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For Pichia pastoris fermentation process with multi-operating conditions, it is difficult to predict the cell concentration under the new operating conditions by the soft sensor model established under the specific operating conditions. Inspired by the idea of transfer learning, a method based on an improved balanced distribution adaptive regularization extreme learning machine (IBDA-RELM) was proposed to solve the problem. The domain adaptation (DA) method in transfer learning is developed to reduce distribution distance by transforming data. However, the joint distribution adaptation (JDA) and the balanced distribution adaptation (BDA) in DA cannot be directly applied to regression problems. The fuzzy sets (FSs) method was proposed to solve this issue. Finally, a soft sensor model of Pichia pastoris cell concentration was realized by inputting the converted data to the RELM model. Simulation verification was carried out with three operating conditions at the scene of fermentation. The transfer effects of three DA methods, including transfer component analysis (TCA), improved joint distribution adaptation (IJDA) as well as IBDA, were compared. The predicted results show that IBDA-RELM had a better performance in the soft sensor of Pichia pastoris cell concentration under multi-operating conditions.

Aquatic microbial communities contribute fundamentally to biogeochemical transformations in natural ecosystems, and disruption of these communities can lead to ecological disasters such as harmful algal blooms. Microbial communities are highly dynamic, and their composition and function are tightly controlled by the biophysical (e.g., light, fluid flow, and temperature) and biochemical (e.g., chemical gradients and cell concentration) parameters of the surrounding environment. Due to the large number of environmental factors involved, a systematic understanding of the microbial community-environment interactions is lacking. In this article, we show that microfluidic platforms present a unique opportunity to recreate well-defined environmental factors in a laboratory setting in a high throughput way, enabling quantitative studies of microbial communities that are amenable to theoretical modeling. The focus of this article is on aquatic microbial communities, but the microfluidic and mathematical models discussed here can be readily applied to investigate other microbiomes.

A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 106 cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry., Graphical Abstract

We present a microfluidic platform for automatic multi-size spheroid formation within constant volume hanging droplets (HDs) from a single inlet loading of a constant cell concentration. The platform introduces three technological improvements over the existing spheroid formation platforms: 1) cell seeding control is achieved by enrichment of a cell solution rather than dilution; 2) cell seeding in each HD is fully independent and pre-programmable at the design stage; 3) the fabricated chip operates well using a hydrophobic PDMS surface, ensuring long-term storage possibility for device usage. Pre-programmed cell seeding densities at each HD are achieved using a "microfluidic funnel" layer, which has an array of cone-shaped wells with increasing apex angles acting as a metering unit. The integrated platform is designed to form, treat, stain, and image multi-size spheroids on-chip. Spheroids can be analyzed on-chip or easily transferred to conventional well plates for further processing. Empirically, enrichment factors up to 37× have been demonstrated, resulting in viable spheroids of diameters ranging from 230-420 μm and 280-530 μm for OV90 and TOV112D cell lines, respectively. We envision that microfluidic funnels and single inlet multi-size spheroid (SIMSS) chips will find broad application in 3D biological assays where size-dependent responses are expected, including chemoresponse assays, photodynamic therapy assays, and other assays involving drug transport characterization in drug discovery.

3D bioprinted vascular constructs have gained increased interest due to their significant potential for creating customizable alternatives to autologous vessel grafts. In this study, we developed a new approach for biofabricating fibrin-based vascular constructs using a novel rotary 3D bioprinter developed in our lab. We formulated a new bioink by incorporating fibrinogen with gelatin to achieve a desired shear-thinning property for rotary bioprinting. The blending of heat-treated gelatin with fibrinogen turned unprintable fibrinogen into a printable biomaterial for vessel bioprinting by leveraging the favorable rheological properties of gelatin. We discovered that the heat-treatment of gelatin remarkably affects the rheological properties of a gelatin-fibrinogen blended bioink, which in turn influences the printability of the ink. Further characterizations revealed that not only concentration of the gelatin but the heat treatment also affects cell viability during printing. Notably, the density of cells included in the bioinks also influenced printability and tissue volumetric changes of the printed vessel constructs during cultures. We observed increased collagen deposition and construct mechanical strength during two months of the cultures. The burst pressure of the vessel constructs reached 1110 mmHg, which is about 52% of the value of the human saphenous vein. An analysis of the tensile mechanical properties of the printed vessel constructs unveiled an increase in both the circumferential and axial elastic moduli during cultures. This study highlights important considerations for bioink formulation when bioprinting vessel constructs. Statement of Significance There has been an increased demand for small-diameter tissue-engineered vascular grafts. Vascular 3D bioprinting holds the potential to create equivalent vascular grafts but with the ability to tailor them to meet patient’s needs. Here, we presented a new and innovative 3D rotary bioprinter and a new bioink formulation for printing vascular constructs using fibrinogen, a favorable biomaterial for vascular tissue engineering. The bioink was formulated by blending fibrinogen with a more printable biomaterial, gelatin. The systematic characterization of the effects of heat treatment and gelatin concentration as well as bioink cell concentration on the printability of the bioink offers new insight into the development of printable biomaterials for tissue biofabrication.

