Cell Mass Concentration Research Articles

Lipid production from sugar beet molasses under non-aseptic culture conditions using the oleaginous yeast Rhodotorula glutinis TR29

In this study the lipid production potential of the isolated yeast Rhodotorula glutinis TR29 in molasses medium under non-aseptic culture conditions was investigated. Different molasses concentrations and initial pH values were tested to make R. glutinis TR29 cells more dominant population in the medium, thereby preventing undesired microbial contaminants. Contamination could be prevented by selecting the high molasses concentration (20%) and low initial pH (5.0). When these parameters were kept constant, the optimum temperature, additional nitrogen source concentration and incubation time for the lipid production were found to be 25 °C, 4 g/L and 168 h, respectively. Under these culture conditions, the cell mass and lipid concentration were determined as 16.2 and 10.5 g/L, respectively. The lipid content was determined as 64.8%. The main cellular fatty acids of the yeast were oleic (63.5%), politic acid (15.4%), stearic acid (9.1%) and palmiteloic acid (7.2%). The yeast lipids seems to be a promising feedstock for biodiesel production due to a high content of C16 and C18 fatty acids. To our best knowledge, this is the first work on lipid production by yeasts in non-sterile molasses medium.

English

Isomaltulose is a low cariogenic sweetener used as a substitute for sucrose in the food industry. In this study, isomaltulose production by Serratia plymuthica ATCC 15928 was performed using free and immobilized cells. Response Surface Methodology was employed to evaluate the influence of temperature, wet cell mass concentration and sucrose concentration during the conversion of sucrose into isomaltulose by free cells. After 2 h of reaction time in shake flasks, a high production of isomaltulose (85.23%) was obtained with a temperature of 25oC, wet cell mass of 20% (w/v) and sucrose solution of 25% (w/v). The free cells were reused during seven successive batches and resulted in efficient isomaltulose conversion between 83.74 and 67.37%. The production of isomaltulose by immobilized cells in calcium alginate was studied in a packed bed bioreactor during seven days in a continuous process. A conversion yield of sucrose into isomaltulose between 81.26 and 70.89% was obtained, using immobilized cells in calcium alginate SynthO 2% (w/v), sucrose solution of 35% (w/v), wet cell mass of 30% (w/v) and temperature of 25oC. The conversion of sucrose into isomaltulose remained high using free cells and using immobilized cells in calcium alginate during the period of execution of the experiments. Key words: Isomaltulose, glucosyltransferase, free cells, immobilized cells, Serratia plymuthica.

Non-water miscible ionic liquid improves biocatalytic production of geranyl glucoside with Escherichia coli overexpressing a glucosyltransferase

Whole cells of Escherichia coli overexpressing a glucosyltransferase from Vitis vinifera were used for the glucosylation of geraniol to geranyl glucoside. A high cell density cultivation process for the production of whole-cell biocatalysts was developed, gaining a dry cell mass concentration of up to 67.6±1.2gL(-1) and a glucosyltransferase concentration of up to 2.7±0.1g protein L(-1) within a process time of 48h. Whole-cell batch biotransformations in milliliter-scale stirred-tank bioreactors showed highest conversion of geraniol at pH 7.0 although the pH optimum of the purified glucosyltransferase was at pH 8.5. The biocatalytic batch process performance was improved significantly by the addition of a water-immiscible ionic liquid (N-hexylpyridinium bis(trifluoromethylsulfonyl)imid) for in situ substrate supply. The so far highest final geranyl glucoside concentration (291±9mgL(-1)) and conversion (71±2%) reported for whole-cell biotransformations of geraniol were achieved with 5% (v/v) of the ionic liquid.

Effects of hydrogen partial pressure on autotrophic growth and product formation of Acetobacterium woodii.

