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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.78, #1-3)
Ethanol production from corn starch in a fluidized-bed bioreactor by Mahesh S. Krishnan; Nhuan P. Nghiem; Brian H. Davison (pp. 359-372).
The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.
Keywords: Ethanol; dry-milled corn starch; Zymomonas mobilis ; glucoamylase; fluidized-bed reactor
Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400(pLNH33) by Mahesh S. Krishnan; Nancy W. Y. Ho; George T. Tsao (pp. 373-388).
Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400 (pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of a 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures was found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product inhibition, and inoculum size. Good agreements were obtained between model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.
Keywords: Recombinant Saccharomyces 1400(pLNH33); ethanol; xylose fermentation; kinetic model
Accumulation of biopolymers in activated sludge biomass by Hong Chua; Peter H. F. Yu; Chee K. Ma (pp. 389-399).
In this study, activated sludge bacteria from a conventional wastewater treatment process were induced to accumulate polyhydroxyalkanoates (PHAs) under different carbon-nitrogen (C:N) ratios. As the C:N ratio increased from 20 to 140, specific polymer yield increased to a maximum of 0.38 g of polymer/g of dry cell mass while specific growth yield decreased. The highest overall polymer production yield of 0.11 g of polymer/g of carbonaceous substrate consumed was achieved using a C:N ratio of 100. Moreover, the composition of polymer accumulated was dependent on the valeric acid content in the feed. Copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was produced in the presence of valeric acid. The 3-hydroxyvalerate (3HV) mole fraction in the copolymer was linearly related tovaleric content in the feed, which reached a maximum of 54% when valeric acid was used as sole carbon source. When the 3HV U in the polymer increased from 0–54 mol%, the melting temperature decreased from 178° to 99°C. Thus, the composition, and hence the mechanical properties, of the copolymer produced from activated sludge can be controlled by adjusting the mole fraction of valeric acid in the feed medium.
Keywords: Activated sludge; carbon-nitrogen ratio; wastewater treatment; butyric-valeric acid ratio; poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
Optimization of L-Lactic acid production from glucose by Rhizopus oryzae ATCC 52311 by Ying Zhou; José M. Domínguez; Ningjun Cao; Jianxin Du; George T. Tsao (pp. 401-407).
The effect of nutrients on L(+)-lactic acid production from glucose was investigated using Rhizopus oryzae ATCC 523 11. From the shake-flask experiments, the optimal medium composition was defined for improved lactic-acid production. In order to enhance lactic-acid production rate and product yield, controlled aeration in a bubble column was conducted under optimal conditions. Results showed a maximum lactic-acid production rate of 2.58 g/L/h was obtained with an initial glucose concentration of 94 g/L. Finallactic-acid concentration of 83 g/L was achieved after 32 h of fermentation with a weight of 0.88 glactic acid/g glucose consumed.
Keywords: Bubble column; lactic acid; Rhizopus oryzae
Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid in a two-zone fermentor-extractor system by Prashant V. Iyer; Y. Y. Lee (pp. 409-419).
Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid was investigated using a two-zone bioreactor. The system is composed of an immobilized cell reactor, a separate column reactor containing the lignocellulosic substrate and a hollow-fiber membrane. It is operated by recirculating the cell free enzyme (cellulase) solution from the immobilized cell reactor to the column reactor through the membrane. The enzyme and microbial reactions thus occur at separate locations, yet simultaneously. This design provides flexibility in reactor operation as it allows easy separation of the solid substrate from the microorganism, in situ removal of the product and, if desired, different temperatures in the two reactor sections. This reactor system was tested using pretreated switchgrass as the substrate. It was operated under a fed-batch mode with continuous removal of lactic acid by solvent extraction. The overall lactic acid yield obtainable from this bioreactor system is 77% of the theoretical.
Keywords: Lactic acid; SSF; cell immobilization; switchgrass; in situ extraction
Microbial dynamics in oil-impacted prairie soil by Kathleen E. Duncan; Ravindra Kolhatkar; Ganesh Subramaniam; Ramesh Narasimhan; Eleanor Jennings; Susan Hettenbach; Amanda Brown; Charles McComas; William Potter; Kerry Sublette (pp. 421-434).