Despite its simplicity, which makes it the most commonly used bioprinting method today, extrusion-based bioprinting suffers from its inability to reproduce the complex tissue architecture found in organs. Generally, this printing method allows for the dispensing of solutions of a predefined cell concentration through a rudimentary needle. Moreover, to avoid cell lysis in the dispensing needle, which is detrimental to the viability of the printed tissue, as well as cell loss in dead volumes of tubing, thereby increasing the cost of printing tissue, a common strategy has been to print with cell concentrations much lower in comparison to the concentrations found in living tissues. As a result, cell-to-cell distance is increased in the dispensed samples impairing communication through cytokines. Here, we present a microfluidic-based print head capable of modulating the printed cell concentration in real-time. This device allows bioprinting at high cell concentrations by concentrating and dispensing fibroblasts at concentrations up to 10 million cells∙mL−1. We also demonstrate that this device can be used to print bladder organoids. As the cell seeding concentration is of major importance for organogenesis in 3D culture, organoid printing allows the user to standardize the process of organoid formation and achieve more reliable and reproducible results.

The effects of important production variables on the biotransformation of sucrose by immobilized cells of Phaffia rhodozyma were investigated. Cell concentration had negative effect on the ...

A non-sterile and buffer-free 2-keto-gluconic acid (2KGA) production method was proposed by using resting cells from an industrial strain of Pseudomonas fluorescens AR4. 2KGA achieved a maximum production performance of 195.00 g/L, which corresponded to 3.05 g/L h of total productivity and 1.07 g/g of yield under optimal conditions, including resting cell aged 20 h, a cell concentration of 3.0 g/L, a reaction temperature of 30 °C, a glucose concentration of 181.82 g/L, and a reaction volume of 20 mL during the 64-h bioconversion process. P. fluorescens AR4 resting cells showed stable bioconversion activity after reuse 3 times or storage for less than 28 d at 4 °C. These findings suggested that the proposed non-sterile and buffer-free bioconversion of glucose to 2KGA using P. fluorescens AR4 resting cells had remarkable advantages, including ease handling and low cost, and they are a potent application for industrial production of 2KGA.

Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells. Biotechnol. Bioeng. 2015;112: 1437–1445. © 2015 Wiley Periodicals, Inc.

Polyunsaturated fatty acids (PUFAs) of the type ω3 and ω6 play important physiological functions in human organism, since they are components of cell membranes and brain cells; they decrease the levels of triglycerides and can prevent the incidence of coronary heart disease. Various parameters, including concentration of the nitrogen source in the cultivation of oleaginous microorganisms have been reported to be essential in the biosynthesis and accumulation of PUFAs. The objective of this work is to study the effect of different concentrations of total nitrogen (TN) in the production of PUFAs, especially DHA, from Thraustochytrium sp. ATCC 26185. The concentrations of TN evaluated were 2.4 and 0.8 g/L (batch) and 0.009 g/L (hourly) under fed-batch process. The content of cell biomass, glucose consumption, TN and production of PUFAs was determined. The major composition of the PUFAs in Thraustochytrium sp. ATCC 26185 cell biomass were DPA ω6 (21-25%) and DHA (69-73%), regardless of the type and time of culture. The maximum cell concentration obtained (30.2 g/L) was using 2.4 g/L TN in 168 h of culture. With this same concentration of TN it was possible to produce the highest concentration of DHA (1.16 g/L) in 120 h of culture, demonstrating that the growth of Thraustochytrium sp. ATCC 26185 and yield of PUFAs are dependent on the concentration TN source available for consumption of this oleaginous microorganism, as well as culture time.

