculum. For an 80% bagasse/20% chicken manure fermentation with marine inoculum at a VSLR of 3.83 g/(L of liquid·d) and an LRT of 12.1 d, total carboxylic acid productivity, total acid selectivity, and yield were 1.38 g/(L of liquid·d), 0.667 g of total acid/g of VS digested, and 0.359 g of total acid/g of VS fed, respectively, at a concentration of 16.2 g of total acid/L.     

Laboratory method for high-solids countercurrent fermentations

Equipment and procedures were developed to study the conversion of lignocellulosic biomass to carboxylic acids using high-solids countercurrent fermentations. Countercurrent fermentations of cattle manure yielded a rapid fermentation (maximum 2.98 g of total acid/[L x d]) with high acid concentrations (maximum of 32.5 g of total acid/L), but the acid yield tended to be low (maximum of 0.24 g of total acid/g of volatile solids). Countercurrent fermentations of a mixture of 80% municipal solid waste/20% sewage sludge fermented more slowly (maximum of 1.98 g of total acid/[L x d]) with a lower acid concentration (maximum of 26.5 g of total acids/L), but higher acid yields were achieved (maximum of 0.34 g of total acid/g of volatile solids).     

Investigation of Nutrient Feeding Strategies in a Countercurrent Mixed-Acid Multi-Staged Fermentation: Experimental Data

Nutrients are essential for microbial growth and metabolism in mixed-culture acid fermentations. Understanding the influence of nutrient feeding strategies on fermentation performance is necessary for optimization. For a four-bottle fermentation train, five nutrient contacting patterns (single-point nutrient addition to fermentors F1, F2, F3, and F4 and multi-point parallel addition) were investigated. Compared to the traditional nutrient contacting method (all nutrients fed to F1), the near-optimal feeding strategies improved exit yield, culture yield, process yield, exit acetate-equivalent yield, conversion, and total acid productivity by approximately 31%, 39%, 46%, 31%, 100%, and 19%, respectively. There was no statistical improvement in total acid concentration. The traditional nutrient feeding strategy had the highest selectivity and acetate-equivalent selectivity. Total acid productivity depends on carbon–nitrogen ratio.     

Fermentation of Sugarcane Bagasse and Chicken Manure to Calcium Carboxylates under Thermophilic Conditions

Sugarcane bagasse and chicken manure were anaerobically fermented to carboxylic acids using a mixed culture of marine microorganisms at 55 °C. Using the MixAlco process— an example of consolidated bioprocessing— the resulting carboxylate salts can be converted to mixed alcohol fuels or gasoline. To enhance digestibility, sugarcane bagasse was lime pretreated with 0.1 g Ca(OH)₂/g dry biomass at 100 °C for 2 h. Four-stage countercurrent fermentation of 80% sugarcane bagasse/20% chicken manure was performed at various volatile solids (VS) loading rates and liquid residence times. Calcium carbonate was used as a buffer during fermentation. The highest acid productivity of 0.79 g/(L day) occurred at a total acid concentration of 21.5 g/L. The highest conversion (0.59 g VS digested/g VS fed) and yield (0.18 g total acids/g VS fed) occurred at a total acid concentration of 15.5 g/L. The continuum particle distribution model (CPDM) predicted the experimental total acid concentrations and conversions at an average error of 10.14% and 12.68%, respectively. CPDM optimizations show that high conversion (>80%) and total acid concentration of 21.3 g/L are possible with 300 g substrate/(L liquid), 30 days liquid residence time, and 3 g/(L day) solid loading rate. Thermophilic fermentation has a higher acetate content (∼63 wt%) than mesophilic fermentation (∼39 wt%).     

