Showing papers in "Biotechnology and Bioengineering in 1972"

Biotechnology report. Solid state fermentations.

Abstract: A unique method is described by which large yields of secondary metabolites arc produced on solid substrates. The process involves the use of moist substrates which are continuously agitated in appropriate fermentation equipment. The amount of agitation, aeration, and moisture can be varied. Extremely high yields of secondary metabolites such as ochratoxin and aflatoxin were obtained using Aspergillus and Penicillium species. The process prevents sporulation of the fungus and because of the nature of the solid substrate makes recovery of the product easier than in conventional liquid media. The substrates include rice, corn, wheat, and other cereals.

Degradation and mineralization of petroleum in sea water: limitation by nitrogen and phosphorous.

Abstract: Biodegradation and mineralization of petroleum, added at 1% (v/v) to freshly collected sea water, were measured using gas–liquid chromatographic, residual weight, and CO2-evolution techniques. Only 3% of the added petroleum was biodegraded and 1% was mineralized in unamended sea water after 18 days of incubation. Added individually, nitrate (10−2M) or phosphate (3.5 × 10−4M) supplements caused little improvement, but when added in combination, they increased petroleum biodegradation and mineralization to 70% and 42%, respectively. Attempts to clean up oil spills with the aid of microorganisms should take into consideration the nutritional deficiencies of sea water.

Degradation and mineralization of petroleum by two bacteria isolated from coastal waters

Abstract: Within the framework of a study on the oil biodegradation potential of the sea the ability of a Flavobacterium sp. and Brevibacterium sp. to metabolize a paraffinic crude oil and a chemically defined hydrocarbon mixture was investigated. Major components of the crude oil were identified by combination gas chromatography and mass spectrometry. The rate and extent of total hydrocarbon biodegradation was measured. In addition, CO2 evolution from the crude oil was continuously monitored in a shaker-mounted gas train arrangement. Degradation started after a 2 to 4 day lag period, and reached its maximum within two weeks. At this time up to 60% of the crude oil and 75% of the model hydrocarbon mixture, each added at the level of 1 ml per 100 ml artificial sea water, were degraded. Mineralization(conversion to CO2) was slightly lower due to formation of products and bacterial cell material. n-Paraffins were preferentially degraded as compared to branched chain hydrocarbons. Biodegradation of n-paraffins in the range of C12 to C20 was simultaneous; no diauxie effects were observed.

Emulsification and degradation of "Bunker C" fuel oil by microorganisms

Abstract: An enrichment culture procedure has been used to isolate mixed culture systems which grow upon “Bunker C” fuel oil. When inoculated into a mineral salts aqueous medium containing Bunker C oil, the mixed cultures initiate oil emulsification. Emulsification usually is observed in 24–48 hr. The role of microbes in this emulsification will be discussed. It appears that certain metabolic products produced by the microbe possess properties of surfactants. Bacteria and fungi have been isolated which possess the ability to cause emulsification. Freeze‐dried biomass is also capable of emulsifying oil. Chromatographic analyses of biodegraded Bunker C fuel oil show that microorganisms selectively metabolize the n‐paraffin fraction.

The effect of carbon dioxide and particle surface area on the microbiological leaching of a zinc sulfide concentrate

Abstract: The effects of carbon dioxide-enriched air on the rate of zinc extraction during the microbiological leaching of a, high-grade zinc sulfide concentrate by Thiobacillns ferrooxidans have been studied. Under normal air-aeration conditions, the leach rate is limited initially by the availability of solid substrate surface area per unit volume of leach liquor, then by availability of carbon dioxide. If carbon dioxide-enriched air is supplied, along with excess substrate, the zinc extraction rate increases as the carbon dioxide content increases until some other, as yet unknown, factor becomes limiting.

Effects of growth rate and influent substrate concentration on effluent quality from chemostats containing bacteria in pure and mixed culture.

Abstract: Studies were performed using pure cultures of A. acrogenes and E. coli and a heterogeneous microbial population growing in carbon‐limited chemostats with glucose as the sole carbon and energy source. A two‐level factorial experimental design was employed to test the hypothesis that the concentration of growth‐limiting substrate in a chemostat is controlled by the growth rate alone and is independent of the concentration of substrate entering the reactor. The pure culture experiments showed that the conclusions depend upon the measurement employed for growth‐limiting substrate. When the concentration of glucose was measured directly, the hypothesis was found to be true within the limits of the study (500–1500 mg/liter). However, if the chemical oxygen demand (COD) test was used as the measure of growth‐limiting substrate the hypothesis was found to be false. When heterogeneous cultures were employed the hypothesis was false regardless of the technique used to measure the concentration of growth‐limiting substrate. Nevertheless, it was possible to generate regression equations which described the interactions among influent COD, growth rate, and effluent COD with a high level of correlation.