Phytase production by Pichiapastoris was used as a case to study the mechanism and strategy for optimization of heterologous protein production. It was found that the ratio of inducer-methanol to cell concentration had a significant influence on phytase production. In this case, we found that the optimum initial cell concentration and methanol concentration were 85 g/L and 10 g/L, respectively. During induction period, an easy-to-control methanol feeding method was proposed according to the optimal ratio of methanol to cell concentration at a range of 0.063 -0.132 g/g, and phytase activity and productivity reached 53984 U/mL and 529.25 U/(mL·h), respectively. The method for optimization of phytase production through controlling the optimal ratio maybe provide an alternative idea to enhance other heterologous protein production with P. pastoris.

A hydrogen peroxide (H2O2)-resistant bacterium, Exiguobacterium oxidotolerans T-2-2T, secretes extracellular catalase. A low shaking frequency (60 rpm) was employed following the observation that a higher amount of extracellular catalase was produced under this condition compared with the previous shaking frequency (120 rpm). The cell concentration reached approximately 4.5 × 106 cells mL−1, at which the initiation of extracellular catalase production was triggered, while the production of the extracellular catalase was largely dependent on that of the intracellular catalase. The best initial aeration condition for extracellular catalase production was 7.5 mg O L−1, which is 95 % of that of the air-saturated state measured under the low shaking frequency. In addition, although the total productivity of catalase was unchanged, introduction of aminolevulinic acid and Tween 60 promoted extracellular production. The extracellular and intracellular catalase productivities were 16,000 and 6,000 U mL−1 of spent culture medium, respectively.

Recombinant alkaline polygalacturonate lyase (PGL) production by recombinant Pichia pastoris GS115 was selected as a model to study as a continuous culture strategy for enhancing heterologous protein production based on controlling methanol feeding (CCCM culture) or on dual carbon source feeding (CCCD culture). Using the CCCM process with a dry cell weight of 75 g/L regulated by controlling methanol concentration in the induction media, the final PGL activity was 441.9 U/mL. The PGL productivity (Qv) and the average specific enzyme production rate (Qx) were 4.65 U mL−1 h−1 and 84.5 U g−1 h−1, an increase of 42.1% and 191.2%, respectively, over what was achieved with traditional fed-batch culture with high cell density. The control strategies also reduced proteolytic degradation by 84.1% in the fermentation broth and increased cell viability by 12.2%.

Aim: The investigation of the different culture systems in order to get high yield of baby hamster kidney (BHK) cell line. Materials and Methods: Different cultivation systems, vibrofermentor and 5-L stirred tank bioreactors, have been used for the production of BHK cell line. The production systems were evaluated with respect to pH, temperature, viable cell number, aeration rate and stirring speed. Results: BHK cells were successfully cultivated in vibrofermentor and stirred tank bioreactor (STR) systems. In addition, higher cell concentration was obtained with low aeration rate (0.005 vvm) comparing to those of 0.02 and 0.05 vvm. The cultivation in STR can be operated with low aeration rate due to decrease the expenditure and improve the cell productivity. Conclusion: In order to improve the cell yield of the anchorage semi-dependent cell line in pilot scale, it should be preferred that the cells are firstly adapted to suspension culture by vibrational stirring and then be cultivated in stirred tank bioreactor which is operated at low aeration rate. These results showed consistency with the previous studies.

Micro-environmental restriction effects to yeast cell growth obtained within Ca-alginate microbeads are considered. It is complex phenomenon influenced by: (1) relaxation of expanded polymer network around the cellular clusters, (2) forces generated by cell growth inside the beads and (3) interactions between solvent, network parts and cells. The resulting effects are measured experimentally by estimating volume of microbeads and yeast cell concentration as function of time of cultivation. Comparative analysis of dynamics of cell growth and increase of microbead volume through four regimes indicates that reversible and irreversible local structural changes of Ca-alginate hydrogel induces micro-environmental restrictions to cell growth. The mechanism of restrictions includes both mechanical and electrostatic effects.

Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of F. lipolytica, we report here that pretreatment of F. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 10(10)/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform F. lipolytica that will be able to yield an efficiency of 2.1 × 10(4) transformants/ug for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for F. lipolytica transformation.

The cross-flow filtration characteristics of bacterial cells (S. bovis) obtained from a lactic acid fermentation broth of fresh cassava roots have been investigated in terms of specific cake resistance. The amount of particles accumulated on the membrane surface was evaluated using a cake filtration model. The effects of the operating conditions (transmembrane pressure, cross-flow velocity and cell concentration) on permeate flux, cake resistance and specific cake resistance were studied. The cake properties in cross-flow filtration were then compared to those in dead-end filtration. The specific cake resistance in the cross-flow filtration shows a higher value than in the dead-end filtration for almost the same cake compressibilities of about 1.0. The specific cake resistance increases with increasing cross-flow velocity and decreases with increasing cell concentration until reaching a concentration at which the specific cake resistance hardly changes. The increasing ratio of the specific cake resistance in the cross-flow filtration over the dead-end filtration is found to be independent of the transmembrane pressure and cell concentration.