A remote site in the Tallgrass Prairie Preserve (Osage County, OK) was contaminated with crude oil by a pipeline break in 1992. In 1996, the contaminated soil was bioremediated by blending with uncontaminated soil, prairie hay, buffalo manure, and commercial fertilizers, and spreading in a shallow layer over uncontaminated soil to create a landfarm. The landfarm was monitored for two years for aerobic and anaerobic bacteria, soil gases indicative of microbial activity, and for changes in the concentration of total petroleum hydrocarbons (TPH). Levels of hydrocarbon degraders and soil gas indicators of aerobic degradation were stimulated in the landfarm during the first warm season relative to uncontaminated prairie soil. However, these same indicators were less conclusive during the second warm season, indicating depletion of the more easily degradable hydrocarbons, although the landfarm still contained 6,800 mg/kg TPH on the average at the beginning of the second warm season. Methane formation and methanogen counts were clearly stimulated in the first warm season relative to uncontaminated prairie soil, in dicating that methanogenesis plays an important role in the mineralization of hydrocarbons even in these shallow soils.
Keywords: Crude oil; bioremediation; TPH; methanogenesis; hydrocarbon-degrading bacteria
Bioconversion of secondary fiber fines to ethanol using counter-current enzymatic saccharification and Co-fermentation by Thomas W. Jeffries; Richard Schartman (pp. 435-444).
This research examined several enzymatic and microbial process for the conversion of waste cellulosic fibers into ethanol. The first was a one-stage process in which pulp fines were contacted with commercial enzyme solutions. The second process used sequential, multistage saccharification. The third used sequential enzyme addition in a countercurrent mode. Experiments compared the results with various feed stocks, different commercial enzymes, supplementation with β-glucosidase, and saccharification combined with fermentation. The highest saccharification (65%) from a 4% consistency pulp and the highest sugar concentration (5.4%) from an 8% consistency pulp were attained when 5 FPU/g plus 10 IU/g of β-glucosidase were used. Sequential addition of enzyme to the pulp in small aliquots produced a higher overall sugar yield/U enzyme than the addition of the same total amount of enzyme in a singledose. In the saccharification and fermentation experiments, we produced 2.12% ethanol from a 5.4% sugar solution. This represents 78% of the theoretical maximum. This yield could probably be increased through optimization of the fermentation step. Even when little saccharification occurred, the enzyme facilitated separation of water, fiber, and ash, so cellulase treatment could be an effective means for dewatering pulp sludges.
Keywords: Cellulase; secondary fiber fines; bioconversion
Conversion of industrial food wastes by Alcaligenes latus into polyhydroxyalkanoates by Peter H. Yu; Hong Chua; Ai-Ling Huang; Kwok-Ping Ho (pp. 445-454).
Broader usage of biodegradable plastics in packaging and disposable products as a solution to environmental problems would heavily depend on further reduction of costs and the discovery of novel biodegradable plastics with improved properties. As the first step in our pursuit of eventual usage of industrial food wastewater as nutrients for microorganisms to synthesise environmental-friendly bioplastics, we investigated the usage of soya wastes from a soya milk dairy, and malt wastes from a beer brewery plant as the carbon sources for the production of polyhydroxyalkanoates (PHA) by selected strain of microorganism. Bench experiments showed that Alcaligenes latus DSM 1124 used the nutrients from malt and soya wastes to biosynthesise PHAs. The final dried cell mass and specific polymer production of A. latus DSM 1124 were 32g/L and 70% polymer/cells (g/g), 18.42 g/L and 32.57% polymer/cell (g/g), and 28 g/L and 36% polymer/cells (g/g), from malt waste, soya waste, and from sucrose, responctively. These results suggest that many types of food wastes might be used as the carbon source for the production of PHA.
Keywords: Polyhydroxyalkanoate (PHA); polyhydroxybutyrate (PHB); malt; soya waste; Alcaligenes; Alcaligenes latus
Bioconversion of mixed solids waste to ethanol by Quang A. Nguyen; Fred A. Keller; Melvin P. Tucker; Charles K. Lombard; Bryan M. Jenkins; David E. Yomogida; Valentino M. Tiangco (pp. 455-472).