Diffusion influences in denaturable insolubilized enzyme catalysis

Abstract: The influence of diffusion on the apparent thermal stability of a reversibly or irreversibly denaturable enzyme is examined theoretically when he enzyme is uniformly distributed in a porous solid. If the overall reaction rate is influenced by diffusion through the catalyst, the insolubilized catalyst is shown to yield an apparently more thermally stable enzyme even though the maximal velocity Vm, and the Michaelis constant, Km, are the same for the free and insolubilized forms of enzyme. Brief consideration is given to the experimental conditions needed to clearly demonstrate whether insolubilization does or does not effect the thermal stability of the enzyme.

Release of protein from Bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill

Abstract: Release of protein from a suspension of bakers' yeast (Saccharomyces cerevisiae) by disruption in an industrial agitator mill has been studied. Protein release on disruption in the mill is a first‐order rate process. The rate constant is dependent on at least six parameters. Increased disruption efficiency was obtained at higher agitator speeds, greater loading of bead attritive elements and lower rates of upward recycle of yeast suspension through the mill. An increase in temperature from 5 to 42°C was accompanied by a reduction in disruption efficiency of approximately 20%. With optimal values of the parameters examined the throughput of the mill is 5.32 kg/hr of soluble protein for 90% disruption.

Invertase covalently coupled to porous glass: Preparation and characterization

Abstract: The enzyme invertase has been covalently coupled to porous glass particles. The product is extremely stable over a long period of time. Kinetic values for the immobilized enzyme are similar to the native enzyme. Excellent enzymatic activity for the immobilized enzyme was exhibited over a broad pH range. The immobilized enzyme when continuously operated for one month was found to have an operational half-life of over 40 days.

Kinetic studies of the lactic acid fermentation in batch and continuous cultures

Abstract: Summary The fermentation kinetics of the homofermentative organism Lactobacillus delbrueckii in a glucose-yeast extract medium is studied in both batch and continuous culture under conditions of controlled pH. From a graphical analysis of the batch data, a mathematical model of the process is derived which relates bacterial growth, glucose utilization, and lactic acid formation. The parameters in the model represent the activity of the organism and are a function of pH, having a maximum value at about 5.90. In a continuous stirred tank fermentor (CSTF), the effect of pH, feed concentration, and residence time is observed. The feed medium is a constant ratio of two parts glucose to one part yeast extract plus added mineral salts. An approximate prediction of the steady-state behavior of the CSTF can be made using a method based on the kinetic model derived for the batch case. In making step changes from one steady state to another, the transient response is observed. Using the kinetic model to simulate the transient period, the calculated behavior qualitatively predicts the observed response.

Effect of mercury on algal growth rates

Abstract: In experiments with one freshwater (Chlorella pyrenoidosa) and three marine organisms (Phaeodactylum tricornutum, Cyclotella nana, and Chaetoceros galvestonensis), mecury was more toxic than the other metals tested (silver, cadmium, lead, and copper); and its toxicity is comparatively irreversible. Growth was monitored by changes in fluorescence of the cultures over a 3-day test period. The toxicity of the mercury varied inversely with the concentrations of nutrients present. Preliminary experiments indicate that mercury in the form of mercuric chloride is more than as dimethylmercury.

Open tubular heterogeneous enzyme reactors: preparation and kinetic behavior.

Abstract: Tubes with immobilized enzymes on the inner wall, called open tubular heterogeneous enzyme reactors, were prepared by binding enzymes either directly to the tube inside surface or to a layer of a porous matrix attached to the inner wall. Kinetic studies of the hydrolysis of N-benzoyl-L-arginine ethylester as a model reaction indicated that the reaction was kinetically controlled in reactors with surface bound trypsin and the kinetic parameters were evaluated by conventional methods. On the other hand, substrate diffusion in both the porous matrix and the bulk substrate solution strongly affected the rate of reaction in porous layer trypsin reactors. The highest overall rates of reaction were obtained when the reaction was bulk diffusion controlled and the measured rates were in agreement with those calculated from expressions derived from heat transfer theory. The design of reactors for the limiting cases of kinetic and bulk diffusion controlled reaction as well as a method for the determination of substrate diffusivity are outlined.

