Maximum Specific Cell Growth Rate Research Articles

Growth kinetics of lead resistant Bacillus infantis isolated from battery industry waste

The current investigation presents an innovative approach to address the challenge of removing heavy metal lead from industrial waste through advanced biological remediation techniques. By amalgamating theoretical insights with empirically acquired data, this research endeavours to develop efficient bioreactor strategies suitable for large-scale applications. Initially, bacteria naturally endowed with lead resistance has been isolated from a native source. Extensive microbiological assessments, including a 16S rDNA study, verified the identity of the lead-resistant bacterial cells as Bacillus infantis 4352-1T. In order to evaluate the efficacy of Bacillus infantis 4352-1T for lead removal an attempt has been made to study the growth dynamics of Bacillus infantis 4352-1T cells in batch mode, using lead amended selective media. It has been observed that Monod's equation effectively defined the cell growth behaviour within the lead concentration range of 0.05–0.25 kg lead/m3. Notably, the experiment was also facilitated the derivation of essential intrinsic kinetic parameters, such as the maximum specific cell growth rate (0.0237 h−1) and substrate saturation constant (0.018 kg/m3). Beyond lead concentrations of 0.25 kg lead/m3, up to 0.43 kg lead/m3, it has also been observed the pronounced influence of substrate inhibition which is quantitatively elucidated by the Haldane equation.

Activation of galactose utilization by the addition of glucose for the fermentation of agar hydrolysate using Lactobacillus brevis ATCC 14869.

To investigate the application of carbon catabolite repression (CCR) relaxed Lactobacillus brevis ATCC 14869 in the utilization of agar hydrolysate to produce bioethanol and lactic acid through fermentation. As a single carbon source, galactose was not metabolized by L. brevis. However, L. brevis consumed galactose simultaneous to glucose and ceased cell growth after depletion of glucose. For complete use of galactose from agar hydrolysis, glucose need to beperiodically replenished into the growth medium. Overall, L. brevis successfully used agar hydrolysate and produced 17.2g/L of ethanoland 31.9g/Lof lactic acid. The maximum specific cell growth rate on galactose and glucose mixture was the same with the glucose-only medium at 0.12h-1. The molar product yields from glucose for lactic acid and ethanol were 1.02 and 0.95 respectively, equal to values obtained from the simultaneous utilization of glucose and galactose. In contribution to the ongoing efforts to utilize marine biomass, the relaxed CCR in Lactobacillus brevis ATCC 14869 was herein exploited to produce bioethanol and lactic acid from red seaweed hydrolysates.

Long-term cell suspension culture of Thymus persicus (Lamiaceae): A novel approach for the production of anti-cancer triterpenic acids

Triterpenic acids (TAs) such as betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA) are well-known natural compounds found in many plant taxa possessive a wide spectrum of biological activities and potential medicinal uses. Thymus persicus has been already reported as a rich source of TAs. Nowadays, long-term culture is an efficient technique in increasing biosynthesis and production of specialized metabolites in the plant cell suspension cultures with the possibility of stimulation, metabolic regulation, permeabilization, and up-scaling to bioreactors. In the present study, both batch and long-term cell suspension culture (CSC) of the plant were developed for the production of these compounds. A maximum specific cell growth rate of 5.7 g day−1, a doubling time of 42 h, and the highest cell viability (94.7%) throughout the growth period were measured. The highest cell fresh weight in CSC and its long-term culture was 100.63 ± 4.2 and 171.6 ± 4.1 g L−1 at day 21 and 35 of culture, respectively. A positive correlation between cell growth and the content of TAs in long-term culture was observed. The plant cells produced the highest levels of TAs in the mid-exponential phase and held their production stability during the long-term culture period. The highest content (mg 100 g−1 DW) of BA (163.11 ± 4.3) was measured in the second week of culture, while the maximum level of OA (10.22 ± 0.1) and UA (11.01 ± 0.4) was recorded in the third week of culture period. The present findings can be interestingly considered for further commercial production of valuable TAs at large-scale cultures in bioreactors.