Low aqueous solubility of the gases for autotrophic fermentations (e.g., hydrogen gas) results in low productivities in bioreactors. A frequently suggested approach to overcome mass transfer limitation is to increase the solubility of the limiting gas in the reaction medium by increasing the partial pressure in the gas phase. An increased inlet hydrogen partial pressure of up to 2.1bar (total pressure of 3.5bar) was applied for the autotrophic conversion of hydrogen and carbon dioxide with Acetobacterium woodii in a batch-operated stirred-tank bioreactor with continuous gas supply. Compared to the autotrophic batch process with an inlet hydrogen partial pressure of 0.4bar (total pressure of 1.0bar) the final acetate concentration after 3.1days was reduced to 50% (29.2gL(-1) compared to 59.3gL(-1)), but the final formate concentration was increased by a factor of 18 (7.3gL(-1) compared to 0.4gL(-1)). Applying recombinant A.woodii strains overexpressing either genes for enzymes in the methyl branch of the Wood-Ljungdahl pathway or the genes phosphotransacetylase and acetate kinase at an inlet hydrogen partial pressure of 1.4bar reduced the final formate concentration by up to 40% and increased the final dry cell mass and acetate concentrations compared to the wild type strain. Solely the overexpression of the two genes for ATP regeneration at the end of the Wood-Ljungdahl pathway resulted in an initial switch off of formate production at increased hydrogen partial pressure until the maximum of the hydrogen uptake rate was reached.

Impact of cyclic pH shifts on carbon monoxide fermentation to ethanol by Clostridium autoethanogenum

Fermentation medium and fermentation pH are among the main parameters that control acidogenesis and solventogenesis in clostridia during CO bioconversion. The approach of cyclic pH shifts from high (pH 5.75) to low (pH 4.75) levels, in continuous gas-fed bioreactors, allowed Clostridium autoethanogenum to consume and convert CO and the produced acetic acid from CO to alcohols using one single bioreactor. Increasing amounts of ethanol accumulate after each pH shift cycle, while acids are used up and converted during each cycle. In this study, a total of three cyclic pH shifts along with partial medium replenishment, resulted in a high ethanol production, reaching a concentration of 7143mg/L. The mixotrophic fermentation using xylose as additional carbon source besides carbon monoxide allowed to conclude that xylose is a preferred carbon source compared to CO and that it allows to increase the cell mass concentration (421mg/L).

Hexavalent Chromium Reduction by Microbacterium oleivorans A1: A Possible Mechanism of Chromate -Detoxification and -Bioremediation.

Hexavalent chromium (Cr(6+)) contamination is one of the mejor problems of environmental protection for its carcinogenic effect on human health. Remediation of Cr(6+) contaminated environment thus becomes highest priority. A Gram positive tiny rod shaped chromate (Cr(6+)) reducing bacterium strain A1 was isolated from uranium ore collected from Jaduguda, East Singhbhum, Jharkhand. The strain was identified and characterized in terms of its potential to reduce more toxic Cr(6+) to its less toxic form for its application in bioremediation of Cr(6+) contaminated environments. 16S rRNA gene based phylogentic analysis identified the strain as Microbacterium oleivorans. Along with Cr(6+), the strain showed resistance to other heavy metals including Ag, Cu, Co, Hg, Ni and Zn also. Complete reduction of Cr(6+) (750 µM) was achieved within 84 h with optimum reduction at pH 9 and 30°C. Effect of different parameters including cell mass concentration, pH, induction with Cr(6+), SO4 = ion, heavy metals, etc. on Cr(6+) reduction were studied thoroughly at resting cell condition to study its potential towards Cr(6+) bioremediation. Chromate reductase gene (chrA) was detected within this strain. Along with presence of appropriate genetic determinant, efficient Cr(6+) reduction ability of the strain indicated its potential for developing redox based Cr(6+) remediation system for varied concentrations of Cr6+ under a wide range of environmental conditions. Patent data have suggested the efficient application of Cr(6+) reducing bacteria in cleaning up of Cr(6+) contaminated environments.