Kinetic behavior of mixed populations of activated sludge

Abstract: The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single-stage isothermal continuous flow completely mixed aeration tank. A series of experiments were carried out at various dilution rates using glucose as the growth limiting substrate. The steady-state behavior of the system was observed at each dilution rate and the results were found to fit fairly well with the steady-state equation bayed on the Monod model with an endogenous respiration term included, i.e., μ = μmS/(Ks + S) − Kd. The growth kinetics of cells harvested at steady state for each dilution rate were studied using batch experiments. The multiple response data of the system as functions of time were used to estimate the parameter values in the above kinetic model. It was found that values of the growth parameters changed significantly and systematically with cell population. For example, values of μm were high at high dilution rates and low at low dilution rates. It was also found that only those batch growth parameters from cells obtained at fairly high dilution rates are comparable with those estimated by the results of steady-state operations. The results of this investigation suggest that (1) different cell populations pre dominated at different steady-state dilution rates, with high dilution rates resulting in predominantly fast-growing organisms and low dilution rates resulting in predominantly slow-growing cells, and (2) risk exists in any randomly picked batch experiment to predict the steady-state behavior of the system when heterogeneous microbial populations must be used.

Continuous fermentation to produce xanthan biopolymer: Effect of dilution rate

Abstract: Single-stage continuous fermentations to produce xanthan gum have been run at dilution rates (D) from 0.023 to 0.196 hr−1. Xanthan production rate (XPR) was a function of D. XPR increased from 0.34 g/hr/kg at D = 0.023 hr−1 to the maximum 0.84 g/hr/kg at D = ca. 0.15 hr−1. At D > 0.15 hr−1 XPR decreased and at the highest D studied (0.196 hr−1) was 0.69 g/hr/kg. Yield of xanthan from glucose consumed was 81–89%. Steady states ended between 6.5 and 8.7 turnovers when a variant strain occurred.

Kinetics of protein release from yeast sonicated in batch and flow systems at 20 kHz

Abstract: The release constant, k, of brewers yeast sonicated at powers up to 200 W at 20 kHz has been shown to be independent of cell concentration up to values of 60 g made up to 100 ml. It is inversely proportional to the volume of the treatment vessel in the range 75 to 450 ml, and almost proportional to the input acoustic power from 60 to 195 acoustic watts. A flow system is described and a relationship linking protein release, flow rate, and the protein release constant, determined from batch experiments, is derived. Good agreement between the theoretical prediction of protein release and experimental results with the flow system was obtained.

Dynamics and control of continuous microbial propagators to subject substrate inhibition

Abstract: Inhibitory substrate levels are common in industrial fermentations and in biological waste-water treatment of many industrial wastes. Continuous microbial cultures are unstable to certain disturbances, such as shock loading by inhibitory substrates. Two feedback proportional control strategies are analyzed and compared for a simple model culture assumed represent able by the culture concentrations of biomass and a single rate-limiting and growth-limiting nutrient (substrate). One control strategy, the well known turbidostat, consists of adjusting culture holding time (e.g., by flow rate adjustment) in response to deviations in turbidity or some other measure of culture biomass concentration. The other control strategy is to adjust holding time in response to deviations in limiting nutrient concentrations in the culture. This second control strategy, termed the nutristat, can be superior to the turbidostat in many applications. The sign and magnitude of the dimensionless group {(X/YD)[dμ/dS]s}, is shown to be an important determinant, in the behavior of the open loop and the two closed loop processes. This characteristic group is positive when the specific growth rate is increased by increases in the nutrient concentration, zero when the growth rate is unaffected by the nutrient concentration, and negative in the presence of nutrient or substrate inhibition. The effects of process modifications and of modeling assumptions on the control of the process are discussed and more sophisticated control schemes are also proposed.

Measurements on the interfacial areas of hydrocarbon in yeast fermentations and relationships to specific growth rates.

