On-line and in-situ monitoring technology for cell density measurement in microbial and animal cell cultures

Commercially available on-line and in-situ devices for monitoring cell density are reviewed in this article. Principles of operation are described as well as capabilities of these probes in specific measurement applications based on literature reports. Pilot-scale experimental observations from three optical density probes, the Cerex, Monitek and Wedgewood designs, have been included for Escherichia coli fermentations. Requirements for future on-line and in-situ instruments are discussed as well as the impact of current limitations on widespread application.     

Application of polydimethylsiloxane-based optical system for measuring optical density of microbial culture

The performance of recently developed polydimethylsiloxane (PDMS)-based optical system was tested for measuring optical density of microbial culture. The data showed that PDMS-based spectrometer is superior to “one drop” spectrometers in the accuracy, and has an advantage over conventional spectrometers in measuring dense culture without dilution.     

A novel device for the assessment of shear effects on suspended microbial cultures

Summary A novel device has been designed for the evaluation of the effects of shear on microorganisms. The device consists of a combination of a coaxial cylinder plus cone and plate viscometers and enables cultures of both eukaryotic and prokaryotic microorganisms to be grown under fully defined and controlled fluid dynamic characteristics for serveral generations. In the preliminary tests performed a change in cell length was observed in Escherichia coli, and with Penicillium chrysogenum the septal length, hyphal diameter, and branching frequency all changes as shear was increased.Symbols L Height of liquid (m) - r Radius of cone/bob (m) - r _m Arithmetic mean of R and r (m) - R Radius of cup (m) - T Torque (Nm) - Shear rate (s^-1) - ₀ Angle of cone (rad) - Shear stress (Nm^-2) - Annular velocity (rad s^-1)     

Real-time monitoring and automatic density control of large-scale microalgal cultures using near infrared (NIR) optical density sensors

Signals from near infrared (NIR) light transmittance sensors were used for both real-time monitoring of algal biomass density in growing mass cultures (200l tubular biofences), and also as feedback in a system that controlled the density of the culture by automatic injection of fresh growth medium. When operated in a semi-continuous production mode between predefined density values, diurnal growth patterns were recorded on-line that provided information on the dynamics of the microalgal cultures with respect to environmental conditions. The bioreactor system was also programmed to operate in constant biomass density mode, thereby maintaining the culture at the optimal population density (OPD), and sustaining high biomass production levels. The system has potential for operating a dynamic density set point for microalgal cultures where the optimal population density varies as a function of ambient growing conditions.     

Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.     

Insect cell density in bioreactor cultures can be estimated from on-line measurements of optical density

Total cell density in growing insect cell suspension cultures was accurately estimated from both off-line and on-line measurements of optical density (OD). The latter measurements were done with an in-situ autoclavable OD sensor. The ability to continuously monitor cell density in insect cell cultures may be useful for the development of a large-scale process for recombinant protein production using baculovirus expression vectors.     

Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures

We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the New Pit Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54°C and 56 to 63°C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adap ted to the habitat temperature (50°C), while populations adapted to 65°C and 70°C were 102- and 104-fold less abundant, respectively. However, enrichment at 70°C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotropic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit.     

The continuously fed batch reactor for measuring microbial growth rates

The contiuously fed batch reactor (CFBR) is proposed as an alternative technique to the traditional chemostat and batch cultures, for measuring microbial growth rates. After reviewing the pitfalls which plague the conventional growth measurement techniques, the methodology for operating the CFBR to generate specific growth-rate-versus-substrate-concentration data is detailed. This information is extracted from the transient state of the CFBR where both the biomass and substrate concentration show extrema in time. It is suggested that the CFBR can be used for measuring microbial growth rates at low rates at low substrate concentrations where the chemostat method normally encounters difficulties.     

Evaluation of lactic acid Streptococci for the preparation of frozen concentrated starter cultures

Eight strains of Streptococcus diacetilactis and Streptococcus lactis were examined for viability, growth rate, lactic acid and diacetyl production in milk and proteolytic activity before and after freezing at --30 degrees C. Concentrations of yeast autolysate, peptone, lactose and citrate as well as the usefulness of milk and whey culture media for active biomass production were investigated. After freezing and storage at --30 degrees C, with the use of non-fat milk as a cryoprotective agent, high survival and endocellular proteolytic activity of the frozen concentrate was achieved. S. diacetilactis sp. and S. lactis 115 were shown to be more biologically active than other strains. Their physiological properties remained unaffected by freezing.     