Validation of a predictive model for fed-batch and continuous lipids production processes from acetic acid using the oleaginous yeast Cryptococcus curvatus

This study focuses on the use of acetic acid as carbon source for cell mass and lipid production using an oleaginous yeast, Cryptococcus curvatus. To improve this cell mass and lipid production on acetic acid, a numerical model was built to predict the yeast behavior. The model was first calibrated and validated on a fed-batch culture of the oleaginous yeast grown on acetic acid. After the validation step, this model was used as a predictive tool to search for optimal dilution rate and C/N ratios for continuous culture. A continuous culture was then launched using these culture parameters, revealing that the experimental data fitted the model predictions. At steady-state, cell mass concentration was stabilized at 26.7g/L containing 48–53% of lipid content. A maximal lipid productivity of 0.54gL−1h−1 was obtained, which was superior to those reported in literature. This model could be qualified as a mechanistic one since it allows understanding reactions underlying the lipid production process. The experimental data fitted the model predictions, which confirmed that the use of a model is a powerful and predictive tool which could help the operator to process parameters to improve the culture.

Computer Aided Design and Performance Analysis of Inverse Fluidized Bed Biofilm Reactors with Special Reference to Bioplastic Synthesis

Poly Laevo Lactic Acid (PLLA), in spite of being an excellent bioplastic, has exorbitantly high market price due to the high cost of raw material (lactose, glucose, sucrose). Hence, its manufacture is being attempted starting from waste effluents such as cheese whey and molasses. Earlier studies on the same in fluidized bed and semifluidized bed biofilm reactors yielded encouraging results. The present study therefore involves design and analysis of inverse fluidized bed biofilm reactors for lactic acid synthesis. The performance features of the bioreactor have been studied both mathematically as well as experimentally. The inverse fluidized bed biofilm reactor has been found to provide more than 75% conversion of sucrose/lactose even at high capacities (high feed flow rates) exceeding 56,000 L/hr, within a reasonably low reactor volume. The fractional substrate conversion increases, though sluggishly, with increase in feed flow rate due to bed expansion and also with increase in cell mass concentration in biofilm due to enhancement in intrinsic rate of bioconversion. The inverse fluidized bed biofilm reactor of proposed design could be safely recommended for the commercial synthesis of polymer grade lactic acid from waste effluents such as cheese whey and molasses. The low operating cost of the bioreactor (due to downflow mode of operation) enhances the economy of the process. This would also help in significantly lowering the market price of the green plastic (PLLA) and shall promote its large scale manufacture and utilisation.

Ferulic acid transformation into the main vanilla aroma compounds by Amycolatopsis sp. ATCC 39116.

The wild strain Amycolatopsis sp. ATCC 39116 was explored in ferulic acid-based media to produce naturally the aroma components of the cured vanilla pod, namely vanillin,vanillic acid, and vanillyl alcohol. Other phenolic compounds(4-vinyl guaiacol, guaiacol, and protocatechuic acid) were also evaluated. The influence of medium composition,fermentation technology (batch or fed-batch), supplementation with vanillic acid, and inoculum concentration on ferulic acid biotransformation were evaluated. The results postulate the initial concentration of cell mass as the variable with the strongest impact on ferulic acid metabolization under the studied conditions. The highest amounts of vanillin and vanillic acid were achieved at intermediate values of cell mass.Vanillyl alcohol and protocatechuic acid were more closely linked to high cell mass concentrations. Conversely, 4-vinyl guaiacol reached its highest amount at the lowest amount of cell mass. Guaiacol was not detected in any case. Therefore,the initial cell concentration must be considered a critical parameter when using Amycolaptosis sp. ATCC 39116 for the production of vanillin and related compounds.