Production of aminopeptidase from soybean meal with nutrients supplementation by Bacillus licheniformis SWJS33: Feasibility and metabolic process

Enhancing aminopeptidase fermentation using cheap substrate is very necessary for industrial production and application of aminopeptidase. The suitability of using raw soybean meal (SBM) as substrate for aminopeptidase production by Bacillus licheniformis SWJS33 in submerged fermentation was first evaluated. SBM alone provided both nitrogen and carbon sources for SWJS33 to produce aminopeptidase. Supplementation of 3 g/L glucose and 15 g/L yeast extract further improved the aminopeptidase yield by 0.63 and 1.3 folds, respectively. Addition of yeast extract altered the metabolic process of SWJS33, including shortening the lag phase, increasing the maximum specific cell growth rate and maximum specific aminopeptidase formation rate, and changing the utilization process of nutrients during the fermentation. What's more, SWJS33 secreted aminopeptidase prior to endoproteases when quick-release nitrogen (e.g. yeast extract) was supplied. High dissolved oxygen was demanded during aminopeptidase fermentation. The aminopeptidase production was successfully up scaled to 20 L fermenter.

Rhodosporidium sp. DR37: a novel strain for production of squalene in optimized cultivation conditions

BackgroundRhodosporidium strain, a well-known oleaginous yeast, has been widely used as a platform for lipid and carotenoid production. However, the production of squalene for application in lipid-based biofuels is not reported in this strain. Here, a new strain of Rhodosporidium sp. was isolated and identified, and its potential was investigated for production of squalene under various cultivation conditions.ResultsIn the present study, Rhodosporidium sp. DR37 was isolated from mangrove ecosystem and its potential for squalene production was assessed. When Rhodosporidium sp. DR37 was cultivated on modified YEPD medium (20 g/L glucose, 5 g/L peptone, 5 g/L YE, seawater (50% v/v), pH 7, 30 °C), 64 mg/L of squalene was produced. Also, squalene content was obtained as 13.9% of total lipid. Significantly, use of optimized medium (20 g/L sucrose, 5 g/L peptone, seawater (20% v/v), pH 7, 25 °C) allowed highest squalene accumulation (619 mg/L) and content (21.6% of total lipid) in Rhodosporidium sp. DR37. Moreover, kinetic parameters including maximum specific cell growth rate (μmax, h−1), specific lipid accumulation rate (qp, h−1), specific squalene accumulation rate (qsq, h−1) and specific sucrose consumption rate (qs, h−1) were determined in optimized medium as 0.092, 0.226, 0.036 and 0.010, respectively.ConclusionsThis study is the first report to employ marine oleaginous Rhodosporidium sp. DR37 for accumulation of squalene in optimized medium. These findings provide the potential of Rhodosporidium sp. DR37 for production of squalene as well as lipid and carotenoids for biofuel applications in large scale.Graphic abstract

