Showing papers in "Biotechnology and Bioengineering in 1981"

Biosorption of uranium and thorium

Abstract: Summary Selected samples of waste microbial biomass originating from various industrial fermentation processes and biological treatment plants have been screened for biosorbent properties in conjunction with uranium and thorium in aqueous solutions. Biosorption isotherms have been used for the evaluation of biosorptive uptake capacity of the biomass which was also compared to an activated carbon and the ion exchange resin currently used in uranium production processes. Determined uranium and thorium biosorption isotherms were independent of the initial U or Th solution concentration. Solution pH affected the exhibited uptake. In general, lower biosorptive uptake was exhibited at pH 2 than at pH 4. No discernible difference in uptake was observed between pH 4 and pH 5 where the optimum pH for biosorption lies. The biomass of Rhizopus arrhizus at pH 4 exhibited the highest uranium and thorium biosorptive uptake capacity (g) in excess of 180 mgig. At an equilibrium uranium concentration of 30 mgiliter, R. arrhizus removed approximately 2.5 and 3.3 times more uranium than the ion exchange resin and activated carbon, respectively. Under the same conditions, R. arrhizus removed 20 times more thorium than the ion exchange resin and 2.3 times more than the activated carbon. R. arrhizus also exhibited higher uptake and a generally more favorable isotherm for both uranium and thorium than all other biomass types examined.

Mechanism of inhibition caused by long‐chain fatty acids in anaerobic digestion process

Abstract: The inhibitory effect of long-chain fatty acids on the anaerobic digestion process was examined in batch experiments using synthetic substrates. The addition of long-chain fatty acids caused the appearance of the lag period in the methane production from acetate and in the degradation of both long-chain fatty acids and n-butyrate. Methane production from hydrogen proceeded without lag period although its rate was lowered. Fermentation of glucose was not inhibited. Neutral fat in the whole milk was easily hydrolyzed to long-chain fatty acids, which brought about the inhibtion. The addition of calcium chloride reduced the inhibitory effect of long-chain fatty acids, but it did not do so after the culture had been exposed to long-chain fatty acids for more than several hours. The addition of calcium carbonate could not reduce the inhibition because of its insolubility. 18 refs.

Bioengineering report: Fouling biofilm development: A process analysis

Abstract: Biofilm development at a surface is the net result of several physical, chemical, and microbial processes including the following: (1)transport of dissolved and particulate matter from the bulk fluid to the surface; (2) firm microbial cell attachment to the surface; (3) microbial transformations (growth, reproduction, etc.) within the biofilm resulting in production of organic matter; (4) partial detachment of the biofilm due primarily to fluid shear stress. This report presents a framework for analyzing the interrelated processes contributing to biofilm development. Some of the available rate and composition data are presented so that the relative process rates can be compared.

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration: CONTINUOUS ENZYMATIC TRANSFORMATION

Abstract: Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 104 [PEG‐10,000‐NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L‐leucine dehydrogenase (LEUDH) was used as catalyze for the reductive amination of α‐ketoisocaproate (2‐oxo‐4‐methylpentanoic acid) to L‐leucine. Format dehydrogenease (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L‐leucine. The kinetic constants Vmax and Km of enzymes are all in the same range regardless of whether native NAD(H) or PEG‐10,000‐NAD(H) is used as coenzyme. L‐leucine was produced continuously out of α‐ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space‐time yield was 324 mmol/L day (or 42.5 g/L day).

Kinetics of phenol oxidation by washed cells

Abstract: The uptake of phenol by pure cultures of Pseudomonas putida growing on phenol in continuous culture has been studied. The purpose of the experiments was to determine the kinetic parameters governing uptake of phenol by organisms growing on phenol in the high‐conversion range by measuring uptake rates per unit biomass per unit time at various phenol concentrations. The microorganisms used were taken from a chemostat at residence times of 8, 5.25, 3.85, 3.2, 3, and 2.7h. The Monod–Haldane model and modifications of it were applied to the data and the best kinetic parameters were determined by nonlinear least‐squares techniques. The best model was a two‐parameters simplification of Monod–Haldane in which μ = K1S/(K2 + S2). The value of K1 was found to increase monotonically with the value of phenol concentration in the original chemostat with an apparent induction “threshold” of 0.1 mg/L.