Conversion of municipal solid wastes to carboxylic acids by thermophilic fermentation

The purpose of this research is to generate carboxylic acids from the biodegradable fraction of municipal solid wastes (MSW) and municipal sewage sludge (MSS) by using a thermophilic (55°C), anaerobic, high-solid fermentation. With terrestrial inocula, the highest total carboxylic acid concentration achieved was 20.5 g/L, the highest conversion obtained was 69%, and the highest acetic acid selectivity was 86.4%. Marine inocula were also used to compare against terrestrial sources. Continuum particle distribution modeling (CPDM) was used to predict the final acid product concentrations and substrate conversions at a wide range of liquid residence times (LRT) and volatile solid loading rates (VSLR). “Maps” showing the product concentration and conversion for various LRT and VSLR were generated from CPDM. The predictions were compared to the experimental results. On average, the difference between the predicted and experimental values were 13% for acid concentration and 10% for conversion. CPDM “maps” show that marine inocula produce higher concentrations than terrestrial inocula.     

Fermentation of “Quick Fiber” produced from a modified corn-milling process into ethanol and recovery of corn fiber oil

Approximately 9% of the 9.7 billion bushels of corn harvested in the United States was used for fuel ethanol production in 2002, half of which was prepared for fermentation by dry grinding. The University of Illinois has developed a modified dry grind process that allows recovery of the fiber fractions prior to fermentation. We report here on conversion of this fiber (Quick Fiber [QF]) to ethanol. QF was analyzed and found to contain 32%wt glucans and 65%wt total carbohydrates. QF was pretreated with dilute acid and converted into ethanol using either ethanologenic Escherichia coli strain FBR5 or Saccharomyces cerevisiae. For the bacterial fermentation the liquid fraction was fermented, and for the yeast fermentation both liquid and solids were fermented. For the bacterial fermentation, the final ethanol concentration was 30 g/L, a yield of 0.44 g ethanol/g of sugar(s) initially present in the hydrolysate, which is 85% of the theoretical yield. The ethanol yield with yeast was 0.096 gal/bu of processed corn assuming a QF yield of 3.04 lb/bu. The residuals from the fermentations were also evaluated as a source of corn fiber oil, which has value as a nutraceutical. Corn fiber oil yields were 8.28%wt for solids recovered following prtetreatment.     

Studies on nutrient requirements and cost-effective supplements for ethanol production by recombinantE. coli

This article describes a systematic study of the nutritional requirements of a patented recombinant ethanologenicEscherichia coli (11303:pLOI297) and provides cost-effective formulations that are compatible with the production of fuel ethanol in fermentations of lignocellulosic prehydrolysate characterized by high xylose conversion efficiency. A complex and nutrient-rich laboratory medium, Luria broth (LB), provided the benchmark with respect to fermentation performance standard. Xylose fermentation performance was assessed in terms of the target values for operational process parameters established by the US National Renewable Energy Laboratory (NREL)—final ethanol concentration (25 g/L), xylose-to-ethanol conversion efficiency (90%), and volumetric productivity (0.52 g/L·h). Biomass prehydrolysates that are rich in xylose also contain acetic acid, and in anticipation of a need to reduce acetic acid toxicity, the fermentors were operated with a pH control set-point of 7.0 Growth and fermentation in the minimal defined salts (DS) medium was only about 15% compared to the reference medium. Amendment of the minimal medium containing 6 wt% xylose with both vitamins and amino acids resulted in improved growth, but the volume productivity (0.59 g/L·h) was still only about 54% of that with LB (1.1 g/L·h). Formulations directed at cost reduction through the use of less expensive commercial complex nutritional supplements were within 90% of the NREL process target with respect to yield and provided a productivity at about 80% of the LB medium, but were not economical. Corn steep liquor (CSL) at about 7–8 g/L was shown to be a complete source of nutritional requirements and supported a fermentation performance approaching that of LB. At a cost of CSL of $50/t (dry wt), the economic impact of using this amount CSL as the sole nutritional supplement in a cellulosic ethanol plant was estimated to be about 4¢/gal of ethanol.

