Production of sophorolipids in high concentration from deproteinized whey and rapeseed oil in a two stage fed batch process using Candida bombicola ATCC 22214 and Cryptococcus curvatus ATCC 20509

High concentrations of 422 g sophorolipids l–1 were produced using a two-stage cultivation process: first deproteinized whey concentrate (DWC) containing 110 g lactose l–1 was used for cultivation of the yeast Cryptococcus curvatus ATCC 20509, resulting in 34 g dry weight l–1, 20 g single-cell oil l–1 and reducing the chemical oxygen demand (COD) from 159 g l–1 to 35 g oxygen l–1. Afterwards cells were disrupted by passing the cell suspension directly through a high pressure laboratory homogeniser. After autoclavation, the resulting crude cell extract containing the single-cell oil served as substrate for growth of Candida bombicola ATCC 22214 and for sophorolipid production in a second stage. When the single-cell oil was consumed, repeated feeding of 400 g rapeseed oil l–1 was started increasing the yield of sophorolipids to 422 g l–1. A simple technique for product isolation, sedimentation, could be used to harvest the crude sophorolipids. © Rapid Science Ltd. 1998     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

Quantifying metabolic activity of filamentous fungi using a colorimetric XTT assay

The filamentous nature and robust cell walls of many fungi render traditional measurements of active biomass (e.g., turbidity, dry cell weight) ineffective for most fungal bioprocesses. To overcome this challenge, an assay for quantification of overall metabolic activity is developed using 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT), which in the presence of active mitochondria is converted to a water-soluble formazan derivative that absorbs light in the visible spectrum (430-490 nm). Tests on the model fungus Aspergillus nidulans show that in actively growing cultures XTT absorbance is linearly related to dry cell weight below 0.2 g/kg broth. Validation through growth rate testing shows the developed XTT assay is able to accurately quantify reductions in culture metabolism during damaging physical treatment (heat, high shear, microwaving). Experiments in batch culture demonstrate that the developed XTT assay is capable of reporting on metabolic activity where dry cell weight is not. The developed assay is inexpensive, relatively rapid, and easy to conduct, making it ideally suited for assessment of fungal processes in the biotechnology industry.     

Pulp and paper mill solid wastes as substrates for single-cell protein production

We have investigated the upgrading of some typical pulp and paper mill solid wastes into protein-enriched animal feed using the cellulolytic fungus Chaetomium cellulolyticum. The waste residues used were six different primary clarifier sludges and a sample of tertiary centricleaner rejects. These were obtained from mills whose modes of operation spanned the range typically in present-day usage: groundwood, sulfite, semichemical, Kraft, and thermomechanical pulping, with and without bleaching. Crude protein production from the solid waste residues is compared to that obtainable from fermentation of untreated or caustic-pretreated sawdusts. Some of these waste residues, especially the Kraft pulp mill rejects, appear to be promising sources of substrate for single-cell protein production. In these preliminary findings, up to 28% dry weight crude protein content of the product has been obtained at specific growth rates of up to 0.12hr^?1 on direct utilization of the wastes.     

The calculation of cellular parameters from the turbidity of yeast cultures may give rise to important errors

Abstract It is shown that cellular parameters of the yeast cultures (e.g. intracellular volume, cellular dry weight, protein content, number of cells, and turbidity) are differently influenced by metabolic changes. Therefore, the cellular parameters change independently of each other. It is hence concluded that whenever quantitation is required, the values of these parameters should be measured independently and not calculated from the turbidity of the cultures or other parameters, as is often done.     

On-line measurement and control of cell concentration of Saccharomyces cerevisiae using a laser turbidimeter

Summary On-line measurement and control of cell concentration of Saccharomyces cerevisiae using a laser turbidimeter was carried out. Effects on the turbidity measurement of operating parameters such as agitation speed, aeration rate, and the concentration of antifoam agent were investigated. Correlations between the on-line-measured turbidity and the off-line dry cell weight concentration were made at various operating conditions. Using the correlations the cell concentration was successfully estimated in a range of up to 8 g/L in batch cultures. The on-line monitoring and regulation of the cell concentration in a range of up to 35 g/L were also satisfactory in continuous and turbidostat cultures with cell recycle.     