Continuous ethanol production from sugarcane bagasse hydrolysate at high temperature with cell recycle and in-situ recovery of ethanol

A continuous process with cell recycle was demonstrated on fermentation of sugarcane bagasse hydrolysate to ethanol having a facility of in-situ stripping of ethanol by air or nitrogen gas. The thermotolerant yeast Kluyveromyces sp. IIPE453, grown in xylose-rich hydrolysate, was used in this study for fermenting the glucose-rich hydrolysate at 50°C. The effect of dilution rate, cell mass concentration, and air or nitrogen gas stripping on ethanol production was evaluated. The maximum volumetric productivity of ethanol was 1.22gl−1h−1 on stripping the bioreactor with nitrogen at a dilution rate of 0.075h−1. An ethanol yield of 96% of its theoretical value and 95% sugar conversion efficiency could be obtained while the ethanol concentration in the condensate of the stripper was found to be 56.3gl−1, which was almost 3.4 times concentrated as compared to ethanol concentration in the broth without stripping. The specific ethanol productivity could be improved due to increase in sugar conversion efficiency and higher ethanol yield in a continuous cell recycle fermentation system applying gas stripping at high temperature.

Continuous gas fermentation by Acetobacterium woodii in a submerged membrane reactor with full cell retention

Acetogenic bacteria like Acetobacterium woodii represent an ancient group of anaerobic microorganisms which use hydrogen and carbon dioxide to produce acetate. Cell concentrations and space–time yields are usually low in gas fermentations. A standard stirred‑tank bioreactor with continuous gas supply was equipped with a customized submerged microfiltration unit. A. woodii showed similar growth behavior with an initial maximal growth rate of 1.2d−1 in continuous gas fermentations with full cell retention and varying dilution rates. A steady increase of cell mass concentrations was observed with the highest biomass formation at the highest dilution rate. By contrast the final acetate concentrations were lowest at the highest dilution rate. The highest final acetate space–time yield of 148gl−1d−1 was measured at the highest dilution rate (increase by factor 8 compared to a standard batch process or by factor 37 compared to published data). The highest reported cell concentration of A. woodii in gas fermentations of nearly 14gl−1 cell dry weight was achieved in the submerged membrane bioreactor with increased yeast extract concentrations in the feed medium. Product inhibition was observed when acetate concentrations exceeded 8–12gl−1 causing a steady decrease in cell mass specific acetate production rates.

Production of butyric acid by Clostridium tyrobutyricum (ATCC25755) using sweet sorghum stalks and beet molasses

Enzymatically liquefied sweet sorghum stalks and beet molasses were evaluated for butyrate production using Clostridium tyrobutyricum in 1L scale fed-batch fermentations. The hydrolysates used for the fermentations were prepared separately by liquefying the sorghum stalks at 50°C, pH 5.0 for 18h, with 30% (w/v) DM content using the enzyme preparation Cellic® CTec2 at an activity of 26.5FPU/gDM. To enhance sucrose consumption, the fermentations were supplemented with invertase at an activity equivalent to 8.3U/gDM. With the hydrolysate as the feedstock, a butyrate concentration of 37.2±0.8g/L, a productivity of 0.86±0.02g/Lh and a yield of 0.39±0.02g/g (p=0.05) consumed sugars were obtained. Finally, a butyrate concentration of 58.8g/L, a productivity of 1.9g/Lh, a butyrate yield of 0.52g/g consumed sugars and a dry cell mass concentration of 15.7g/L were obtained with fed-batch cultivation and a constant feed consisting of 64% sorghum hydrolysate juice and 36% molasses. Evidence for inducible saccharolytic activity was also proven, as the cellulase activity in the culture supernatant was found more than double during feed with limiting sugar concentrations. The present study clearly demonstrates that combinations of low cost raw materials can be used for efficient butyrate production, also without cell immobilization.