D-psicose exits in an extremely small amount in nature and is difficult to be chemically synthesized. Only three bacteria have been used in the biotransformation of D-psicose from D-fructose. In this paper, another bacterium which could convert D-fructose to D-psicose was isolated and identified as Rhodobacter sphaeroides. The process parameters of D-psicose production using permeabilized cells of Rhodobacter sphaeroides SK011 were optimized, including the permeabilization procedure: 0.1% (w/v) CTAB, 10 min, and reaction conditions: cell concentration, 30 g dry cell wt/L; concentration of substrate, 50 g/L; 40°C, pH 9.0; reaction time, 8 h. Under the optimized conditions, the permeabilized cells produced approximately 6.5 g/L D-psicose with a D-psicose productivity of 0.82 g·L−1·h−1. This is the first report of bioproduction of D-psicose using permeabilized cells of Rhodobacter sphaeroides.

Baker's yeast number 6 was selected by screening. It showed good catalytic activity and enantioselectivity for asymmetric reduction of 2,5-hexanedione to produce (2S,5S)-2,5-hexanediol. Gas chromatography-mass spectrometry (GC-MS) revealed that the intermediate was (S)-5-hydroxyhexane-2-one. Reduction of 2,5-hexanedione proceeded in a two-step reaction. The hydroxyketone was initially formed, and this intermediate was further reduced to the diol. Factors influencing the product yield and the enantiomeric excess of the reduction of 2,5-hexandione catalyzed by baker's yeast number 6 were investigated. Higher concentration (≤100 mmol·L−1) of 2,5-hexandione did not influence 5-hydroxyhexane-2-one production, but 2,5-hexanediol production was inhibited by excess accumulation (>30 mmol·L−1) of intermediate. The optimal conditions were glucose as the co-substrate at an initial glucose concentration of 20 g·L−1, 34°C, pH 7.0 and cell concentration 60 g·L−1 (cell dry mass). Under the optimal condition and an initial substrate concentration of 30 mmol·L−1, the yield of 2,5-hexandiol was 78.7% and the enantiomeric excess of (2S,5S)-2,5-hexandiol was 94.4% for 24-h reduction.

Polyunsaturated aldehydes (PUAs) have been extensively studied for their potential role in phytoplankton defence against grazers, but recently a potential infochemical role has been identified as well. We present here the first evidence of a synchronised release of PUAs from intact cells of a culture of the diatom Skeletonema marinoi. The release occurs transiently before the culture changes to the decline phase, providing a time-limited potential signal. When cultures are exposed during different stages of growth to PUAs in concentrations found in the medium, no general responses were found. However, a weak effect on cell concentration is observed exclusively when cultures in the late stationary phase are challenged with PUAs. Our results support for the first time the experimentally postulated hypothesis that PUA can play a role as infochemicals in mediating plankton interactions.

The clinical application of bioartificial liver support systems (BALS) is still limited because of technical problems associated with the storage, transport and scale-up of common systems. The encapsulation of primary hepatocytes could solve these problems since the scale-up depends only on the number of the beads and encapsulation leads to protection of the cells during the process of freezing and thawing. Many efforts have been made to find an appropriate material for the encapsulation of primary hepatocytes in terms of mechanical resistance as well as appropriate bio- and hemo-compatibility This study focuses on the improvement of the metabolic functionality of encapsulated primary hepatocytes. A comparison between two different cultivation models showed that dynamic cultivation conditions lead to a 20.4-fold increase in the albumin production and a 5.21-fold increase in the urea synthesis of encapsulated hepatocytes. Furthermore, the influence of different ratios of the number of the cells to the volume of the media was analyzed. Encapsulated hepatocytes cultured with a high amount of medium were characterized by a significantly higher metabolic activity compared to encapsulated hepatocytes cultured with a low level of medium. Interestingly, the cell concentration per mL alginate has no significant influence on the metabolic activity of encapsulated hepatocytes. In conclusion, different optimization strategies are discussed and, finally, the functionality of encapsulated hepatocytes is compared to the standard model of hepatocyte culture, the collagen sandwich.

Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v). The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration of storage at -70 degrees C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO. Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option.

The production of C595 diabody fragment (dbFv) in Escherichia coli ( E. coli ) HB2151 clone has been explored. The comparison of fermentation processes mode demonstrated that a higher biomass inoculum operation enhanced C595 dbFv production. It was demonstrated that a concentration of 12.1 mg l −1 broth of dbFv and a cell concentration of 23.6 g l −1 broth were achieved at the end of 75 l fermentation.

The development of fermentation conditions for the production of C595 diabody fragment (dbFv) inE. coli HB2151 clone has been explored. Investigations were carried out to study the effect of carbon supplements over the expression period, the comparison of C595 dbfv production in synthetic and complex media, the influence of acetic acid upon antibody production, and comparison of one-stage and two-stage processes operated at batch or fed-batch modes in bioreactor. Yeast extract supplied during expression yielded more antibody fragment than any other carbon supply. The synthetic medium presented higher specific productivity (0.066 mg dbFv g−1 dry cell weight) when compared to the complex medium (0.044 mg dbFv g−1 DCW). The comparison of fermentation strategies demonstrated that (1) one-stage fed-batch fermentation performed higher C595 dbFv production than that operated in batch mode which was significantly affected by acetate concentration; (2) a two-stage batch operation could enhance C595 dbFv production. It was found that a concentration of 12.3 mg L−1 broth of C595 dbFv and a cell concentration of 10.8 g L−1 broth were achieved at the end of two-stage operation in 5-L fermentation.

Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h.

In order to achieve an effective decolorization with Funalia trogii, fungal immobilization and repeated batch experiment were investigated. The activities of three enzymes, namely laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP), were reported during decolorization of acid black 52 under the condition of immobilized and suspended fungal mycelia. When F. trogii were immobilized in Na-alginate, the cell viability increased as alginate concentration was increased, although the cell concentration decreased. Na-alginate of 4% (w/v) was an optimum concentration. Decolorization rate by fungal immobilization was higher than that by suspended cultivation. Decolorization could be stably maintained with the repeated batch experiments during long period (at least for 300 h).

We present a centrifugal process for the extraction of plasma from sediment by a decanting structure, terminating with metered plasma which is readily available for subsequent on-disk processing. Our technique supplies 2 microl plasma from 5 microl of whole blood at moderate spinning frequencies of 40 Hz within 20 s, only. The residual cell concentration in the purified plasma amounts to less than 0.11%, independent of the frequency of rotation. A capillary duct connects the extracted plasma to subsequent on-disk processing units.

Lyophilized non-proliferating microbial cells of Pseudomonas sp. 42A2 showed their ability to catalyze the bioconversion of oleic acid into (E)-10-hydroxy-8-octadecenoic acid (MHOD) and (E)- 7,10-dihydroxy-8-octadecenoic acid (DHOD). The reaction conditions were optimized in order to produce the maximum amount of MHOD. At 30°C and using phosphate buffer (0.1 M and pH=8), an initial substrate concentration of 10 mg mL� 1 and a cell concentration of 8 mg mL� 1 , practically all the oleic acid in the medium was biotransformed within 42 h, with hardly any DHOD produced (1.4 mg mL� 1 ).

Delftia acidovorans (IFO13582) was grown in fed-batch and chemostat cultures with n-butyric acid and 1,4-butanediol as mixed carbon substrates. In fed-batch cultures, the synthesis rate of the polymer was considerably increased with the increment of the cell growth. The polymer content increased and reached to a maximum of 27 wt% with increasing the cell dry-weight of ca. 4.4-5.2 g/l at 26 °C for 72 h and the maximum 4HB unit composition of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] was slightly low value of ca. 66 mol%. Whereas the maximum 4HB unit composition of P(3HB-co-4HB) obtained by the fed-batch culture with lower cell concentration of ca. 2.5 g/l reached to 94 mol% though the content of P(3HB-co-4HB) was always low, at ca. 13 wt%.