Turbidimetric measurement of plant cell culture growth

Turbidimetric measurement of cultures grown in sidearm flasks was used to measure the growth of plant cells. The turbidity was shown to vary proportionally with cell number and dry weight over time. The effect of different freezing conditions on the growth of the culture was presented to demonstrate the application of the sidearm-turbidity method.     

Growth of Chaetomium cellulolyticum on Alkali-Pretreated Hardwood Sawdust Solids and Pretreatment Liquor

The treatment of a hardwood sawdust with 1% NaOH solution at 121°C dissolved 19.7% of the dry matter, mainly hemicellulose and lignin. Fermentation of the treated solids by Chaetomium cellulolyticum for 48 h gave a product containing 12.5% crude protein (total N × 6.25) on a dry weight basis. The in vitro rumen digestibility of the 48-h fermentation product was 30%, compared to 24% for the alkali-treated but unfermented sawdust. Growth was independent of sawdust particle size in the range 40 to 100 mesh. Fermentation of the pretreatment liquor gave a product containing up to 50% crude protein (dry weight basis) with an in vitro rumen digestibility of 65 to 76%. Approximately 6.7 g of crude protein was obtained from the treated solids and 2.2 g from the pretreatment liquor per 100 g of sawdust treated. The product from the pretreatment liquor fermentation has potential as a high-protein animal feed supplement but could not be produced economically without an outlet for the relatively indigestible product from the solids fermentation. Growth on the pretreatment liquor was strongly pH dependent; there was a considerable increase in the lag phase when the pH was lowered from 7.5 to 5.2. This effect appears to be due to an inhibitor whose toxicity is reduced at high pH.     

On-line monitoring of cell concentration of Perilla frutescens in a bioreactor

This article demonstrates the successful in situ real-time monitoring of the cell concentration of Perilla frutescens in a bioreactor by using a laser turbidimeter. It was found that turbidity measurements at 780 nm with the laser sensor were hardly affected by the red color of the anthocyanin produced by P. frutescens cells, nor by the aeration rate or agitation speed within the ranges investigated. There was an excellent linear relationship, with a correlation coefficient (r(2)) higher than 0.99, between the sensor's response and the cell concentration. The whole growth stage of the cells, i.e., lag, logarithmic, and stationary phases, in bioreactor cultivations, could be satisfactorily estimated on-line by means of the in situ turbidimeter. However, during the declining phase of the cells, an apparent deviation was observed between the on-line estimations and off-line measurements of cell concentrations by dry cell weight, while the wet cell weight could be estimated by the same turbidimeter system. We found that this deviation was caused by a decrease in the cell density due to an increase of the individual cell volume and a decrease of the cell dry weight during the declining phase. (c) 1993 John Wiley & Sons, Inc.     

Phytase production by fermentation of recombinant Pichia pastoris in monosodium glutamate wastewater

A novel method is proposed to produce both phytase and single-cell protein in recombinant Pichia pastoris fermentation using monosodium glutamate wastewater (MSGW) as the basal medium. Recombinant P. pastoris MR33 transformed with a phytase gene (AppA-m) from Escherichia coli was constructed and showed capability to utilize ammonium as the only nitrogen source. The fermentation medium was optimized in shake flasks by single-factor test and response surface methodology. A fed-batch system containing 30% MSGW, 50 g/l glucose, 1.58 g/l CaSO₄, 5.18 g/l MgSO₄ and 6.67 g/l KH₂PO₄ was developed in a 3.7-l bioreactor. The maximum phytase activity in the MSGW medium reached 3,380 U/ml, 84.2% of that in chemically defined medium, and the dry cell weight was 136 g/l. The single-cell protein (SCP; 46.66% dry cell weight) contains a variety of amino acids and is low in fat, which is ideal for utilization in animal feed. Thus, it is feasible to use MSGW medium for the production of enzymes that can be expressed in P. pastoris.     