High-cell-density cultivations to increase MVA virus production

Increasing the yield and the productivity in cell culture-based vaccine manufacturing using high-cell-density (HCD) cultivations faces a number of challenges. For example, medium consumption should be low to obtain a very high concentration of viable host cells in an economical way but must be balanced against the requirement that accumulation of toxic metabolites and limitation of nutrients have to be avoided. HCD cultivations should also be optimized to avoid unwanted induction of apoptosis or autophagy during the early phase of virus infection. To realize the full potential of HCD cultivations, a rational analysis of the cultivation conditions of the appropriate host cell line together with the optimal infection conditions for the chosen viral vaccine strain needs to be performed for each particular manufacturing process.We here illustrate our strategy for production of the modified vaccinia Ankara (MVA) virus isolate MVA-CR19 in the avian suspension cell line AGE1.CR.pIX at HCD. As a first step we demonstrate that the adjustment of the perfusion rate strictly based on the measured cell concentration and the glucose consumption rate of cells enables optimal growth in a 0.8 L bioreactor equipped with an ATF2 system. Concentrations up to 57 × 106 cells/mL (before infection) were obtained with a viability exceeding 95%, and a maximum specific cell growth rate of 0.019 h−1 (doubling time = 36.5 h). However, not only the cell-specific MVA-CR19 virus yield but also the volumetric productivity was reduced compared to infections at conventional-cell-density (CCD).To facilitate optimization of the virus propagation phase at HCD, a larger set of feeding strategies was analyzed in small-scale cultivations using shake flasks. Densities up to 63 × 106 cells/mL were obtained at the end of the cell growth phase applying a discontinuous perfusion mode (semi-perfusion) with the same cell-specific perfusion rate as in the bioreactor (0.060 nL/(cell d)). At this cell concentration, a medium exchange at time of infection was required to obtain expected virus yields during the first 24 h after infection. Applying an additional fed-batch feeding strategy during the whole virus replication phase resulted in a faster virus titer increase during the first 36 h after infection. In contrast, a semi-continuous virus harvest scheme improved virus accumulation and recovery at a rather later stage of infection. Overall, a combination of both fed-batch and medium exchange strategies resulted in similar cell-specific virus yields as those obtained for CCD processes but 10-fold higher MVA-CR19 titers, and four times higher volumetric productivity.

Mathematical modeling of continuous ethanol fermentation in a membrane bioreactor by pervaporation compared to conventional system: Genetic algorithm

In this paper, genetic algorithm was used to investigate mathematical modeling of ethanol fermentation in a continuous conventional bioreactor (CCBR) and a continuous membrane bioreactor (CMBR) by ethanol permselective polydimethylsiloxane (PDMS) membrane. A lab scale CMBR with medium glucose concentration of 100gL−1 and Saccharomyces cerevisiae microorganism was designed and fabricated. At dilution rate of 0.14h−1, maximum specific cell growth rate and productivity of 0.27h−1 and 6.49gL−1h−1 were respectively found in CMBR. However, at very high dilution rate, the performance of CMBR was quite similar to conventional fermentation on account of insufficient incubation time. In both systems, genetic algorithm modeling of cell growth, ethanol production and glucose concentration were conducted based on Monod and Moser kinetic models during each retention time at unsteady condition. The results showed that Moser kinetic model was more satisfactory and desirable than Monod model.

Isolation and growth kinetics of a novel phenol-degrading bacterium Microbacterium oxydans from the sediment of Taihu Lake (China).

Seven phylogenetically diverse phenol-degrading bacterial strains designated as P1 to P7 were isolated from the industry-effluent dump sites of an industrial area near Taihu Lake, China. Through the 16S rDNA sequence analysis, these strains were widely distributed among five different genera: Rhodococcus (P1), Pseudomonas (P2-P4), Acinetobacter (P5), Alcaligenes (P6), and Microbacterium (P7). All seven isolates were capable of growing with phenol as the sole carbon source. Strain P7 was found to be a novel phenol-degrading strain by detailed morphological, physiological and biochemical characteristic analysis as well as the 16S rDNA sequence analyses, and was named Microbacterium oxydans LY1 (M. oxydans LY1 in its short form). Degradation experiments of phenol at various initial concentrations (20-1,000 mg/L) revealed that phenol is an inhibitory substrate to M. oxydans LY1. In a batch culture experiment, more than 95% of the phenol (500 mg/L) was degraded by M. oxydans LY1 at 30°C, pH 7.0 and 120 rpm within 88 h. Phenol concentration higher than 200 mg/L was found to inhibit the bacterial growth. The growth kinetics correlated well with the Haldane model with μmax (maximum specific cell growth rate) = 0.243 h(-1), Ks (saturation constant) = 25.7 mg/L, and Ki (self-inhibition constant) = 156.3 mg/L. This is the first report of the ability of M. oxydans to degrade phenol, and the results could provide important information for bioremediation of phenol-contaminated environments.

