Viable Biomass Concentration Research Articles

Steady state modeling of autotrophic membrane bioreactor – a new approach to quantify biomass

PurposeThe main purpose of this study resides essentially in the development of a new tool to quantify the biomass in the bioreactor operating under steady state conditions.Design/methodology/approachModeling is the most relevant tool for understanding the functioning of some complex processes such as biological wastewater treatment. A steady state model equation of activated sludge model 1 (ASM1) was developed, especially for autotrophic biomass (XBA) and for oxygen uptake rate (OUR). Furthermore, a respirometric measurement, under steady state and endogenous conditions, was used as a new tool for quantifying the viable biomass concentration in the bioreactor.FindingsThe developed steady state equations simplified the sensitivity analysis and allowed the autotrophic biomass (XBA) quantification. Indeed, the XBA concentration was approximately 212 mg COD/L and 454 mgCOD/L for SRT, equal to 20 and 40 d, respectively. Under the steady state condition, monitoring of endogenous OUR permitted biomass quantification in the bioreactor. Comparing XBA obtained by the steady state equation and respirometric tool indicated a percentage deviation of about 3 to 13%. Modeling bioreactor using GPS-X showed an excellent agreement between simulation and experimental measurements concerning the XBA evolution.Originality/valueThese results confirmed the importance of respirometric measurements as a simple and available tool for quantifying biomass.

Determining the linear correlation between dielectric spectroscopy and viable biomass concentration in filamentous fungal fermentations

ObjectivesDielectric spectroscopy is commonly used for online monitoring of biomass growth. It is however not utilized for biomass concentration measurements due to poor correlation with Cell Dry Weight (CDW). A calibration methodology is developed that can directly measure viable biomass concentration in a commercial filamentous process using dielectric values, without recourse to independent and challenging viability determinations.ResultsThe methodology is applied to samples from the industrial scale fermentation of a filamentous fungus, Acremonium fusidioides. By mixing fresh and heat-killed samples, linear responses were verified and sample viability could be fitted with the dielectric Delta C values and total solids concentration. The study included a total of 26 samples across 21 different cultivations, with a legacy at-line viable cell analyzer requiring 2 ml samples, and a modern on-line probe operated at-line with 2 different sample presentation volumes, one compatible with the legacy analyzer, a larger sample volume of 100 ml being compatible with calibration for on-line operation. The linear model provided an {R}^{2} value of 0.99 between Delta C and viable biomass across the sample set using either instrument. The difference in ∆C when analyzing 100 mL and 2 mL samples with an in-line probe can be adjusted by a scalar factor of 1.33 within the microbial system used in this study, preserving the linear relation with {R}^{2} of 0.97.ConclusionsIt is possible to directly estimate viable biomass concentrations utilizing dielectric spectroscopy without recourse to extensive and difficult to execute independent viability studies. The same method can be applied to calibrate different instruments to measure viable biomass concentration. Small sample volumes are appropriate as long as the sample volumes are kept consistent.

Evaluation of the Growth Kinetics of Lactobacillus Plantarum ATCC 8014 on a Medium Based on Hydrolyzed Bovine Blood Plasma at Laboratory and Bench-Scale Levels and Its Application as a Starter Culture in a Meat Product

Lactobacilli are used in food because of their beneficial effect on human health and their biopreservative activity in matured meat products. The objective of this work was to study the growth kinetics of Lactobacillus plantarum ATCC 8014 by submerged fermentation at laboratory and bench scales, using a culture medium based on bovine blood plasma (BBP) with hydrolyzed proteins, and to evaluate the maturational effects and sensory properties conferred by the obtained biomass on a matured meat product (pepperoni). At bench scale, it was found that the maximum viable biomass concentration of L. plantarum was 9.58 log CFU/mL, which was higher than what was found in the MRS culture medium (9.53 log CFU/mL). The mathematical model proposed appropriately described the L. plantarum growth kinetics and carbohydrate dynamics during fermentation at laboratory and bench scales in hydrolyzed BBP medium. The application of viable L. plantarum biomass propagated on this medium did not show statistically significant differences during pepperoni maturation compared to the product made with the commercial starter culture. The sensory panel found no differences in the evaluated sensory attributes between these two products. The L. plantarum biomass obtained on this medium can be used successfully in maturation processes in different meat matrices.