Enhanced production of cellulase, hemicellulase, and b-glucosidase by trichoderma reesei, (rut c-30)

Abstract: LBL-11 074 Prepri nt Submitted to Biotechnology and Bioengineering ENHANCED PRODUCTION OF CELLULASE, HEMICELLULASE AND S-GLUCOSIDASE BY TRICHODERMA REESEI (RUT-C-30) S. Kishen Tangnu, Harvey W. Blanch and Charles R. Wilke June 1980 TWO-WEE LOAN Y This is a Library Copy which may borrowed for two weeks. For a personal Tech. In Divis call Ext. 6782. Prepared for the U.S. Department of Energy under Contract W-7405-ENG-48

Influence of engineering variables upon the morphology of filamentous molds

Abstract: A model has been described for the influence of growth rate and shear stresses in the fermentor upon the morphology of filamentous molds. The main concept of this model is the dynamic equilibrium between growth and breakup of the hyphae. The latter has been approached according to well-known engineering theories for dispersion of physical systems. Experiments to verify the model with a strain of Tenicillium chrysogenum in batch and continuous culture revealed that the length of the mycelial particles increased with increasing' growth rate and decreased with increasing power input per unit mass in the fermentor. Although this was qualitatively in agreement with the presented model, quantitatively the model had to be rejected. Variation of the tensile strength of the hyphae with age and culturing conditions could have been one of the causes of disagreement. Oxygen tension, varied independently from stirrer speed, in the range of 12-300 mm Hg was shown to have no influence upon the morphology. With respect to the question of possibly using high-energy inputs in industrial mold fermentation in order to decrease hyphal length and suspension viscosity, it was concluded that this is of little practical value. A substantial decrease in hyphal length requires an enormousmore » increase in energy input.« less

The influence of major structural features of cellulose on rate of enzymatic hydrolysis

Abstract: An attempt was made to determine the effect of two structural features of cellulose on its enzymatic hydrolysis: its crystallinity and its specific surface area. Solka Floc SW40 was pretreated in various ways prior to hydrolysis: ball milling, gamma radiation, pyrolysis, treatment by sodium hydroxide, treatment by CMCS, and treatment by sulfuric acid. Microcrystalline cellulose (Sigmacell 50) was also employed in the test. The culture filtrate of Trichoderma reesei QM9414 was the source of the enzyme. It was observed that, independent of the methods of treatment, the rate of hydrolysis would tend to increase with an increase in specific surface area and with a decrease in crystallinity index.

Acidogenic fermentation of lignocellulose–acid yield and conversion of components

Abstract: Corn stover was fermented with a mixed culture of anaerobic microorganisms to form simple (C2-C6), volatile organic acids. Alkaline pretreatment allowed a greater fermentation of the pectin and hemicellulose than of the cellulose and lignin, but all components were utilized. The percent fermentation of the soluble fraction, hemicellulose, cellulose, and lignin was 79.6, 74.1, 36.9, and 20.9%, respively. The yield of acid (as acetate) with respect to material fermented was 84%.

Bioenergetic analysis of mixotrophic growth in Chlorella vulgaris and Scenedesmus acutus

Abstract: The specific growth rate of Chlorella vulgaris in the presence of glucose (mixotroph) was always larger than that in autotroph when the light intensity was less than 10 klux. However, Scenedesmus acutus behaved differently, i.e., when the light intensity was more than 6 klux, the specific growth rate in mixotroph became smaller than that in autotroph. Cellular contents of chlorophyll a and b in Scenedesmus acutus that deteriorated more markedly in mixotroph than those of Chlorella vulgaris could account for these different growth behaviors. In other words, mechanisms relevant to the photosynthesis and the oxidative phosphorylation of glucose seem to function independently with respect to Chlorella Vulugaris.

The production of ethanol by immobilized yeast cells

Abstract: Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to a 0.05M CaCl2 solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature,pH, ethanol concentration), cell densities, and gel concentrations. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells were examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentrations were monitored at different feedstock flow rates. (Refs. 13).

Alcoholic fermentation: On the inhibitory effect of ethanol

Abstract: The inhibitory effect of ethanol was studied during alcoholic fermentation under strictly anaerobic conditions assured by stripping dissolved oxygen with pure nitrogen. It is shown that ehthanol produced during batch fermentation is more inhibitory than added ethanol in the range of 0 to 76 g/liter. Thus, the inhibition constant is 105.2 and 3.8 g/liter for added and produced ethanol, respectively. The measurement of the intracellular alcohol concentration can explain the difference in inhibitory effects of added and produced oxygen. 15 refs.