Comparative testing of tangential microfiltration for microbial cultures

In an attempt to extend and intensify the productive periods of bioprocesses, a self-cleaning tangential filtration device was examined. Built into a special-design bioreactor, its cell retention was evaluated for continuous-flow operation with selected examples of bacteria (Escherichia coli), yeasts (Sacharomyces cerevisiae), and filamentous fungi (Aspergillus niger). Performance characteristics such as filtration rates and cell accumulation were assessed as a function of filter rotational speed, operating pressure, cultivation time, and microfilter type (i.e., membrane or porous metallic). The highest flux of cell-free filtrate for each culture type was achieved using a 0.45-micron membrane-covered microfilter. While the respective yeast (S. cerevisiae) and bacterial (E. coli) cell concentrations were enhanced by as much as 16- and 8-fold over the batch growth levels, the representative A. niger fungal cultivation was less satisfactory because of progressively declining filtration rates limited by hydraulically resistant layers of microbial surface growth quite resistant to in situ filter backflushing with gas. Maximum steady-state flux was independent of operating pressure, yet was enhanced at rotational speeds up to about 800 rpm. Higher speeds offered no further improvements. The overall fermentation process was limited by the moderate levels of attainable flux which restricted the feed and dilution rates. The maximum attainable stabilized fluxes were 26-40 L/m(2) x h.     

Simple device for automatically transferring broth cultures

A simple device is described for automatically transferring broth cultures from one tube to another by means of a tilting platform.     

Evaluation of Streptococcus cremoris for preparing frozen concentrated starter cultures.

Five strains of Streptococcus cremoris were investigated with respect to their usefulness for frozen concentrated biomass production. APLC medium assured high growth of 3 strains and highest cell concentration i.e. 4--6.7 g of fresh biomass from 1 litre of this medium. Two strains were sensitive to citrate present in APLC medium requiring a lowering of its dose to 0.5% in order to assure the highest biomass accumulation. The viability, endocellular proteolytic activity against casein and acidifying ability of frozen concentrates revealed that the physiological features of cells were preserved and remained unchanged during 2 and 12 weeks of storage at -30 degrees.     

Nondestructive monitoring of carotenogenesis in Haematococcus pluvialis via whole-cell optical density spectra

We investigated the feasibility of rapid, nondestructive assay of carotenoid-to-chlorophyll (Car/Chl) ratio and total carotenoids (Car) in cell suspensions of the carotenogenic chlorophyte Haematococcus pluvialis Flotow under stressful conditions. Whole-cell spectra are characterized by variable nonlinear contributions of Car and chlorophylls (Chl), with a strong influence of Car packaging and sieve effect inherent to stressed H. pluvialis cells. Nevertheless, nondestructive assay of Car/Chl in the range of 0.55–31.2 (Car content up to 188 mg?L^?1; 5.4 % of the cell dry weight) turned to be achievable with a simple spectrophotometer lacking an integrating sphere upon deposition of the cells on glass fiber filters. The scattering-corrected optical density (OD) in the blue–green region of the whole-cell spectrum, normalized to that in the red maximum of Chl absorption (OD₅₀₀/OD₆₇₈), was tightly related (r ²?=?0.96) with the Car/Chl ratio found in extracts. Some features such as the amplitude and position of the minimum of the normalized first-derivative OD whole-cell spectra also exhibited a strong (r ²?>?0.90) nonlinear correlation with Car/Chl. These spectral indices were also tightly related with Car, but the slope of the relationship varied with the stressor intensity. The importance of calibration over the widest possible range of pigment contents and a correct choice of biomass load per filter are emphasized. The advantages and limitations of nondestructive monitoring of carotenogenesis in H. pluvialis are discussed in view of its possible application in optical sensors for laboratory cultivation and mass production systems of the algae.     

In situ fiber‐optical monitoring of cytosolic calcium in tissue explant cultures

We present a fluorescence‐lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber‐optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green‐5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Equipment for the automatic aseptic inoculation and sampling of microbial cultures

Equipment has been designed and constructed for the automatic aseptic inoculation and sampling of microbial cultures. It enables fermentations to be started and samples to be taken round-the-clock, according to a predetermined program. The equipment is suitable for microbial processes on both a laboratory and a factory scale.