Assessment of Serratia sp. isolated from iron ore mine in hexavalent chromium reduction: kinetics, fate and variation in cellular morphology

ABSTRACTSerratia sp. strain SU.ISM.1 was isolated from Noamundi iron ore mines for the first time and was observed for hexavalent chromium reduction, and growth kinetics modelling was applied for bacterial chromium reduction. For 4–8 ppm of hexavalent chromium concentration, complete reduction was observed within 36 h when the selected isolate was applied, and for 12–20 ppm chromium concentration, complete reduction was achieved within 48 h. The viable biomass concentration increased up to 36 h of treatment time, after which the biomass concentration gradually declined. The Aiba model of product inhibition growth kinetics best described the growth of biomass in the presence of hexavalent chromium. The total mass conversion of Cr(VI) to Cr(III) for 4, 8, 12, 16 and 20 ppm was found to be 94.9%, 88.5%, 74.66%, 70.75% and 78.8%, respectively. The AFM and FESEM studies showed that the roughness of the cell surface increased with increasing concentration of hexavalent chromium, probably due to adsorption of chromium.

Evaluation of an online biomass probe to monitor cell growth and cell death

Background The estimation of cell density and cell viability of mammalian cell lines in cell culture has traditionally been performed using the exclusion dye trypan blue that stains “dead” cells when their cell membrane is damaged. In large scale cell cultures using bioreactors this estimation is performed off-line. The online biomass probe is based on the principle that under the influence of an electric field between two electrodes, ions in suspension migrate toward the electrodes. The cell plasma membrane is non-conductive so that the cells with intact plasma membranes are polarized and act as tiny capacitors and it has been shown that capacitance increases as the cell concentration does. The measurement is based on the linear relationship between the permittivity difference e1e2 and the viable biomass concentration as it has been described by Ansorge et al. [1]. This study compares the data obtained using the biomass probe against the cell counts and viability determined by trypan blue exclusion with two GS-CHO cell line productions in bioreactors. Apoptosis determination measurements using rhodamine-123 and pancaspase activation by flow cytometry will be compared to permittivity values.

Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 muL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96-well microtiter plate, to preserve compatibility with the current high-throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO(2) production rate was obtained from the online measurement of cumulative CO(2) production during the cultivation. The online data obtained by the sensor array and the CO(2) production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench-scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro-scale fermentations, e.g. using the 96-well microtiterplate format.

Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h(-1)) was fitted with a 0.22-microm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h(-1) (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g(-1) of biomass h(-1) calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h(-1). This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, d-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae.

Lab‐scale fermentation tests of microchip with integrated electrochemical sensors for pH, temperature, dissolved oxygen and viable biomass concentration

This article shows the development and testing of a microchip with integrated electrochemical sensors for measurement of pH, temperature, dissolved oxygen and viable biomass concentration under yeast cultivation conditions. Measurements were done both under dynamic batch conditions as well as under prolonged continuous cultivation conditions. The response of the sensors compared well with conventional measurement techniques. The biomass sensor was based on impedance spectroscopy. The results of the biomass sensor matched very well with dry weight measurements and showed a limit of detection of approximately 1 g/L. The dissolved oxygen concentration was monitored amperometrically using an ultra-microelectrode array, which showed an accuracy of approximately 0.2 mg/L and negligible drift. pH was monitored using an ISFET with an accuracy well below 0.1 pH unit. The platinum thin-film temperature resistor followed temperature changes with approximately 0.1 degrees C accuracy. The dimensions of the multi sensor chip are chosen as such that it is compatible with the 96-well plate format.