Effects of the surfactant tween 80 on enzymatic hydrolysis of newspaper

Abstract: The effects of Tween 80 on the enzymatic hydrolysis of newspaper were tested. By monitoring sugar production it was found that the surfactant (0.1%) increased the rate and extent of cellulose saccharification. Consequently, the rate of enzyme usage in the hydrolysis reactor was improved by 33%. In addition, in the presence of surfactant the recovery of enzymes was higher. Analysis of the enzyme solution showed that with Tween 80 present larger fractions of enzyme remained in solution throughout hydrolysis. Thus, it appears that the surfactant hindered the immobilization of the enzymes on the substrates by reducing the strength of adsorption.

Microbial transformation of racemic hydantoins to D‐amino acids

Abstract: It has been shown that resting cells of Agrobacterium radiobacter catalyze a sequence of two stereospecific hydrolytic reactions leading to the complete transformation of racemic hydantoins to D‐amino acids. This report describes some properties of this new biocatalyst and its potential application for the production of some D‐amino acids, which are used as intermediates in the preparation of semisynthetic penicillin and cephalosporins.

A macroscopic model describing yield and maintenance relationships in aerobic fermentation processes

Abstract: Summary The present paper presents a generalized treatment of the principles of elemental and enthalpy balances which are applied to aerobic fermentation processes. It is shown that strict relations do exist between the various yield factors of biomass or product on substrate, oxygen, carbon dioxide, and between the various maintenance coefficients. These relations are confirmed from the existing body of literature data on yield and maintenance coefficients. Another consequence of the application of elemental balances is the existence of limits for the maximum biomass yield on substrate and oxygen, which depend on the degree of reduction of the substrate. Furthermore a simple model is proposed for the estimation of yield coefficients on substrates with different degree of reduction. It appears from this model that substrates with a high degree of reduction are C limited and substrates with a low degree of reduction are energy limited. Finally the effects of temperature on yield and maintenance coefficients are analyzed from the existing body of literature data. It can be concluded that the maintenance coefficients follow an Arrhenius type of relationship and that yield is temperature independent. The literature data seem to indicate that a degree of reduction of about 4 is optimal for the carbon and energy needs for biomass formation.

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Abstract: Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 hours. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given time was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration. (Refs. 28).

Production of ethanol from raw cassava starch by a nonconventional fermentation method

Abstract: Raw cassava root starch was transformed into ethanol in a one-step process of fermentation, in which are combined the conventional processes of liquefaction, saccharification, and fermentation to alcohol. Aspergillus awamori NRRL 3112 and Aspergillus niger were cultivated on wheat bran and used as Koji enzymes. Commercial A. niger amyloglucosidase was also used in this experiment. A raw cassava root homogenate-enzymes-yeast mixture fermented optimally at pH 3.5 and 30/degree/C, for five days and produced ethanol. Alcohol yields from raw cassava roots were between 82.3 and 99.6%. Fungal Koji enzymes effectively decreased the viscosity of cassava root fermentation mashes during incubation. Commercial A. niger amyloglucosidase decreased the viscosity slightly. Reduction of viscosity of fermentation mashes was 40, 84, and 93% by commercial amyloglucosidase, A. awamori, and A. niger enzymes, respectively. The reduction of viscosity of fermentation mashes is probably due to the hydrolysis of pentosans by Koji enzymes. 12 refs.

Evaluation of feeding strategies in carbon‐regulated secondary metabolite production through mathematical modeling

Abstract: There is now growing evidence that the production of many secondary metabolic by microorganisms is subjected to carbon-catabolite regulation. Even though the exact mode of this regulation is not yet clear, an engineering analysis of the production process is still possible based upon a suitable hypothesis. By way of simulation of penicillin fermentation data obtained from the literature, a mechanistic model involving a substrate inhibition kinetics of product formation has been verified in this paper. Such a model has been found successful not only in predicting simple sugar-feeding strategy, but also a complicated computer guided strategy based upon controlling biomass growth rates in the tropo and idiophases. Using this model, for strategies for sugar feeding into penicillin fermentation have been investigated. These results show that similar penicillin productivities can be obtained using any of these strategies provided fermentations are carried out under optimal conditions corresponding to the strategy chosen. Effect of maximum oxygen transfer capacity of the fermentor under the conditions of fungal growth has been incorporated using an upper limit of biomass concentration on achievement of which the fermentations must be stopped due to serious oxygen limitations. Results of model simulations with such limits throw light upon the way in which different fermentors may behave with respect to product formation.