This study proposes lumostatic operations using a specific light uptake rate ( q e ) as a control parameter in order to maintain a desirable light condition. As a model system, bubble column photobioreactors have been operated lumostatically for photoautotrophic cultivations of Haematococcus pluvialis . Lumostatic operations were conducted at three levels of specific light uptake rates ( q e =1.5×10 −8 , 3×10 −8 , and 4.5×10 −8 μE cell −1 s −1 ) and compared with constant light energy supply ( I 0 =40, 90, and 140 μE m −2 s −1 ). In the case of the lumostatic operation at 4.5×10 −8 μE cell −1 s −1 , the cell concentration was enhanced over 250% than that obtained under constant light energy, without inducing astaxanthin accumulation. For the lumostat at q e of 3.0×10 −8 μE cell −1 s −1 , the cell concentration increased almost 200% than that of the control experiments. These results indicate that a specific light uptake rate can be a useful parameter for lumostatic operation of photobioreactors and that the productivity of vegetative stage can be greatly improved by regulating the light condition based on the specific light uptake rates.

Chlorella VT-1 a pentachlorophenol-tolerant microalga was exposed to a wide range of chlorophenols. The alga showed some tolerance to all the chlorophenols at a concentration of 10 mg/l except for 2,4,5-trichlorophenol, which was toxic at all concentrations tested. The chlorophenols inhibited the initial growth of the alga but after 5–10 days the culture resumed growth at a rate comparable with that of the control. The resumption of growth was not due to adaptation to the chlorophenols but was a feature of the cell concentration.

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.

The substantial concern with the possible use of immobilized yeast cells for beer production is reduction of internal mass transfer resistance during continuous fermentation. One way to minimise this problem is to use small-diameter beads. The effects of bead diameters in the range 0.3–2.0 mm on yeast cell immobilization and growth over a short-term cultivation were investigated. Bead diameters in the range 0.5–0.6 mm were optimal and provided rapid cell growth and the highest final cell concentration (2.33×10 9 cells/ml of beads). Electrostatic droplet generation was investigated as a technique for production of alginate microbeads. The effects of applied potential, internal needle diameter and electrode position on bead diameter were assessed. The results have shown that this method can be used for controlled production of small-size microbeads loaded with yeast. Depending on applied conditions it was possible to produce the beads in the range 250 μm–2.0 mm in diameter.

An effect of chromium(VI) ions on the growth and bioaccumulation properties of growing cells of Candida Utilis was studied. Molasses media for yeast growth containing 20 g glucose l −1 and 50+500 mg Cr(VI) l −1 were used in batch cultivation. Addition of 100 mg Cr(VI) l −1 resulted in a threefold decrease in the cell concentration, as compared with the culture grown without metal. Cr(VI) inhibited culture growth in a concentration-dependent manner, this dependence was not linear. Glucose consumption by growing cells depended on the initial Cr(VI) concentration in the medium and correlated with growth activity. No inhibitory effect of high Cr(VI) concentrations on the activity of some exo-enzymes of C. utilis cells was observed. During C. utilis cultivation, Cr(VI) was partially reduced to an ultimate Cr(III) in all tested ranges of the Cr(VI) concentrations. The specific chromium uptake by cells was detected in biomass that grew in the presence of 100+300 mg Cr(VI) l −1 in a period of 48–96 h. The highest values were achieved after 96 h growth in the presence of 200 and 300 mg Cr(VI) l −1 , and were 7.3 and 7.2 mg Cr g dw l −1 , respectively. Electron microscopic observations showed morphological changes in yeast cells to be more pronounced upon culture growth with 100–300 mg Cr(VI) l −1 than in the cells subjected to higher metal concentrations. The conclusion has been made that the mechanism of the Cr(VI) toxic effect on the growing yeast cells can vary in dependence of the metal concentration.

We investigated the possibility that human erythrocytes can reduce extracellular ascorbate free radical (AFR). When the AFR was generated from ascorbate by ascorbate oxidase, intact cells slowed the loss of extracellular ascorbate, an effect that could not be explained by changes in enzyme activity or by release of ascorbate from the cells. If cells preserve extracellular ascorbate by regenerating it from the AFR, then they should decrease the steady-state concentration of the AFR. This was confirmed directly by electron paramagnetic resonance spectroscopy, in which the steady-state extracellular AFR signal varied inversely with the cell concentration and was a saturable function of the absolute AFR concentration. Treatment of cells N-ethylmaleimide (2 mM) impaired their ability both to preserve extracellular ascorbate, and to decrease the extracellular AFR concentration. These results suggest that erythrocytes spare extracellular ascorbate by enhancing recycling of the AFR, which could help to maintain extracellular concentrations of the vitamin.