A mixed solids waste (MSW) feedstock, comprising construction lumber waste (35% oven-dry basis), alm ond treeprunings (20%), wheat straw (20%), office waste paper (12.5%), and newsprint (12.5%), was converted to ethanol via dilute-acid pretreatment followed by enzymatic hydrolysis and yeast fermentation. The MSW was pretreated with dilute sulfuricacid (0.4% w/w) at 210°C for 3 min in a 4-L stea mexplosion reactor, then washed with water to recover the solubilized hemicellulose. The digestibility of water-washed, pretreated MSW was 90% in batch enzymatic hydrolysis at 66 FPU/g cellulose. Using an enzyme-recycle bioreactor system, greater than 90% cellulose hydrolysis was achieved at a net enzyme loading of about 10 FPU/g cellulose. Enzyme recycling using mebrane filtration and a fed-batch fermentation technique is a promising option for significantly reducing the cost of enzyme in cellulose hydrolysis. The hexosesugars were readily fermentable using a Saccharomyces cerevisiae yeast strain that was adapted to the hydrolysate. Solid residue after enzyme digestion was subjected to various furnace experiments designed to assess the fouling and slagging characteristics. Results of these analyses suggest the residue to be of a low to moderate slagging and fouling type if burned by itself.
Keywords: Biomass; ethanol; pretreament; MSW; bioconversion; hydrolysis; enzyme recycle
Separation optimization for the recovery of phenyl ethyl alcohol by Sarah A. Priddy; Thomas R. Hanley; W. Thomas Effler (pp. 473-484).
Phenyl ethyl alcohol is a compound that occurs naturally in flower petals and in many common beverages, such as beer. Desire for the floral, rose-like notes imparted by phenyl ethyl alcohol has created a unique niche for this chemical in flavor and fragrance industries. Phenyl ethyl alcohol can be produced by Saccharomyces cerevisiae via bioconversion. Often this method of production results in extremely low yields, thus placing a great deal of importance on recovery and purification of the valuable metabolite.To determine the best method for recovering the chemical, a primary recovery step and a secondary recovery step were developed. The primary recovery step consisted of comparing dead-end filtration with crossflow ultrafiltration. Crossflow ultrafiltration was ultimately selected to filter the fermentation broth because of its high flow rates and low affinity for the product. The secondary recovery step consisted of a comparison of liquid-liquid extraction and hydrophobic resin recovery. The hydrophobic resin was selected because of its higher rate of recovery and a higher purity than the liquid-liquid extraction, the current practice of Brown-Forman.
Keywords: Phenyl ethyl alcohol; Saccharomyces cerevisiae ; crossflow ultrafiltration; hydrophobic resin; bioconversion
Electrodialysis of acetate fermentation broths by U. N. Chukwu; M. Cheryan (pp. 485-499).
Electrodialysis (ED) shows good potential for downstream processing of acetate fermentation broths, to separate acetic acid while unreacted glucose and other nutrients are partially recycled back to the fermenter. With conventional anion- and cation-exchange membranes, higher current increased acetate flux, water flux, and energy consumption. Multiple ED stacks connected in series with unequal initial volumes for a batch process maximized acetate concentration in the concentrating stream to 134g/L calcium-magnesium acetate (CMA) in the fermentation broth at pH 6.8. Back-transport of acetate from the product into the feed stream and water transport limit the maximum concentration possible. Cost of ED is about $295/ton acetate for the CMA broth.
Keywords: Acetate; electrodialysis; membranes; separation
Partitioning invertase between a dilute water solution and generated droplets by Samuel Ko; Veara Loha; Liping Du; Ales Prokop; Robert D. Tanner (pp. 501-510).
Water droplets or mist occur naturally in the air at seashores. These water droplets carry inorganic and organic substances from the sea to the land via the air, creating fertile land in sandy coastal areas (1). The same phenomenon occurs in an air-fluidized bed bioreactor (2). In an air-fluidized bed reactor, proteins can be transferred from the bioreactor semisolid bulk phase to an enriched droplet phase. This protein transfer process (droplet fractionation) can be experimentally simulated by shaking a separatory funnel containing a dilute solution of a given protein, which can be an enzyme like invertase. The created droplets become richer in invertase (protein) than that of the original dilute solution. The droplets can then be coalesced by tranpping them and recovering the concentrated protein in the new liquid phase. Typically, in such a droplet fractionation process a collected enzyme can be degraded in its ability tocatalyze a chemical reaction. In this article, we explore whether the initial solution pH control variable can be adjusted to minimize the decrease of enzyme activity in this process. The protein droplet recovery problem is one in which the recovered amount of desired protein (enzyme) in the droplet is maximized, subject to the minimization of the enzyme activity loss. The partition coefficient, which is the ratio between the protein concentration in the droplets and the residual solution, is maximized at approx 4.8 and occurs at pH 3.0. Here, the partition coefficient for invertase decreases as the initial solution pH increases, between pH 3.0 and 8.0. Interestingly, the initial solution surface tension seems to be inversely proportional to the partition coefficient. The partition coefficien treachesa maximum value at a surface tension value of approx 63 mN/m at pH 3.0. The enzymatic activity of the initial, the residual, and the droplet solutions all decrease as the bulksolution pH increases. Adecrease of enzymatic activity was observed in the residual bulk solution when compared with that in the initial bulk solution at all pH levels. Also, up to 90% of the invertase activity was lost in the droplets when compared to the initial bulk solution.