Effect of light intensity on the correlation between cell mass concentration and optical density in high density culture of a filamentous microorganism

We investigated the effect of light intensity on the correlation between optical density (OD) and cell mass concentration in high cell density culture of Anabaena variabilis. When the light intensity was gradually increased to maintain specific irradiation rate above 10 mmol/s/g dry cell, the dry cell mass concentration to OD ratio changed when the cell density became higher than OD 10, while the correlation was linear when light intensity remained constant after 2.5 days. When the OD was below 10, the dry cell concentration to OD ratio in unicells was the same as that of filaments, indicating that filament length, unicell size, and unicell dry cell concentration did not affect the dry cell concentration to OD ratio. Scanning electron microscope pictures revealed the differences in morphological structures between filaments below and above OD 10. Therefore, we propose that the morphological structures of filaments affected the dry cell concentration to OD ratio.

English

The present work attempts to investigate the ability of fusariumoxysporum and neurosporacrassa to ferment cellulose to ethanol directly. Batch experiments were performed for both the strain at 30.c in 250ml conical flasks.1000ml of production medium was distributed equally in 7 sterile,250 ml conical flasks, each containing various initial concentrations of cellulose 20,30,40,50,60,70 and 80g/l respectively.20ml of inoculum medium was transferred to each 100ml production medium in sterile conditions. Initial values of ethanol concentration, cellulose concentration and cell mass were determined. The inoculated flasks were maintained at 300 C in a rotary shaker operating at 200 rev min-1.Analyses were carried out for every 24 hours using the contents of the various conical flasks.Experiments were conducted for mixed cultures of different compositions 80:20, 60:40, 40:60, and 20:80by volume respectively.The cultures fusariumoxysporum and neurosporacrassa were mixed in proportion 80:20,60:40,40:60,and 20:80 respectively mixed cultures were used for inoculation.Based on the kinetic models, equations have been developed for the determination cell mass, product and substrate concentration as a function of time. Using the experimental data, constants of these equations have been evaluated. These mathematical relationships can be used with reasonable accuracy.

Cell mass energetic yields of fed-batch culture by Lipomyces starkeyi.

Estimation of the energy capacity of a microbial cell mass on the basis of its lipid content and elemental composition can be used for the comparative evaluation of different microbial sources of biodiesel. Lipomyces starkeyi cell mass concentration reached 94.6 g/L with 37.4% of lipids in a fed-batch process using xylose and urea as substrates. The fatty acid composition of the yeast oil was quite similar to that of palm oil. L. starkeyi converted more than 80% of the energy contained in xylose into cell mass energy yield. The approach used in this study makes it possible to determine the energy of a cell mass by its elemental composition. A heat of combustion (Q c) of 25.7 (kJ/g) was obtained for the cell mass after 142 h of fed-batch cultivation, which represents approximately 56% of the energy content of diesel oil (45.4 kJ/g). The Q c of the triacylglycerols produced was 48.9 (kJ/g), indicating the potential of this oleaginous yeast for biodiesel production. Our work developed here provides a simple and efficient tool for characterization of this cell mass to further our understanding of its use as a feedstock for bioenergy production.

Ultrasound enhanced ethanol production from Parthenium hysterophorus: A mechanistic investigation

This study presents mechanistic investigations in ultrasound-assisted bioethanol fermentation using Parthenium hysterophorus biomass. Ultrasound (35kHz, 10% duty cycle) has been used for sonication. Experimental results were fitted to mathematical model; the kinetic and physiological parameters in the model were obtained using Genetic Algorithm (GA) based optimization. In control experiments (mechanical shaking), maximum ethanol titer of 10.93g/L and cell mass concentration of 5.26g/L was obtained after 18h. In test experiments (mechanical shaking and intermittent sonication), ethanol titer of 12.14g/L and cell mass concentration of 5.7g/L was obtained in 10h. This indicated ∼2× enhanced productivity of ethanol and cell mass with sonication. Trends in model parameters obtained after fitting of model to experimental data essentially revealed that beneficial influence of ultrasound on fermentation is a manifestation of enhanced trans-membrane transportation and dilution of toxic substances due to strong micro-convection induced by ultrasound.