To produce polyhydroxyalkanoate (PHA) copolymer which consists of 3-hydroxy-butyrate (3HB) and 3-hydroxyhexanoate (3HHx) by cultivation ofAeromonas hydrophila, fed-batch cultures were done under several nutrient limiting conditions. With the results from flask cultures, fed-batch cultures were carried out to produce large amounts of PHA. In the fed-batch culture, firstly glucose was fed to grow cell, and then, oleic acid fed to stimulate PHA in the cell. The final cell concentration, PHA content, PHA concentration, and 3-hydroxy-hexanoate fraction in 38 hr were 48.9 g/L, 15.05 wt%, 7.36 g/L and 12.2 wt%, respectively, resulting in the productivity of 0.19 g/L-h under phosphate-limiting condition.

To maximize the production of virginiamycin (VM and VS) which is commercially used as an antibiotic mixed in animal feed, an empirical approach was employed in the batch culture of Streptomyces virginiae . Here, effects of Dissolved Oxygen (DO) concentration and/or DO supplying condition such as pure oxygen supply and agitation speed changes on the maximum cell concentration at the production phase as well as production activity of virginiamycin were investigated. In order to keep DO concentration in the fermenter at a certain level, either agitation speed or inlet oxygen concentration of supplying gas can be manipulated. It was found that the increase of agitation speed gave positive effect on antibiotics productivity apart from the controlled DO concentration. Optimum DO concentration and agitation speed including VB-C addition was determined for maximize virginiamycin production.

A variety of disruption techniques are available for the release of intracellular proteins. Analysis of present day equipment in terms of possible cavitating conditions indicates that, to get efficient disruption of cells, the equipment must operate very near or below the cavitation inception number. A simple set-up has been described which generates cavitating flow. The effect of various parameters such as discharge pressure, and the number of passes through the cavitating zone were studied. Also, the effect of cell type, growth stage of cell and cell concentration on the cell disruption were investigated. The effect of these cavitating conditions on the activity of the macromolecules was also studied. The energy effciency of the hydrodynamic cavitation was compared with that of established techniques such as ultrasonication and mixer-blender methods. It was observed that the hydrodynamic cavitation set-up described is almost an order of magnitude more energy effcient than the established techniques.

An indirect conductimetric method was developed for the rapid detection of heterotrophic growth of Haematococcus pluvialis. Selective HKU medium was superior to Whitley Impedance Broth. Acetate was better than glucose not only for the biomass production (30,200 cells ml on 1 g acetate l vs. 14,800 cells ml on 1 g glucose l ) but also for the time needed to detect significant growth (10.9 h on 1 g acetate l vs. 36.9 h on 1 g glucose l ). The largest maximum conductance change of -1455 μS and the shortest detection time of 10.9 h were found in HKU medium containing 1 g acetate l , and the cell concentration increased by 5.5 times after 4 days of cultivation.

The effects on the growth of Gambierdiscus toxicus Adachi et Fukuyo (Dinophyceae) cultures by conditioned media from dense-growing cultures were examined and enhanced growth was observed. The increase in Gambierdiscus toxicus cell concentration with the addition of the conditioned media suggested the existence of endogenously produced growth enhancer(s). Gambieric acid-A, a polyether compound, is known to be excreted by G. toxicus, therefore we examined the effects of gambieric acid-A of G. toxicus growth. The growth of G. toxicus clones responded maximally at 1.89 nM gambieric acid-A. Gambierdiscus toxicus growth followed a dose-dependent saturation curve with inhibition at 18.9 nM gambieric acid-A. The results of this study strongly suggest the potential biological function of gambieric acid-A to be an endogenous growth enhancer of G. toxicus. To our knowledge, no endogenously produced factor(s) enhancing the growth of any dino-flagellate have been reported.

International audience; Dilution needed for plate counts (CFU/ml) is known to disrupt streptococci chains, and therefore the validity of this method is controversial. A novel apparatus was designed to disrupt Leuconostoc mesenteroides chains in pairs, by forcing them to pass through a narrow capillary tube. When Leuconostoc mesenteroides occurs in pairs it appears that the real cell concentration can be simply deduced by doubling the CFU/ml.

After incubation of Maillard reaction polymers (MRPs) with rumen fluid from wethers neither volatile fatty acids nor lactate were produced. Soluble polymeric products of the Maillard reaction were nonmetabolizable by a mixed culture of rumen microorganisms. MRPs added at 0.5 and 2 g/L inhibited the growth of seven ruminal Gram-negative bacteria by 20 and 30%, respectively. In four strains of Gram-positive bacteria, MRPs lowered the cell concentration by 11% (0.5 g/L) and 25% (2 g/L). The rumen fungusOrpinomyces jojonii also did not metabolize soluble MRPs.