Keywords: Droplet fractionation; droplets; invertase; protein separation; decrease in enzyme activity
Bioconversion of fumarate to succinate using glycerol as a carbon source by Hwa-Won Ryu; Kui-Hyun Kang; Jong-Sun Yun (pp. 511-520).
In this study, a facultative bacterium that converts fumarate to succinate at a high yield was isolated. The yield of biocon version was enhanced about 1.2 times by addition of glucose into culture medium at an initial concentration of 6 g/L. When the initial cell density was high (2 g/L), the succinate produced at pH 7.0 for initial fumarate concentrations of 30, 50, 80, and 100 g/L were 29.3, 40.9, 63.6, and 82.5 g/L, respectively, showing an increase with the initial fumarate concentration. The high yield of 96.8%/mole of fumarate in just 4 h was obtained at the initial fumarate concentration of 30 g/L. Comparing these values to those obtained with low cell culture (0.2 g/L), we found that the amount of succinate produced was similar, but the production rate in the high cell culture was about three times higher than was the case in the low cell culture. This strain converted fumarate to succinate at a rate of 3.5 g/L·h under the sparge of CO2.
Keywords: Bioconversion; succinate; fumarate; Enterococcus sp. RKY1; fumarate reductase
Biosorption of actinides from dilute waste actinide solution by egg-shell membrane by Shin-Ichi Ishikawa; Kyozo Suyama; Isamu Satoh (pp. 521-533).
Removal of radioactive elements from the effluent and waste aqueous solutions is an important problem. In previous laboratory batch experiments, hen egg-shell membrane (ESM) was stable as an insoluble protein and was very capable of binding heavy metal ions from aqueous solution. Batch laboratory pH profile, time dependency, and capacity experiments were performed to determine the binding of uranium (U) and thorium (Th) to ESM. Batch pH profile experiments indicated that the optimum pH for binding these actinides was approx 6.0 (U) or 3.0 (Th). The adsorption isotherms were developed at pH 5.0 (U) or 3.0 (Th) at 25°C, and the adsorption equilibrium data fitted both Langmuir and Freundlich models. The maximum uptakes by the Langmuir model were about 240 mg U/g and 60 mg Th/g dry weight ESM. In addition, their adsorption capacities increased as salt concentration increased. ESM could also accumulate uranium from dilute aqueous solution by adjusting to the optimum pH. These results showed that ESM was effective for removing actinides from solution and would be useful in filtration technology to remove actinides from aqueous solution.
Keywords: Biosorption; egg-shell membrane; actinide; uranium; thorium
Ethanol production by a flocculant yeast strain in a CSTR type fermentor with cell recycling by Ossamu Hojo; Carlos Osamu Hokka; Ana Maria Souto Maior (pp. 535-545).
Tests were performed in a continuous stirred tank reactor (CSTR), with and without cell recycling, to produce ethanol. The reactor without cell recycling produced the kinetic model of ethanol production, whereas the reactor with cell recycling allowed for a study of process stability.The Levenspiel kinetic model was adopted; however, in the case of fermentation with cell recycling, the coefficient of cell death was added. It was observed that cellular viability varied greatly throughout the fermenting process and that microaeration is of fundamental importance in maintaining the stability of the process.
Keywords: Ethanol; flocculant yeast
Ethanol production using concentrated oak wood hydrolysates and methods to detoxify by Woo Gi Lee; Jin Suk Lee; Chul Seung Shin; Soon Chul Park; Ho Nam Chang; Yong Keun Chang (pp. 547-559).