Use of a Fiber Optic Probe for Spectral Measurements and the Continuous Recording of the Turbidity of Growing Microbial Cultures

This paper describes the properties and use of a fiber optic probe as an attachment to a spectrophotometer and its use for measurements in solutions and turbid suspensions. Measurements of a standard were identical when a spectrophotometer equipped with the probe was used or when a spectrophotometer was used in a conventional manner. The probe was calibrated for turbidimetric measurements with microorganisms by relating the apparent absorbancy measured on the spectrophotometer to the dry weight of each species of organism. Continuous measurements were made of the turbidity of growing cultures of Escherichia coli, Streptococcus mutans, and Saccharomyces cerevisiae. Transient changes in cell mass were observed in some cultures during continuous monitoring of growth. The data were recovered in a manner which allowed direct computer processing.     

A finite representation model for an asynchronous culture of E. coli

A computer model is described which models an asynchronous population of E. coli by using a large, but finite number of representative single cells. Asynchrony generation and maintenance occurs at the single cell level by modulating the activity of an enzyme responsible for septum formation. Such modulation introduces cycle time imprecision and does not require the introduction of any new parameters into the single-cell model. Based on comparisons to experiment, reasonable predictions are possible for changes of cellular dry weight during exponential growth and turbidostat washout, and overall chemostat cell yields and changes in cell number, glucose concentration, and cell size distribution for a chemostat subject to a step change in dilution rate. Additionally, a correlation between cell RNA content and size is predicted as is an inertial effect when chemostat residence time is decreased under conditions of initially high glucose concentrations. Limitations imposed by the model's finite nature and their solutions are discussed.     

Aflatoxin B1 Induction of Lysogenic Bacteria

A technique for biological verification of aflatoxin B(1) was developed based on toxin-mediated induction of lysis in a lysogenic strain of Bacillus megaterium NNRL B-3695. Reduction of culture turbidity was determined at various concentrations of toxin. Incubation of 1.1 x 10(-4) g (dry weight) of cells/ml of growth medium containing 25 mug of B(1) per ml at 37 C reduced initial turbidity 0.20 absorbance units in 4 hr. If the bacterial lysate of the lysogenic strain, after a 2-hr incubation with 25 mug of B(1) per ml, was plated with a sensitive B. megaterium strain (NRRL B-3694), plaque-forming units increased approximately 150 times relative to the control. Comparable testing of the effects of aflatoxin on the nonlysogenic, sensitive strain demonstrated that 75 mug of B(1) per ml neither induced lysis nor plaque-forming units. Although induction is not an exclusive property of aflatoxin B(1), the differential response of the lysogenic and sensitive Bacillus strains to B(1) offers a unique and rapid technique for biological verification of the toxin.     

Microfluidic chip-based single-cell cloning to accelerate biologic production timelines

Cell line development (CLD) represents a critical, yet time-consuming, step in the biomanufacturing process as significant resources are devoted to the scale-up and screening of several hundreds to thousands of single-cell clones. Typically, transfected pools are fully recovered from selection and characterized for growth, productivity, and product quality to identify the best pools suitable for single-cell cloning (SCC) using limiting dilution or fluorescence-activated cell sorting (FACS). Here we report the application of the Berkeley Lights Beacon Instrument (BLI) in an early SCC process to accelerate the CLD timeline. Transfected pools were single-cell cloned when viabilities reached greater than 85% or during selection when viabilities were less than 30%. Clones isolated from these accelerated processes exhibited comparable growth, productivity, and product quality to those derived from a standard CLD process and fit into an existing manufacturing platform. With these approaches, up to a 30% reduction in the overall CLD timeline was achieved. Furthermore, early process-derived clones demonstrated equivalent long-term stability compared with standard process-derived clones over 50 population doubling levels (PDLs). Taken together, the data supported early SCC on the BLI as an attractive approach to reducing the standard CLD timeline while still identifying clones with acceptable manufacturability.     

