Production of l-lactic acid byRhizopus oryzae in a bubble column fermenter

Two distinctive forms of growth (mycelial filamentous and mycelial pellets) ofRhizopus oryzae were obtained by manipulating the initial pH of the medium with the controlled addition of CaCO₃ in a bubble fermenter. In the presence of CaCO₃, diffused filamentous growth was obtained when the initial pH of the substrate was 5.5. In the absence of CaCO3, mycelial pellet growth was obtained when the initial pH was 2.0. The fermentation study indicated that the mycelial growth has a shorter lag period before the onset of acid formation. Both physical forms of growth ofRhizopus exhibited a high yield of L-lactic acid in the bubble fermenter when the initial glucose concentration exceeded 70 g/L. A final lactic acid concentration of 62 g/L was produced by the filamentous form ofRhizopus from 78 g/L glucose after 27 h. This showed a weight yield of 80% of glucose consumed, with an average specific productivity of 1.46 g/h/g. Similarly, the pellet form ofRhizopus produced a final lactic acid concentration of 66 g/L from 76 g/L glucose after 43 h, with a weight yield of 86% and an average specific productivity of 1.53 g/h/g.     

Fermentation of xylose to glycerol byRhizopus javanicus

Glycerol production from xylose fermentation usingRhizopus javanicus (ATCC 22581) has been investigated in shake flasks. The medium composition (xylose concentration, nitrogen sources), aeration rate, and temperature have been found to affect the accumulation and yield of glycerol. Some of these effects are explained in terms of the critical parameters, osmotic pressure, and dissolved oxygen levels in the medium. Relatively high glycerol yields and concentrations have been obtained at high sugar concentration with high level of aeration at room temperature. The addition of polyethylene glycol or sulfite can improve the yield and accumulation of glycerol.     

An integrated bioconversion process for production of l-lactic acid from starchy potato feedstocks

The potential market for lactic acid as the feedstock for biodegradable polymers, oxygenated chemicals, and specialty chemicals is significant. L-lactic acid is often the desired enantiomer for such applications. However, stereospecific lactobacilli do not metabolize starch efficiently. In this work, Argonne researchers have developed a process to convert starchy feedstocks into L-lactic acid. The processing steps include starch recovery, continuous liquefaction, and simultaneous saccharification and fermentation. Over 100 g/L of lactic acid was produced in less than 48 h. The optical purity of the product was greater than 95%. This process has potential economical advantages over the conventional process.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Effect of pretreatment of molasses and posttreatment of fermented broth in industrial production of ethanol

The aim of preclarification is to minimize sludge going to yeast separators. This purpose is partially fulfilled. However, it has been measured during the plant trial runs that preclarification does not noticeably improve fermentation. The aim of postclarification is to minimize sludge going to distillation. This purpose is well served as noted from the fact that cycle run of distillation columns using postclarification is three times longer (9–12 mo) as compared to the normal one (3–4 mo).     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.     

Biosolubilization and liquid fuel production from coal

Recently, several microorganisms have been shown to be capable of directly solubilizing low-rank coals. This bioextract has a high molecular weight and is water soluble, but is not useful as a liquid fuel. This paper presents the results of studies to biologically solubilize coal and convert the solubilized coal into more useful compounds. Preliminary experiments have been conducted to isolate cultures for the serial biological conversion of coal into liquid fuels. Coal particles have been solubilized employing an isolate from the surface of Arkansas lignite. Natural inocula, such as sheep rumen and sewage sludge, are then employed in developing cultures for converting the bioextract into fuels. This paper presents preliminary results of experiments in coal solubilization and bioextract conversion.     