Ethanol production from concentrated oak wood hydrolysate was carried out to obtain a high ethanol concentration and a high ethanol yield. The effect of added inhibitory compounds, which are typically produced in the pretreatment step of steam-explosion on ethanol fermentation, was also examined. p-Hydroxybenzoic aldehyde, a lignin-degradation product, was the most inhibitory compound tested in this study. Compounds with additional methyl groups had reduced toxicity and the aromatic acids were less toxic than the corresponding aldehydes. The lignin-degradation products were more inhibitory than the sugar-derived products, such as furfural and 5-hydroxymethylfurfural (HMF). Adaptation of yeast cells to the wood hydrolysate and detoxification methods, such as using charcoal and overlime, had some beneficial effects on ethanol production using the concentrated wood hydrolysate. After treatment with charcoal and low-temperature sterilization, the yeast cells could utilize the concentrated wood hydrolysate with 170 as well as 140 g/L glucose, and produce 69.9 and 74.2 g/L ethanol, respectively, with a yield of 0.46–0.48 g ethanol/g glucose. In contrast, the cells could not completely utilize untreated wood hydrolysate with 100 g/L glucose. Low-temperature sterilization, with or without charcoal treatment, was very effective for ethanol production when highly concentrated wood hydrolysates were used. Low-temperature sterilization has advantages over traditional detoxification methods, such as using overlime, ion exchange, and charcoal, because of the reduction in the total cost of ethanol production.
Keywords: Ethanol; concentrated wood hydrolysate; oak; detoxification; inhibitory compounds; low-temperature sterilization
Performance of fibrous bed bioreactor for treating odorous gas by Hong Chua; Xiang Z. Li; Peter H. F. Yu; Chung Y. Tam; Yu. L. Huang; Shang T. Yang (pp. 561-569).
A fibrous bed bioreactor was used for treatment of odorous volatile fatty acid (VFA). The effect of gaseous VFA (acetic, propionic, and butyric acids) mass loading on the bioreactor performance was investigated. The VFA degrading microbial culture was selected from activated sludge by the three VFAs using a shake-flask culture. The selected microorganisms were then immobilized in a biofilter using cotton fabric as packing material. In the biofiltration experiment, the inlet gas flow rates ranged from 1 to 4 L/min, the total VFA concentrations ranged from 0.10 to 0.43 g/m3, and the resulting total mass loadings of VFA studied ranged from 9.7 to 104.3 g/m3/h. At total mass loading of 104.3 g/m3/h, the VFA removal efficiency was 87.7%. Higher removal efficiencies (>90%) were achieved at mass loadings below 50.3 g/m3/h.
Keywords: Biofiltration; odor control; VFA; fibrous bed bioreactor
A preliminary information about continuous fermentation using cell recycling for improving microbial xylitol production rates by Silvio S. Silva; Adolfo Q. Chanto; Michele Vitolo; Maria G. A. Felipe; Ismael M. Mancilha (pp. 571-575).
Xylitolis a sugar-alcohol with important technological properties, such as anticariogenicity, low caloric value, and negative dissolution heat. It can be used successfully in food for mulations and pharmaceutical industries. Its production is therefore in great demand. Biotechnological xylitol production has several economic advantages in comparison with the conventional process based on the chemical reduction of xylose. The efficiency and the productivity of this fermentation chiefly depends on the microorganism and the process conditions employed. In this article a simple continuous culture with cell recycling was evaluated to enhance the capability of Candida guilliermondii FTI 20037 to produce xylitol. The fermentation was initiated batchwise by directly inoculating the grown seed culturein a 2-L bench-scale fermentor. Continuous feeding was begun at a dilution rate (D) of 0.060/h after the xylose concentration had completely consumed and the cell concentration was a bout 4.0 g/L. At a dilution rate of 0.060/h the xylitol concentration was about 15g/L and in creased by about 35%, whereas the dilution rate decreased by about 58%. Furthermore, the volumetric productivity, Qp, markedly depended on the dilution rate, diminishing by about 37% as D was changed from 0.060 to 0.025/h. These preliminary results show us that the continous fermentation with cell recycling is a good way to study the xylitol production by xylose-fermenting yeasts.
Keywords: Xylitol; yeast; continous fermentation; recycling