     

Fermentation of rice straw/chicken manure to carboxylic acids using a mixed culture of marine mesophilic micoorganisms

Countercurrent fermentation of rice straw and chicken manure to carboxylic acids was performed using a mixed culture of marine mesophilic microorganisms. To increase the digestibility of the biomass, rice straw, and chicken manure were pretreated with 0.1 g Ca(OH)₂/g biomass. Fermentation was performed for 80% rice straw and 20% chicken manure at various volatile solid loading rates (VSLR) and liquid residence times (LRT). The highest acid productivity of 1.69 g/(L·d) occurred at a total acid concentration of 32.4 g/L. The highest conversion (0.69 g VS digested/g VS fed) and yield (0.29 g total acids/g VS fed) were at a total acid concentration of 25 g/L. A Continuum Particle Distribution Model of the process predicted the experimental total acid concentration and conversion results with an average error of 6.41% and 6.15%, respectively. Results show how total acid concentrations, conversions, and yields vary with VSLR and LRT in the MixAlco process.     

Thermostability of Selected Enzymes in Organic Wastes and in their Humic Extract

The objective of this study was to evaluate the thermostability up to 70 degrees C for 1 h of selected enzymes present in fresh and composted sewage sludge (SS and SSC) or municipal solid wastes (MSW and MSWC) and their humic extract. After a thermal treatment at 70 degrees C, no beta-glucosidase activity in any humic extract was detected, whereas in SS, SSC, MSW, and MSWC, it was respectively, 35%, 68%, 17%, and 12% compared to thermally untreated samples. By contrast, o-diphenol oxidase activity was even stimulated by thermal treatment in SS samples, but in the humic extracts, this activity decreased by 75-81%. Urease activity in all humic extracts decreased by 70% or more just at 40 degrees C, whereas for organic wastes, this decrease was observed after treatment at 70 degrees C. Alkaline phosphatase (AP) activity was affected by thermal treatment only in MSW and MSWC. In humic extracts, AP activity decreased gradually to zero except for the MSW extract, where 45% activity was retained after treatment at 70 degrees C. In general, thermostability of enzymes in humic extracts was lower than the materials they were extracted from.     

Production of fuel ethanol from rye and triticale by very-high-gravity (VHG) fermentation

Very-high-gravity (VHG) rye and triticale mashes, containing about 28.5 g dissolved solids/100 mL of mash supernatant, were prepared by adjusting water:grain ratios to 2:1. Because of high viscosity, which develops during mashing, it was necessary to pretreat ground rye-water slurries with viscosity-reducing enzymes. There were no viscosity problems during the preparation of triticale mashes. Fermentations were conducted at 20°C, with and without 16 mM urea as a nitrogenous supplement. All fermentations were completed within 120–144 h. Supplementation with urea shortened the times required for completion of fermentation by 33% for triticale and by 40% for rye. The fermentation efficiencies for both grains ranged between 90 and 93%. These values are comparable to those reported for wheat, implying competitiveness of rye and triticale as fermentation feedstocks to replace wheat. The final ethanol yields were 409 L for rye and 417–435 L for triticale/t (dry basis). For a given size of fermentation vessel, 33% more grain was used in the VHG fermentation process than in normal gravity fermentation. This resulted in a 35–56% increase in ethanol concentration in the beer, when fermentors were filled to a constant volume. The corresponding reduction in water use by about one-third would result in savings in energy consumption in mash heating, mash cooling, and ethanol distillation. Fermentation efficiencies and final ethanol yields obtained per unit weight of grain fermented were not significantly different from the normal gravity fermentations.     