Establishment and characterization of a Satureja khuzistanica Jamzad (Lamiaceae) cell suspension culture: a new in vitro source of rosmarinic acid.

An in vitro approach to the production of rosmarinic acid (RA), a medicinally important caffeic acid ester, in a cell suspension culture (CSC) of Satureja khuzistanica Jamzad (Lamiaceae) has been investigated for the first time. The CSC was established from friable calli derived from shoot tip explants in Gamborg's B5 liquid medium supplemented with 30g/L sucrose, 20mg/L L-glutamine, 200mg/L casein hydrolysate, 5mg/L benzyladenine (BA) and 1mg/L indole-3-butyric acid (IBA). The effect of nitrogen source (KNO3 and (NH4)2SO4) and their different concentrations on the fresh and dry weight (g/L), as well as RA content (mg/g dry weight) were measured. CSC growth measurements indicated a maximum specific cell growth rate of 1.5/day, a doubling time of 7.6days and a high percentage of cell viability (96.4%) throughout the growth cycle. Maximum cell fresh weight (353.5g/L), dry weight (19.7g/L) and RA production (180.0mg/g) were attained at day 21 of culture. Cell growth and RA content were affected by nitrogen deficiency. Media containing 8.3mM of total nitrogen (¼ of B5 standard medium) led to a minimum cell fresh weight (243.0g/L), dry weight (17.4g/L) and RA content (38.0mg/g) after 21 days. The established CSC provided useful material for further optimization experiments aimed at a large-scale production of RA.

Evaluation of performance of Planococcus sp. TRC1 an indigenous bacterial isolate monoculture as bioremediator for tannery effluent

An indigenous bacterial isolate Planococcus sp. TRC1 was found to tolerate Cr(VI) solution up to 500 mg/L concentration when grown in mineral salt media. Gene sequencing of the isolated strain using 16S rDNA technique and phylogenetic analysis confirmed that the species was 96% close to Planococcus maritimus (KP8). Studies on cell dynamics in batch bioreactor showed the maximum specific cell growth rate (μmax) to be 0.276 h−1. Removal of Cr(VI) was observed to be dependent on initial chromium concentration and the maximum removal of Cr(VI) (75 ± 3%) was obtained at 25 mg/L Cr(VI) solution for an incubation period of 72 h. Above this value, the removal of Cr(VI) was declined. Scanning electron microscope and Fourier transform infrared spectroscopic studies indicated that the Cr(VI) removal took place by the adsorption process on the cell outer membrane. It is observed that the adsorption process follows the Freundlich adsorption isotherm (R2 = 0.987). Planococcus sp. TRC1 reduced 80 ± 5% chemical oxygen demand (COD) of tannery effluent (COD 7,270 ± 45 mg O2/L) within 48 h of batch treatment. The elimination of cytotoxicity and genotoxicity imparted by the raw tannery effluent and Cr(VI) solution after bacterial treatment is an important observation which reflects the eco-friendly behavior of the Planococcus sp. TRC1 mediated tannery effluent and aqueous Cr(VI) solution treatment. The results revealed the applicability of Planococcus sp. TRC1 in both tannery waste and Cr(VI) reduction without affecting our environment.