Production of fumaric acid with immobilized biocatalysts

Immobilization ofRhizopus arrhizus mycelium improved fumaric acid production. The optimum conditions for fumaric acid production with immobilized cells were investigated using a statistical experimental design. Substrate concentration, carbon:nitrogen ratio, and residence time were chosen as independent variables. In the repeated batch shake flask fermentation, the fumaric acid yield from xylose was as much as 3.5 times higher with immobilized mycelium than with free mycelium. Polyurethane foam cubes, in this case, gave better results than nylon net cubes as a carrier.     

Development of xylose-fermenting yeasts for ethanol production at high acetic acid concentrations

Mutants resistant to comparatively high levels of acetic acid were isolated from the xylose-fermenting yeastsCandida shehatae andPichia stipitis by adapting these cultures to increasing concentrations of acetic acid grown in shake-flask cultures. These mutants were tested for their ability to ferment xylose in presence of high acetic acid concentrations, in acid hydrolysates of wood, and in hardwood spent sulfite liquor, and compared with their wild-type counterparts and between themselves. TheP. stipitis mutant exhibited faster fermentation times, better tolerance to acid hydrolysates, and tolerance to lower pH.     

Fuel ethanol production from corn fiber current status and technical prospects

Corn fiber, which consists of about 20% starch, 14% cellulose, and 35% hemicellulose, has the potential to serve as a low cost feedstock for production of fuel ethanol. Currently, the use of corn fiber to produce fuel ethanol faces significant technical and economic challenges. Its success depends largely on the development of environmentally friendly pretreatment procedures, highly effective enzyme systems for conversion of pretreated corn fiber to fermentable sugars, and efficient microorganisms to convert multiple sugars to ethanol. Several promising pretreatment and enzymatic processes for conversion of corn fiber cellulose, hemicellulose, and remaining starch to fermentable sugars were evaluated. These hydrolyzates were then examined for ethanol production in bioreactors, using genetically modified bacteria and yeast. Several novel enzymes were also developed for use in pretreated corn fiber saccharification. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Xylanase recovery by ethanol and Na2SO4 precipitation

Xylans are the major components of the hemicellulosic fraction of lignocellulosic biomass and their hydrolysis can be obtained using xylanases fromPenicillium janthinellum. In this work, sugarcane bagasse hemicellulosic hydrolysate was used as the substrate for producing xylanase. The precipitation of these enzymes was studied using ethanol and Na₂SO₄ as precipitating agents. Ethanol precipitation experiments were performed batchwise in concentrations ranging from 10 to 80%, pH 4.0 to 7.0, at 4áC. The concentrations used in the precipitations with Na₂SO₄ were from 5 to 60% at pH 5.5 and 25áC. Solubility curves as a function of xylanase activity and total protein for both precipitating agents were made. According to the results, Na₂SO₄ is not appropriate for precipitating xylanases in this medium since at salt concentrations higher than 25%, the enzyme was denaturated and at this concentration less than 80% of the enzyme and total protein were precipitated. Because of differences in xylanase and total protein solubility, a fractionated precipitation using ethanol can be performed, since with 40% ethanol, 49% of the total protein was precipitated and more than 95% of the enzyme was kept in solution. On the other hand approx 100% of the xylanases were recovered by precipitation after adding 80% ethanol.     

Continuous fermentation of cellulosic biomass to ethanol

Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification and fermentation (SSF) system comprised ofTrichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation bySaccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised ofClostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H₂SO₄) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H₂SO₄). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC. The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time. At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system (77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in the range of 5–60 g/L cellulose in the feed.     

Ethanol production from AFEX-treated forages and agricultural residues

Lignocellulosic materials derived from forages, namely timothy grass, alfalfa, reed canary grass, and agricultural residues, such as corn stalks and barley straw, were pretreated using ammonia fiber explosion (AFEX) process. The pretreated materials were directly saccharified by cellulolytic enzymes. Sixty to 80% of theoretical yield of sugars were obtained from the pretreated biomasses. Subsequent ethanolic fermentation of the hydrolysates byPachysolen tannophilus ATCC 32691 resulted in 40-60% of theoretical yield after 24 h, based on the sugars present in the hydrolysates. The uptake of sugars was not complete, indicating a possible inhibitory effect onP. tannophilus during the fermentation of these substrates.     