Production of Ammonium Lactate by Fed-batch Fermentation of Rhizopus oryzae from Corncob Hydrolysate

IntroductionThere is an increased demand for lactic acid asa starting material for the synthesis of poly- lacticacid ( PLA) ,a biodegradable polymer,which cansubstitute for petrochemical derivatives.However,an optically pure L- ( + ) - lactic acid( LLA) is need-ed[1] .Industrial lactic acid fermentations are usual-ly carried out with bacteria,which produces a mix-ture of L- ( + ) - and D- ( - ) - lactic acid in a richmedium[2 ,3 ] .The rich nutrient supplements re-quired for bacterial ferm…     

Early phase process scale-up challenges for fungal and filamentous bacterial cultures

Culture pelleting and morphology has a strong influence on process productivity and success for fungal and filamentous bacterial cultures. This impact is particularly evident with early phase secondary metabolite processes with limited process definition. A compilation of factors affecting filamentous or pelleting morphology described in the literature indicates potential leads for developing process-specific control methodologies. An evaluation of the factors mediating citric acid production is one example of an industrially important application of these techniques. For five model fungal and filamentous bacterial processes in an industrial fermentation pilot plant, process development strategies were developed and effectively implemented with the goal of achieving reasonable fermentation titers early in the process development cycle. Examples of approaches included the use of additives to minimize pelleting in inoculum shake flasks, the use of large-volume frozen bagged inoculum obtained from agitated seed fermentors, and variations in production medium composition and fermentor operating conditions. Results were evaluated with respect to productivity of desired secondary metabolites as well as process scalability. On-line measurements were utilized to indirectly evaluate the cultivation impact of changes in medium and process development. Key laboratory to pilot plant scale-up issues also were identified and often addressed in subsequent cultivations.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Enhancement of Fumaric Acid Production by Rhizopus oryzae Using a Two-stage Dissolved Oxygen Control Strategy

Batch fermentative production of fumaric acid by Rhizopus oryzae ME-F12 was investigated in a 7-l stirred tank fermentor under different dissolved oxygen (DO) concentrations. High fumaric acid yield on glucose (0.56 g/g) was achieved under high DO concentration (80%), but the glucose consumption rate and fumaric acid productivity were rather low (0.91 and 0.51 g/l/h). Fumaric acid productivity was enhanced under low DO concentration (30%), but the fuamric acid yield on glucose decreased to 0.52 g/g. In order to achieve the high fumaric acid yield and productivity simultaneously, a two-stage dissolved oxygen control strategy was proposed, in which the DO concentration was controlled at 80% in the first 18 h and then switched to 30%. This was experimentally proven to be successful. Relatively high fumaric acid production (56.2 g/l), high fumaric acid yield on glucose (0.54 g/g), and high glucose consumption rate (1.3 g/l/h) were achieved by applying this strategy. The productivity (0.7 g/l/h) was improved by 37%, 21%, and 9%, respectively, compared with fermentations in which DO concentrations were kept constant at 80%, 60%, and 30%.     

Optimization of Fermentation Conditions for the Biosynthesis of <Emphasis Type="SmallCaps">l</Emphasis>-Threonine by <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this study, the fed-batch fermentation technique was applied to improve the yield of l-threonine produced by Escherichia coli TRFC. Various fermentation substrates and conditions were investigated to identify the optimal carbon source, its concentration and C/N ratio in the production of l-threonine. Sucrose was found to be the optimal initial carbon source and its optimal concentration was determined to be 70 g/L based on the results of fermentations conducted in a 5-L jar fermentor using a series of fed-batch cultures of E. coli TRFC. The effects of glucose concentration and three different feeding methods on the production of l-threonine were also investigated in this work. Our results showed that the production of l-threonine by E. coli was enhanced when glucose concentration varied between 5 and 20 g/L with DO-control pulse fed-batch method. Furthermore, the C/N ratio was a more predominant factor than nitrogen concentration for l-threonine overproduction and the optimal ratio of ammonium sulfate to sucrose (g/g) was 30. Under the optimal conditions, a final l-threonine concentration of 118 g/L was achieved after 38 h with the productivity of 3.1 g/L/h (46% conversion ratio from glucose to threonine).     