Integrated Electrochemical Sensor Array for On-Line Monitoring of Yeast Fermentations

This paper describes the design, modeling, and experimental characterization of an electrochemical sensor array for on-line monitoring of fermentor conditions in both miniaturized cell assays and in industrial scale fermentations. The viable biomass concentration is determined from impedance spectroscopy. As a miniaturized electrode configuration with high cell constant is applied, the spectral conductivity variation is monitored instead of the permittivity variation. The dissolved oxygen concentration is monitored amperometrically using an ultramicroelectrode array, which is shown to have negligible flow dependence. pH is monitored using an ion-sensitive field effect transistor (ISFET), and a platinum thermistor is included for temperature measurements. All sensors were shown to be sufficiently accurate within the range relevant to yeast fermentations. The sensor array is shown to be very stable and durable and withstands steam-sterilization.

Evaluation of a new annular capacitance probe for biomass monitoring in industrial pilot-scale fermentations

The four-pin electrode capacitance probe has already shown to be a valuable tool for on-line monitoring viable biomass concentration in industrial-type fermentations. A new prototype annular probe was developed and its performance in real-time monitoring the concentration of viable cells during industrial pilot-scale fermentation for the production of an Active Pharmaceutical Ingredient (API) was investigated and compared to the four-pin probe. A set of 14 fermentations was monitored on-line: four of them with the four-pin probe, the remaining with the annular probe. The performance of both the annular and the four-pin electrode probe were compared against each other and against off-line measurements (viscosity and packed mycelial volume). The prototype annular probe showed to have higher signal intensity and sensitivity than the standard four-pin probe, with higher signal-to-noise ratio. Furthermore, its new design and construction proved to be easier to handle in an industrial environment.

Biomass measurement by inductive permittivity

An electrodeless, inductive permittivity probe is shown to reliably indicate fermentation biomass. By designing the probe based on electromagnetic induction, the electrode polarization artifact-common to electrode-based permittivity measurements of conductive solutions-is eliminated. The measurement of viable biomass concentration (X(V)) is precise and linear, with negligible interference, as shown for two bacteria, two yeasts, and two mammalian cells. The permittivity spectra often can be described by the three parameters of the Cole-Cole equation: the maximum (low-frequency) permittivity, the characteristic frequency (f(C)), and the Cole-Cole alpha. The f(C) and alpha have potential as relative indicators of cell size and morphology, respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 289-304, 1997.

Studies of on‐line viable yeast biomass with a capacitance biomass monitor

A commercially available biomass monitor has been employed in a number of applications. For capacitance monitors, a relationship between capacitance measurement and cell counts or colony forming units has been reported in the literature. However, for use as an online instrument, a more practical correlation with the biomass concentration is needed. In this study, we followed the batch growth of brewer's yeast and a correlation with viable biomass concentration (g DW/L) was demonstrated. This correlation was utilized with the capacitance biomass monitor in a control loop to maintain setpoint biomass levels in a cyclic reactor under perturbations. Not only did the system demonstrate the capability of the biomass monitor to control biomass in such a system, but it also confirmed the correlation reported in our earlier work. (c) 1994 John Wiley & Sons, Inc.

Measurements of the viable biomass concentration in activated sludge by respirometric techniques

The parameters required to calculate the concentration of variable bacteria in activated sludge can be determined by using two different respirometers, viz. the Sapormat for long-run batches and an open respirometer, described earlier, for short-run batches. Sludges with different sludge loads were found to contain widely different amounts of bacteria, varying from 8 to 60% of the dry weight. A temporarily higher sludge load on a low loaded system has little influence on the clarification because of the rapid increase of the viable bacteria concentration. A temporarily lower sludge load, however results in a decreased efficiency due to the decay of the viable biomass. On the basis of calculations it can be concluded that the effluent BOD merely consists of cell decay products.