Mathematical modeling of a single-cell enzyme assay

A quantitative assay of beta-galactosidase activity in single cells of Saccharomyces cerevisiae has been developed using a fluorogenic substrate and flow cytometry [reported in Wittrup & Bailey, Cytometry, 9,394 (1988)]. The beta-galactosidase activity is expressed in yeast from the Escherichia coli lacZ gene under the control of the yeast GAL10 promoter, and is used as a marker for multicopy plasmid content. A nonfluorescent fluorogenic substrate is enzymatically cleaved by intracellular beta-galactosidase to form a fluorescent product. The accumulation of fluorescent product in single cells was found to depend on bulk substrate concentration and single-cell enzyme activity in a fashion that could not be described by a Michaelis-Menten kinetic rate form. It has been demonstrated that diffusion limitation rather than enzyme activity can determine the level of single-cell fluorescence under certain assay conditions, and a mathematical model has; been formulated which accounts for substrate and product diffusion. Guided by the mathematical model, the assay conditions were modified to allow measurement of single-cell enzyme activity rather than diffusion rates.     

Detection of enteric viruses in treated drinking water.

The occurrence of viruses in conventionally treated drinking water derived from a heavily polluted source was evaluated by collecting and analyzing 38 large-volume (65- to 756-liter) samples of water from a 9 m3/s (205 X 10(6) gallons [776 X 10(6) liters] per day) water treatment plant. Samples of raw, clarified, filtered, and chlorinated finished water were concentrated by using the filter adsorption-elution technique. Of 23 samples of finished water, 19 (83%) contained viruses. None of the nine finished water samples collected during the dry season contained detectable total coliform bacteria. Seven of nine finished water samples collected during the dry season met turbidity, total coliform bacteria, and total residual chlorine standards. Of these, four contained virus. During the dry season the percent removals were 25 to 93% for enteric viruses, 89 to 100% for bacteria, and 81% for turbidity. During the rainy season the percent removals were 0 to 43% for enteric viruses, 80 to 96% for bacteria, and 63% for turbidity. None of the 14 finished water samples collected during the rainy season met turbidity standards, and all contained rotaviruses or enteroviruses.     

Enhanced biotransformations and product recovery in a membrane bioreactor through application of a direct electric current

The simultaneous enhancement of biotransformation coupled to product recovery, purification and concentration is presented. The nitrilase of Rhodococcus rhodochrous LL100-21 catalyses the single-step hydrolytic biotransformation of benzonitrile to benzoic acid and ammonia. When a direct electric current is applied across a bioreactor containing the bacterium and benzonitrile, the charged product (benzoic acid) can be removed in situ across an anion exchange membrane and recovered in a separate compartment. Over the course of a 24-hour biotransformation, benzonitrile was converted to benzoic acid which was completely removed from the bioreactor chamber and concentrated 3-fold in a separate chamber. The rate of production of benzoic acid increased by 42% when the current was applied (0.044 mmol/min/g dry cell weight in the presence of current as compared to 0.03 mmol/min/g dry cell weight in its absence). The enhanced reaction rate was achieved irrespective of product separation and therefore appears to be a direct effect upon the bacterial cells. This process has potential for enhanced productivity from biotransformations through a simultaneous increase in metabolic activity and in situ product recovery.     

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli

Abstract: The production of the fusion protein staphylococcal protein A/E. coli β-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g 1^-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g 1^-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g 1^-1.     

Simulation of CFSTR through development of a mathematical model for anaerobic growth of Escherichia coli cell population

A computer model is described that simulates a population of Escherichia coli B/r-A cells growing under anaerobic conditions. This population model is an ensemble of single-cell models. The ability of the model of predict the dynamic response of a cell population in a CFSTR to a change in feed flowrates or concentrations was investigated. With glucose as the limiting nutrient the feed concentration of glucose was shifted from 1.0 to 1.88 g/L. With a fixed concentration of glucose (1.0 g/L) step changes in residence time (4.1-1.95 h) were examined. The predicted changes of cell size distribution, substrate concentration, RNA content, and cell dry weight during the transition period compared reasonably well to those observed experiementally. We believe this model is the only model currently available that can make such predictions on an a priori basis.