Non-Structured Kinetic Model of Aspergillus niger Growth and Substrate Uptake in a Batch Submerged Culture

Aims: To investigate cell growth profile of Aspergillus niger in a batch submerged culture medium and then evaluation of cell kinetic behavior using some different non-structured kinetic models. Methodology: Experiments of cell growth and substrate utilization were conducted in batch submerged cultures with identified medium composition. Fitness assessment of experimental data on the cell growth and glucose consumption by models was performed using the curve-fitting tool in Mat Lab software. This report is the first in the kinetic investigation of Aspergillus niger PTCC 5010 with the studied models. This work was performed in Department of Chemical Engineering, Islamic Azad University, Qaemshahr Branch between April 2013 to September 2013. Results: Based on the obtained results; Moser kinetic model with R2 equal to 0.913 and Gompertz kinetic model with R2 equal to 0.949 were the best fitted models to describe the growth behavior of Aspergillus niger PTCC 5010 in the applied culture condition. Maximum specific cell growth rate with Moser and Gompertz kinetic models were 0.024 and 0.003h-1, respectively. Other kinetic constants for all studied models were also Original Research Article British Biotechnology Journal, 4(9): 970-979, 2014 971 determined at the applied culture conditions. The consistency of the experimental data with Monod, Verhulst and Contois kinetic models wasn't in an acceptable range. Conclusion: In scale up biochemical projects by Aspergillus niger PTCC 5010 for some industrial products, Moser and Gompertz kinetic models are able to demonstrate cell growth behavior and its substrate uptake profile. In continuous processes, dilution rate could be determined based on obtained maximum specific cell growth rate equal to 0.024 h-1.

Aerobic granulation of aggregating consortium X9 isolated from aerobic granules and role of cyclic di-GMP

This study monitored the granulation process of an aggregating functional consortium X9 that was consisted of Pseudomonas putida X-1, Acinetobacter sp. X-2, Alcaligenes sp. X-3 and Comamonas testosteroni X-4 in shaken reactors. The growth curve of X9 was fit using logistic model as follows y=1.49/(1+21.3*exp(-0.33x)), the maximum specific cell growth rate for X9 was 0.33 h(-1). Initially X9 consumed polysaccharides (PS) and secreted proteins (PN) to trigger granulation. Then X9 grew in biomass and formed numerous micro-granules, driven by increasing hydrophobicity of cell membranes and of accumulated extracellular polymeric substances (EPS). In later stage the intracellular cyclic diguanylate (c-di-GMP) was at high levels for inhibiting bacteria swarming motility, thereby promotion formation of large aerobic granules. The findings reported herein advise the way to accelerate granule formation and to stabilize operation in aerobic granular reactors.

Integrated utilization of grape skins from white grape pomaces

An approach for the integrated utilization of grape skins from white grape pomaces has been proposed. This consists of consecutive or simultaneous extraction of grape skins with neutral organic solvent and water under reflux. Organic extract is a valuable raw material for the isolation of oleanolic acid. The aqueous extract (ca 50%, w/w) is composed of essentially hexoses and suitable for the high yield (till 51%) bioethanol production at a maximum specific cell growth rate (μmax) of 0.29h−1. The remained extracted grape skins are the complex of structural polysaccharides embedded into cutinous matrix. Extracted grape skins were shown to be a prospective raw material for the production of low-density boards (d≤0.40) for insulation needs. The boards produced from grape skins and bind of 8% urea–formaldehyde resin revealed reasonable tensile strength (0.4MPa) and a low thermal conductivity (0.09–0.12W(mK)−1) over a wide range of temperatures (40–200°C).

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l−1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH4NO3 (24.7 mM), 3.0× KNO3 (56.4 mM), 3.0× MgSO4 (4.5 mM) and 3.0× KH2PO4 (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day−1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.

Xylitol production from DEO hydrolysate of corn stover by Pichia stipitis YS-30

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a D-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-∆1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. D-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose )l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

Cellulase production by continuous culture of Trichoderma reesei Rut C30 using acid hydrolysate prepared to retain more oligosaccharides for induction