Abstract: The transport of insoluble substrates such as hydrocarbons to microorganisms is often postulated to be dictated by the availability of the hydrocarbon surface area. Many publications, qualitative and quantitative, have appeared to substantiate this hypothesis. Experiments have been performed in our laboratory to assess the absolute values of the interfacial area of hexadecane as the carbon source for the growth of Candida intermedia. A sedimentometer, mounted directly in the fermentor, was used to measure the interfacial hydrocarbon area during active growth of this organism. The specific hydrocarbon interfacial area was found to be directly related to the impeller speed, hydrocarbon concentration and surfactant concentration in a 1-liter working volume, turbine-agitated fermentor. The specific growth rate was in turn found to be directly related to the specific hydrocarbon interfacial area. Lastly, cessation of logarithmic growth and onset of linear growth was found at all instances to be governed by the specific hydrocarbon surface area.

Reduction of the nucleic acid content of single‐cell protein concentrates

Abstract: Methods for reducing the content of nucleic acid in protein concentrates from disintegrated yeast and microalgae were investigated. Protein concentrates were prepared by acid precipitation of extracted protein after cell wall separation. The influence of alkaline protein extraction on the content of RNA in isoelectrically precipitated protein concentrates was studied. It was found that when a strong decrease in the RNA content was obtained, this was followed by a decrease in the yield of protein concentrate. Protein concentrates were also prepared without cell wall separation by precipitation with different agents after cell disintegration. In the precipitates from microalgae, a RNA reduction was obtained. Precipitation of yeast, protein gave no essential reduction with the precipitants used. Precipitation of yeast protein by heating at an alkaline pH gave a protein concentrate with a low content of RNA. A slightly lower RNA content was obtained when the precipitation was performed in the presence of NaCl. The yield of amino acid nitrogen was 70–80% and the RNA content was 1–2%. A process with precipitation at alkaline pH for the production of microbial protein concentrates with a low content of nucleic acid is suggested.

A kinetic model for microbial growth on liquid hydrocarbons

Abstract: A kinetic model is presented to explain microbial growth using liquid n-alkanes as substrate. The model is based on the assumption that growth occurs on the soluble alkane and that the metabolite produced by the growing cells helps the dissolution of liquid alkanes in the aqueous medium. Growth curves based on that model fit well with growth data for batch and continuous culture reported by various authors. The model also explains the differences between the relative length of exponential and linear phases of growth reported earlier.

Kinetic model identification in mixed populations using continuous culture data

Abstract: The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single-stage, isothermal, continuous flow, completely mixed aeration tank. A series of experiments were carried out at various dilutions rates using glucose as the limiting substrate. The cell dry weight and substrate concentration in terms of chemical oxygen demand (COD) were continuously monitored. The results indicate that reproducible steady-state conditions can generally be obtained; however, multiple steady states were observed at dilution rates near washout. At low dilution rates (below about 0.1 hr−1) the contribution of microorganism decay became appreciable. Using the multiresponse data of cell dry weight and COD, the parameter values in various existing growth models were estimated. The analyses of variance and residuals revealed that models proposed by Moser, Monod, and Contois, each with a decay term added, were significantly better than the other models which were tested.

Bacteria which attack petroleum hydrocarbons in a saline medium.

Abstract: Bacterial strains were isolated from California coastal areas which showed the ability to oxidize normal paraffins, iso-paraffins, and aromatic hydrocarbons in a synthetic seawater medium. The ability to utilize a particular hydrocarbon was established not only on the basis of visible bacterial growth but also through a chromatographic technique which was standardized and which could define the amount of each hydrocarbon consumed by the bacteria in a mixture. Some of the strains exhibited vigorous hydrocarbon oxidation when exposed to synthetic mixtures of hydrocarbons as well as crude oil. Under conditions of aeration and agitation, mixed cultures could destroy approximately 50% of a South Louisiana crude oil in a period of 48 hr.

The supply of oxygen to submerged cultures of BHK 21 cells.

Abstract: Oxygen solution rates were measured in 4, 30, and 100 liter culture vessels, and the oxygen demand of growing BHK 21 cells estimated. This data was used to calculate the minimal sparged air rates necessary to satisfy oxygen demand throughout the cell growth cycle, and in this way adequate oxygen was supplied without the damaging effects of excessive sparging. Comparable results were obtained when oxygen was supplied by this method and when pO2 was controlled at 80 mmHg, but both cell growth rate and maximum cell density were reduced when pO2 was controlled at other values.

The fermentation of L-sorbose by Gluconobacter melanogenus. I. General characteristics of the fermentation.

Abstract: Growing cultures, washed cells, and cell-free preparations of Gluconobacter melanogecnus IFO 3293 converted L-sorbose to 2-keto-L-gulonic acid, to D-sorbitol (which was metabolized further) and to 5-keto-D-fructose.