Disruption of Candida utilis cells in high pressure flow devices

Abstract: The disruption of Candida utilis cells in suspensions subjected to different types of stress was investigated. Stresses caused by impingement of a high velocity jet of suspended cells against a stationary surface were found to be significantly more effective for disruption than either shear or normal stresses. The fraction of cells disrupted by impingement is a first order function of the number of passes through the disruptor and, over a prescribed range of operating pressures, is a power function of pressure. These results indicate that impingement is the predominant mechanism causing cells disruption in high pressure flow devices such as Manton–Gaulin homogenizers. The impingement results suggest that cells grown in cyclic batch culture are easier to disrupt than cells grown at a lower specific growth rate in continuous culture. In addition to determining the fraction of cells disrupted, the release of invertase activity was determined for the impingement experiments. The fraction of total invertase activity released was found to be somewhat greater than the fraction of cells disrupted.

The effect of combined chemical and enzyme treatments on the saccharification and in vitro digestion rate of wheat straw

Abstract: Wheat straw was pretreated with sodium hydroxide, ozone, and sulfur dioxide, and subsequently treated with four sources of cellulase, T. reesei, T. reesei, T. viride, A. niger, and Oxyporus sp. The effect of the combined chemical + enzyme treatments on the extent of saccharification and on the digestion rate by rumen microorganisms was studied. Cellulases were applied at an equal but low concentration (0.5 FPU/ml) on the pretreated straw. The combined treatments, SO2 + T. reesei cellulase nd SO2 + T. viride cellulase, produced the highest and significant levels of reducing sugars (RS), 577 and 597 mg RS/g straw organic matter. The highest enzyme efficiency, 44.7 mg RS/mg enzyme, was found with T. reesei cellulase when applied on SO2 pretreated straw. The in vitro organic matter digestibility was affected significantly only by the chemical pretreatments, whereas the effect of the cellulases was expressed mainly in increasing the fermentability of the hydrolyzed straw. The in vitro digestion pattern of the saccharified straw was found to be typical of a highly fermentable feed and comparable to a starchy mixture such as used in concentrate ruminant diets.

Methods for quantitative representation of the morphology of molds

Abstract: A semiautomatic method for the quantitative representation of mold morphology is described.

Development of a hollow‐fiber system for large‐scale culture of mammalian cells

Abstract: A flat‐bed hollow‐fiber cell culture system has been developed which maximizes the utilization of the large fiber surface while diminishing significantly the problems inherent in a cartridge‐type reactor. The reactor core consists of a shallow bed of hollow fibers sandwiched between two stainless‐steel microporous filter plates through which the media flow is directed normal to the plane of the fiber bed. Reactors with both 930 and 9300 cm2 of fiber surface have been successfully constructed and operated. A variety of cells has been grown in these reactors including SV3T3 cells, baby hamster kidney cells, Vero cells, and rhesus money kidney cells, and cell products such as plasminogen activator and migration inhibition factor (MIF) were produced. This system offers an excellent prototype for scaleup design.

Acidogenic fermentation of corn stover

Abstract: Corn stover was fermentd by anaerobic acidogenic bacteria to produce volatile (C2-C6) organic acids Mild pretreatment with dilute alkali solutions produced a two-fold increase in fermentability A mixture of lime and sodium carbonate was found to be a better pretreatment agent than sodium hydroxide Methane generation was inhibited by low temperature less than or equal to 25 degrees Celcius and high solids greater than or equal to 25% (w/v) fermentation Volatile acid yields of 05-055 g acetic acid equivalent/g dry ash-free (DAF) stover could be obtained in batch fermentations Several extractants and extraction solvents for organic acids were found to be nontoxic to acidogenic fermentation The data show that acidogenic fermentation can produce useful volatile fatty acids in high yields from a complex lignocellulosic feedstock These fermentation are nonsterile, need no stirring, and are easy to run Moreover, cellulose, pentosans, and other carbohydrates are directly utilized by acidogenic bacteria Hence, acidogenic fermentation could be useful in converting biomass to chemical feedstocks and fuel

Conversion of the enzymatic hydrolysate of shellfish waste chitin to single‐cell protein

Abstract: The conversion of the enzymatic hydrolysate of shellfish chitin waste to single-cell protein was investigated as part of a comprehensive waste treatment program. Forty-two yeasts were screened for ability to assimilate the monomer of chitin, N-acetylglucosamine, which has been shown to be the sole product of enzymatic hydrolysis of chitin. The Yeast Pichia Kudriavzevii was selected for study, based on ability to grow at high temperature (37°C and above), low pH (4.0 ± 0.5), and in a nutritionally simple medium. Growth rates of P. kudriavzevii were similar on N-acetylglucosamine and on the chitin hydrolysate. Dependencies of specific growth rate on temperature, pH, medium composition, and oxygen tension were studied. The variations of yield, protein content, and total nucleic acid content with the specific growth rate were evaluated. The amino acid distribution of the protein of P. kudriavzevii was obtained.