An acid hydrolysate was prepared by a procedure chosen for retaining more oligosaccharides to improve the cellulase-inducing capability when used as substrate in the fungal fermentation for cellulase production. The effect was evaluated with continuous culture of Trichoderma reesei Rut C30 at the dilution rates of 0.03–0.08 h −1. The specific cellulase production rates were found to be relatively constant at 8.9 ± 0.3 (FPU/g dry cells-h), except for the lower rate, i.e., 7.2 (FPU/g-h), at the lowest dilution rate investigated (0.03 h −1). The former value was slightly higher than the rate obtained with a lactose-based medium, i.e., 8.2 (FPU/g-h). The maximum specific cell growth rate supported by the hydrolysate-based medium was 0.096 (h −1) and the apparent cell yield increased from 0.44 to 0.57 (g dry cells)/(g consumed reducing sugars) with increasing dilution rates. The best-fit maximum/ideal cell yield (without endogenous metabolism) was 0.68 (g/g), the endogenous substrate consumption rate was 0.023 (g reducing sugars)/(g dry cells-h), and the specific cell death rate was 0.016 h −1.

Kinetic response of a Drosophila melanogaster cell line to different medium formulations and culture conditions

In the past few years, Drosophila melanogaster cells have been employed for recombinant protein production purposes, and a comprehensive knowledge of their metabolism is essential for process optimization. In this work, the kinetic response of a Schneider S2 cell line, grown in shake flasks, in two different culture media, the serum-free SF900-II((R)) and the serum-supplemented TC-100, was evaluated. Cell growth, amino acids and glucose uptake, and lactate synthesis were measured allowing the calculation of kinetic parameters. The results show that S2 cells metabolism was able to adjust to different environmental situations, as determined by medium formulation, as well as by the particular situation resulting from the culture conditions. Cells attained a 163% higher final cell concentration (1.4 x 10(7) cells mL(-1)) in SF900 II((R)) medium, when compared to serum-supplemented TC-100 medium. Also, a maximum specific cell growth rate 52% higher in SF900 II((R) )medium, when compared to serum-supplemented TC-100 one, was observed. Glutamine was the growth limiting factor in SF900 II((R)) medium, while glucose, sometimes associated with glutamine, controlled growth in serum-supplemented TC-100 medium based formulation. The different pattern of lactate production is an example of the versatility of the metabolism of these cells. This by-product was produced only in glutamine limitation, but the amount synthesized depended not only on the excess glucose, but on other medium components. Therefore, in serum-supplemented TC-100 medium a much smaller lactate amount was generated. Besides, glucose was identified not only as a growth limiting factor, but also as a viability limiting factor, since its depletion accelerated cell death.

Wastepaper hydrolysate as soluble inducing substrate for cellulase production in continuous culture of trichoderma reesei

The enzymatic hydrolysate of wastepaper was evaluated for its cellulase-inducing capability and production characteristics in continuous culture of Trichoderma reesei RUT C30. Under the study conditions, i.e., pH 5.0, temperature 25 degreesC, and typical medium C:N ratio, the apparent cell yield constant was found to be 0. 76 (g of dry cell weight/g of reducing sugar), and the maximum specific cell growth rate was 0.26 h-1. The study on the effects of medium C:N ratio confirmed an important role of N sources in the cellulase synthesis. The cellulase production decreased significantly when the feed concentrations of N sources were reduced. An experiment at pH 7.5 with 4-fold N source concentrations also led to poorer cellulase production. When compared with cellulose, the wastepaper hydrolysate was found to have similar cellulase-inducing strength and to induce an apparently complete set of cellulase components. The hydrolysate was also concluded to be a better soluble inducer than sophorose. While comparable at a low dilution rate (0.012 h-1), the specific cellulase productivities of the hydrolysate-supported and the sophorose-induced systems exhibited opposite trends with increasing dilution rates. The specific productivity in sophorose-induced systems decreased with an increase in the dilution rate. On the other hand, with increasing dilution rate the specific productivity in the hydrolysate-supported systems increased from 2.2 FPU/g.h at D = 0.012 h-1 to 12.2 FPU/g.h at D = 0.122 h-1 before beginning to decline. The initial increasing trend was attributed to the higher concentrations of inducing oligomer intermediates at larger dilution rates.