Sedimentation of hardened erythrocytes in a centrifugal field was studied by time recording of the current chamber in the longitudinal and the transversal directions relative to the cells' movement. The results clearly indicate the existence of an erythrocyte concentration profile during centrifugation. The rates of both longitudinal and transversal current alteration increase with centripetal acceleration and with falling cell concentration. The pellet formed from hardened cells represents virtually incompressible body. It is shown that erythrocyte shape affects the pellet conductivity. Analysis of the data using the modified Stokes' law enables calculation of the cell size distribution. The modal size of macrocytes, normal erythrocytes and two samples of microcytes thus measured was 3.40, 3.01, 2.63 and 2.83 μm, respectively. These data demonstrate that conductometric analysis is useful for investigating abnormalities in erythrocyte size.

The time variation in bioprinter output, i.e. the number of cells per printed drop, was studied over the length of a typical printing experiment. This variation impacts the cell population size of bioprinted samples, which should ideally be consistent. The variation in output was specifically studied in the context of cell settling. The bioprinter studied is based on the thermal inkjet HP26A cartridge; however, the results are relevant to other cell delivery systems that draw fluid from a reservoir. A simple mathematical model suggests that the cell concentration in the bottom of the reservoir should increase linearly over time, up to some maximum, and that the cell output should be proportional to this concentration. Two studies were performed in which D1 murine stem cells and similarly sized polystyrene latex beads were printed. The bead output profiles were consistent with the model. The cell output profiles initially followed the increasing trend predicted by the settling model, but after several minutes the cell output peaked and then decreased. The decrease in cell output was found to be associated with the number of use cycles the cartridge had experienced. The differing results for beads and cells suggest that a biological process, such as adhesion, causes the decrease in cell output. Further work will be required to identify the exact process.

A cell-recycled air-lift bioreactor was studied for its performance in cultivation of Mentha piperita cells producing essential oils. The reactor system sustained a stable operation over 30 days with the aid of a cell settler. The maximum cell concentration reached 50% packed cell volume and it occurred at the dilution rate of 0.27 day-1. Volumetric productivity of essential oils in this system was substantially higher than that obtainable from batch culture.

A novel technique of using hydrodynamic cavitation for the large-scale disruption of yeast cells is described. Baker's yeast and brewer's yeast cells in a pressed yeast form were used. Cell disruption was monitored in the form of increase in soluble protein content. Disruption by hydrodynamic cavitation is compared with that obtained by established techniques such as blade blender and acoustic cavitation (ultrasonication). The effect of cell concentration, time of treatment and number of passes in the flow loop system on the extent of cell disruption is reported. The energy efficiency of the hydrodynamic cavitation setup is compared with that of established techniques. Hydrodynamic cavitation was found to be at least an order of magnitude more energy efficient than established techniques such as ultrasonication or blade blender (mixer).

Immobilized yeast was encapsulated with cell-free calciumalginate gel by two-step preparation procedure. The volume of coated film decreased with increasing cell concentration. The encapsulation did not affect ethanol production and could prevent cell leakage from the gels.

We have established a two-step strategy for the modeling of cell cultures in suspension and applied it to Escherichia coli cultures. Cell concentration, pH, and sugar consumption (glucose) were involved. In the first step, graphical analysis of the experimental kinetic data allowed an approach to the kinetic cell behavior and led to the mathematical dynamic model. An approximation of the corresponding parameters was thus obtained. Then, a more precise estimation of these parameters was obtained using a numerical simulation, including a general modeling of pH effects in acid fermentations. The results show that a simple graphical analysis is able to give a very good estimation for most parameters characterizing the culture at a macroscopic level.

Lactococcus lactis subsp. cremoris was microencapsulated within alginate/poly-L-lysine (alg/PLL), nylon or crosslinked polyethyleneimine (PEI) membranes. Toxic effects were observed with solvents and reagents used in nylon and PEI membrane formation. Alg/PLL encapsulation resulted in viable and active cell preparations which acidified milk at a rate proportional to the cell concentration, but at rates less than that of free cell preparations. At 4 x 10(8) colony-forming units (cfu/ml milk), encapsulated cells took 17 per cent longer than free lactococci to reduce the pH of milk to 5.5. Similar activities of free and micro-encapsulated cells may be attained at higher cell concentrations (10(9) cfu/ml milk). The rate of lactic acid production was approximately 2 mmol/h at an encapsulated cell concentration of 4 x 10(8) cfu/ml.