Lactate and ethanol as intermediates in two‐phase anaerobic digestion

Abstract: The thermodynamics of the various anaerobic digestion patterns of hexose to methane are compared. It appears that by directing the hexose-hydrolysis phase towards ethanol and lactic acid production, methanogenesis can be enhanced because the synthrophic bacteria are allocated more potentially available energy. This hypothesis was confirmed in a series of laboratory tests runs. They revealed that lactic acid and ethanol as intermediates, in comparison to lower volatile fatty acids, give rise to a considerably high effluent quality and a slightly larger biogas production. 15 refs.

Raw materials evaluation and process development studies for conversion of biomass to sugars and ethanol

Abstract: A range of cellulosic raw materials in the form of agricultural crop residue was analyzed for chemical composition and assessed for potential yields of sugars through chemical pretreatment and enzymatic hydrolysis of these materials. Corn stover was used as a representative raw material for a preliminary process design and economic assessment of the production of sugars and ethanol. With the process as presently developed, 23 gallons of ethanol can be obtained per ton of corn stover at a processing cost of about $1.80 per gallon exclusive of by-product credits. The analysis shows the cost of ethanol to be highly dependent upon (1) the cost of the biomass, (2) the extent of conversion to glucose, (3) enzyme recovery and production cost and (4) potential utilization of xylose. Significant cost reduction appears possible through further research in these directions.

Immobilization of microbial cells in crosslinked, prepolymerized, linear polyacrylamide gels: Antibiotic production by immobilized Streptomyces clavuligerus cells

Abstract: A mild new method for the immobilization whole microbial cells has been developed. Cells were suspended in a solution of preformed, linear, water-soluble Polyacrylamide chains, partially substituted with acylhydrazide groups. The Prepolymerized backbone polymer was crosslinked, in the presence of viable cells, by stoichiometric amounts of dialdehydes such as glyoxal, glutardialdehyde, and period ate-oxidized polyvinyl alcohol. The crosslinking reaction, carried out in cold, neutral physiological conditions resulted in cells entrapped in gels with physical properties similar to those of the common Polyacrylamide gels. However, cell damage generally caused by the acrylamide monomer was avoided. Resting Streptomyces clavuligerus cells, possessing a high capacity for antibiotic production, were entrapped according to this procedure. These immobilized cells produced cephalosporins continuously for 96 h with yields similar to those of free resting cells. The same cells, when immobilized by direct polymerization acrylamide monomers, yielded significantly lower amount of antibiotics.

A mathematical model for the aerobic growth of Saccharomyces cerevisiae with a saturated respiratory capacity

Abstract: A mathematical model for the aerobic growth of Saccharomyces cerevisiae in both batch and continuous culture is described. It was based on the experimental observation that the respiratory capacity of this organism may become saturated and exhibit a maximum specific oxygen uptake rate after suitable adaptation. This experimental observation led to the possibility that transport into and out of the mitochondrion was of major importance in the overall metabolism of S. cerevisiae and was subject to long-term adaptation. Consistent with this observation a distributed model was proposed which, as its basis, assumed the control of respiration and fermentation to be the result of saturation of respiration without any specific repression or inhibition of the uptake rates of other substrates. No other regulation of fermentation and respiration was assumed. The model provided a suitable structure allowing precise quantification of the changes in rate and stoichiometry of energy production. The model clearly indicated that growth under the wide range of experimental conditions reported could not be predicted using constant values for the maximum specific respiratory rate or constant values of Yatp (g biomass/mol ATP) and PO ratio of (mol ATP/atom oxygen). The causes of the variation in the respiratory rate were notmore » determined and it was concluded that a more detailed analysis (reported subsequently) was required. The variation of Y atp and PO ratio with specific growth rate implied that the efficiency of ATP generation or ATP utilization decreased with increasing specific growth rate. It was concluded that it was not possible to quantify the individual effect of Yatp and PO ratio until independent means for their reliable estimation is available. (Refs. 84).« less

Estimation of heats of combustion of biomass from elemental analysis using available electron concepts

Abstract: The method of Thornton and Dulong's formula for estimating heats of combustion are compared in this work. Heats of combustion predicted by Thornton's method for renewable resources such as wood, straw, and municipal solid wastes are considerably closer to experimentally measured values compared to values predicted by Dulong's formula. Thornton's method states that the heat of combustion is directly proportional to the quantity of oxygen consumed in the combustion process. A method which utilizes the weight fraction carbon on a dry basis and the reductance degree to predict the heat of combustion of renewable resources is presented. (Refs. 12).