Ester fuels and chemicals from biomass

Bench-scale research demonstrated that using an efficient esterification step to integrate an ethanol with a carboxylic acid fermentation stream offers potential for producing valuable ester feedstocks and fuels. Polar organic acids from bacterial fermentations are difficult to extract and purify, but formation of the ammonium salts and their conversion to esters facilitates the purifications. An improved esterification procedure gave high yields of esters, and this method will lower the cost of ester production. Fuel characteristics have been determined for a number of ester-gasoline blends with promising results for lowering Reid vapor pressure and raising octane numbers.     

Biotechnological Production of Xylitol: Enhancement of Monosaccharide Production by Post-Hydrolysis of Dilute Acid Sugarcane Hydrolysate

Dilute-acid hydrolysis pretreatment of sugarcane bagasse resulted in release of 48% (18.4 g/L) of the xylan in the hemicellulose fraction into the hydrolysate as monomeric xylose. In order to enhance the recuperation of this monomer, a post-hydrolysis stage consisted of thermal treatment was carried out. This treatment resulted in an increase in xylose release of 62% (23.5 g/L) of the hemicellulose fraction. Original and post-hydrolysates were concentrated to the same levels of monomeric xylose in the fermentor feed. During the fermentation process, cellular growth was observed to be higher in the post-hydrolysate (3.5 g/L, Y _x/s?=?0.075 g cells/g xylose) than in the original hydrolysate (2.9 g/L, Y _x/s?=?0.068 g cells/g xylose). The post-treated hydrolysate required less concentration of sugars resulting in a lower concentration of fermentation inhibitors, which were formed primarily in the dilute acid hydrolysis step. Post-hydrolysis step led to a high xylose–xylitol conversion efficiency of 76% (0.7 g xylitol/g xylose) and volumetric productivity of 0.68 g xylitol/L h when compared to 71% (0.65 g xylitol/g xylose and productivity of 0.61 g xylitol/L h) for the original hemicellulosic hydrolysate.     

Ethanol fermentation in a tower fermenter using self-aggregatingSaccharomyces uvarum

A self-aggregating strain ofSaccharomyces uvarum (U4) was used as a biocatalyst to carry out continuous ethanol fermentation in a tower fermentor equipped with a cell separator. Cell aggregates (2–3 mm) formed a stable packed bed in the fermentor, and the cell separator retained yeast cells effectively. Corn steep liquor was used as a nitrogen source for the fermentation of corn syrup and black strap molasses. An ethanol productivity of 54 g/L/h was reached using corn syrup at a dilution rate of 0.7/h, and sugar concentration in the feed was 15% (w/v). For molasses fermentation, an ethanol productivity of 22 g/L/h was obtained at a dilution rate of 0.7/h, and sugar concentration in the feed was 12.5% (w/v). Ethanol yields obtained from tower fermentation are higher than those obtained from flask fermentation (96% for corn syrup fermentation and 92% for molasses fermentation). No significant loss in fermentation activity was observed after 3 mo of operation.     

Kinetic Analysis and pH-Shift Control Strategy for Propionic Acid Production with <Emphasis Type="Italic">Propionibacterium Freudenreichii</Emphasis> CCTCC M207015

The production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 was investigated in a 7.5-l stirred-tank fermentor. Batch fermentations by P. freudenreichii CCTCC M207015 at various pH values ranging from 5.5 to 7.0 were studied. Based on the analysis of the time course of specific cell growth rate (μ _x) and specific propionic acid formation rate (μ _p), a two-stage pH-shift control strategy was proposed. At first 48 h, pH was controlled at 6.5 to obtain the maximal μ _x, subsequently pH 6.0 was used to maintain high μ _p to enhance the production of propionic acid. By applying this pH-shift control strategy in propionic acid fermentation, the maximal propionic acid and glucose conversion efficiency had a significant improvement and reached 19.21 g/l and 48.03%, respectively, compared with those of constant pH operation (14.58 g/l and 36.45%). Fed-batch fermentation with pH-shift control strategy was also applied to produce propionic acid; the maximal propionic acid yield and glucose conversion efficiency reached 25.23 g/l and 47.76%, respectively.