Model for the growth of aerobic microorganisms under oxygen limiting conditions.

Abstract: A simple dynamic model is proposed which will allow fermenters to be run at throughputs which fully utilize the mass transfer capabilities of the fermenters while not decreasing the yield from the substrate. The model is compared with one previously proposed, which was originally formulated for double substrate limitation when both substrates were supplied in the feed. Computer solutions of the model are given which show the effects of the parameters used. Experimental results from growing Candida utilis on a high concentration of glucose were found to be similar to those predicted by the model.

Preparation and characteristics of microcapsules containing asparaginase

Abstract: Conditions for the preparation of microcapsules containing asparaginase by interfacial polymerization were investigated.

Effect of wall growth on scale‐up problems and dynamic operating characteristics of the biological reactor

Abstract: If inhibitory substrates are being utilized in a well-stirred biological reactor, microbiological growth on the walls of the reactor can create a scale-up problem. A simple model is proposed which shows that without such growth, of the three existing steady states only one is stable and nontrivial, but with wall growth the trivial, stable, steady state (washout) is impossible. In addition, wall growth reduces the region over which three steady states are feasible and reduces the minimum residence time for which there is only one steady state that corresponds to a high conversion. Thus, a laboratory process with a high surface area to volume ratio can give an over optimistic prediction of both necessary residence; time and stability of the full scale process unless wall growth is accounted for.

Growth models of cultures with two liquid phases. VIII. Experimental observations on droplet size and interfacial area

Abstract: Because of the importance of the drop she distribution and interfacial area of the dispersed liquid phase in hydrocarbon fermentations, experiments were carried out to determine the drop size distribution and the interfacial area during batch fermentations of Candida lipolytica on gas oil and on n-hexadecane dissolved in dewaxed gas oil. The effects of cell concentration and dispersed phase volume fraction on size distribution and interfacial area were investigated. Measurements of interfacial tensions, densities, viscosities, and fatty acid concentrations were also made. The results show that the size distribution is skewed and that the Sauter mean diameter is in the range of 10 to 30 μ. Both the Sauter mean diameter and the interfacial area increased during the course of a batch fermentation; however, they decreased at the end of the fermentation. The interfacial area also increased with inoculum size.

Covalent coupling of pullulanase to an acrylic copolymer using a water soluble carbodi-imide.

Abstract: Pullulanase (EC 3.2.1.9) prepared from a culture of Acrobacter aerogeneshas been covalently bound to an inert crosslinked copolymer of aerylamide-acrylic acid by using a water-soluble carbodi-imide. The binding yield based on the amount of added pullulanase was 34%. The residual enzymic activity was 43%, of that of free enzyme. Coupling in the presence of the substrate pullulan gave a 5-fold increase in activity over that obtained when substrate was lacking. The effect of different carbodi-imide concentrations on the coupling has been investigated. The isoelectric point of the pullulanase preparation (3.5–4.0) was determined using isoelectric, focusing, in order to find optimal pH conditions for the coupling procedure. The immobilized pullulanase in a packed bed column was used to debranch amylopeetin to low molecular weight amylose.

Continuous production of extracellular protease by Bacillus subtilis in a two-stage fermentor.

Abstract: Growth and protease production of Bacillus subtilis in semisynthetic and synthetic media were studied in batch culture and in a two-stage, laboratory scale, continuous fermentor. The amount, of extracellular protease production was measured under specific growth conditions in both stages of the ferment or. At the dilution rates employed, the cells in the first stage of the ferment or produced negligible quantities of protease, and the culture primarily functioned as a continuous inoculum for the second stage of the fermentor. The culture effluent from the second stage of the fermentor contained extracellular protease, on the average, equal to 60 per cent, of the activity that had been found in the supernatant of a 48-hr batch culture grown in a medium having the same composition as that in the continuous fermentor. Extracellular protease was produced in semisynthetic medium by B. subtilis in the two-stage fermentor for as long as 20 days without culture degeneration. Additional studies indicated that continuous protease production could also be achieved in a synthetic medium. The RNA/ protein ratios of cells grown in semisynthetic medium in batch culture and in each stage of the two-stage fermentor were examined. There was a positive correlation between the amount of protease produced by the cells and their RNA/ protein ratio. Techniques employed for continuous production of protease by B. subtilis and the potential use of the method for investigating the control of secondary metabolite synthesis are discussed.