Technical and economic evaluation of different reactors for methanotrophic cultures for propylene oxide production

A two-stage process for the manufacture of propylene oxide is described. The preliminary economics based on use of methanol as a regeneration factor has resulted in a production cost of $12.10/lb of propylene oxide based on propylene oxide production rate of 40 mg/g-cell/h in conventional reactor. Increasing the propylene oxide production from 40 to 500 mg/g-cell/h resulted in a cost reduction from $12.10 to 5.8/lb of propylene oxide. The granular-activated, carbon-fluidized bed reactor (GAC-FBR) absorbs the propylene oxide and when saturated is eluted with ethyl acetate, and the bed is regenerated by steam to drive off the residual solvents. The estimated manufacturing costs are approx 59% lower (from $12.10/lb in conventional reactors to $5.00/lb for GAC-FBRs) for products that are highly inhibitory such as epoxides. In the GAC-FBR reactor, enhancing the propylene oxide production rate from 120 to 1500 mg/g-cell/h has resulted in the cost reduction to $2.00/lb. Enhancing the production capacity from 1 million lb to 10 million lb/yr has further reduced the cost of production to $1.00/lb.     

β-Cyclodextrin production by simultaneous fermentation and cyclization

Production of β-cyclodextrin (CD) with high-dextrose equivalent (DE) starch hydrolysates by simultaneous fermentation and cyclization (SFC) gives higher yields than using only the enzyme CGTase, because fermentation eliminates glucose and maltose that inhibit CD production, while at the same time, produces ethanol that increases yield. A 10% (w/v) solution of cassava starch, liquefied with α-amylase, was incubated with CGTase using: only the enzyme, added ethanol (from 1 to 5%), and added yeast,S. cerevisiae (12% w/v), plus nutrients, the latter being the SFC process. Reaction conditions were: 38αC, pH 6.0, DE from 2 to 25, and 3.3 mL of CGTase/L. The yield of β-CD has decreased with an increase in DE, and maximum reaction yields were found for DE equal to 3.54, reaching 5.6, 14.7, and 11.5 mM β-CD, respectively. For an increase of DE, of approx 6 times (from 3.54 to 23.79), β-CD yield decreased 6 times for the first, and second reaction media with 3% (v/v) ethanol, and only approx 3 times for SFC (from 11.5 to 3.73 mM), showing that this process is less sensitive to variations in the DE     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

Bacillus stearothermophilus for thermophilic production of l-lactic acid

A process for the continuous production of high purityL-lactic acid in a membrane bioreactor at 65°C has been developed. Two differentBacillus stearothermophilus strains have been tested in batch experiments. Lactic acid yields are between 60 and more than 95% of theoretical yields. The amounts of ethanol, acetate, and formate formed varied between 0 and 0.4, 0 and 0.1, and 0 and 0.5, respectively (mol/mol glucose). All byproducts are valuable and may be separated easily by rectification of the fermentation broth. Complete cell retention enables high volumetric productivity (5 g/Lh), and a minimum of growth supplements. The high temperature of 65°C allows the autoselective fermentation without problems with contamination.     

The effect of sodium sulfite and cobalt chloride on the oxygen transfer coefficient

An experiment was conducted in an aeration system made from 2000 mL Erlenmeyer Flask to investigate the effect of sodium sulfite with cobalt chloride and the sparging nitrogen gas on the measurement of the oxygen transfer coefficient. Twice the theoretical quantity of sodium sulfite that is required to react with dissolved oxygen, or nitrogen sparging at a rate equivalent to that of air used during the aeration process, was used to deoxygenate the water. The results obtained from the study were consistent and repeatable and showed no significant differences in the values of the oxygen transfer coefficient obtained by the two methods.