Infl uence of selenium deficiency on neural tube defects

Objective: As for the role of selenium on human fetal development, a little data is available in literature. The purpose of this manuscript was to study the influence of selenium on neural tube defects. This study will be helpful in planning strategies for prevention of neural tube defects.Methodology: After collection of the venous blood the same was immediately centrifuged and after immediate centrifugation, the clear serum was transferred to deionized plastic vials, stored and frozen at –20º C until determination of the analysis was carried out. The selenium levels as ng/ml were determined on GBC 932 spectrophotometer by fluorometery.Results: The mean maternal blood serum and cell mass concentrations in NTD group (306.4 ± 10.95 ng/ml, 192.44 ± 6.12 ng/ml, 165.8 ± 16.99 ng/g respectively) were significantly lower than those of control mothers (363.75 ± 17.1 ng/ml, 242.34 ± 15.7 ng/ml, 260.0 ± 20.57 respectively). A significant decrease in concentration of selenium in newborns with NTD (298.4 ± 12.3 ng/ml, 96.3 ± 7.15 ng/ml, 139.8 ± 27.5 ng/g respectively) as compared with healthy babies (358.1 ± 16.11 ng/ml, 122.44 ± 6.03 ng/ml, 268.6 ± 31.37 ng/g respectively).Conclusion: Selenium deficiency in mothers during pregnancy thought to be one of the factors responsible for NTDs. However, the lowered selenium concentrations in blood, serum and cell mass can be secondary cause of an abnormal pregnancy and didn’t contribute to its production. More investigations on selenium status in mothers during antenatal period, especially in prenatal development and antenatal selenium status including normal babies and NTD babies are required.DOI: http://dx.doi.org/10.3126/ajms.v6i2.11151Asian Journal of Medical Sciences Vol.6(2) 2015 62-65

Assessment of pectinase production by Aspergillus oryzae in growth-limiting liquid medium under limited and non-limited oxygen supply

The cultivation of Aspergillii in liquid media is often hindered by the intense consumption of oxygen by the fungi. In this work, pectinase production by Aspergillus oryzae IPT-301 was assessed in cultures grown with limiting and non-limiting oxygen supplies to evaluate the effects of pH and the concentration of dissolved oxygen on the process. Different strategies – such as the use of a growth-limiting medium and the delayed addition of inducer (citrus pectin) to the bioreactor – were employed to provide an effective oxygen supply to the microorganisms and to avoid excessively high impeller speeds that could damage the fungal hyphae. In a limited oxygen environment, it was observed that the maintenance of a constant pH value of 4.0 throughout the cultivation resulted in high cell mass concentration but no enzyme activity. However, when the pH was kept at 4.0 only during the period of intense cell growth followed by sustained control at pH 2.7, significant enzyme activity was detected (43UmL−1). It was observed that this low pH favors both the production of enzyme and their stability. Under a non-limiting dissolved oxygen concentration, over 30% of saturation, the delayed pH control approach resulted in the maximum pectinase activity of 120UmL−1.

Microbial transformation of quinic acid to shikimic acid by Bacillus megaterium

Abstract Background Biotransformation of quinic acid to shikimic acid was attempted using whole cells of Bacillus megaterium as a biocatalyst. Results Physico-chemical parameters such as temperature (37°C), pH (7.0), agitation (200 rpm), substrate (5 mM) and cell mass concentrations (200 kg/m 3) and reaction time (3 h) were found optimum to enhance the bioconversion. Maximum conversion (89%) of quinic acid to shikimic acid was achieved using the above optimized parameters. Shikimic acid was extracted from the reaction mixture by a pH-dependent method and maximum recovery (76%) was obtained with petroleum ether. Conclusions Biotransformation of quinic acid to shikimic acid seems to be a better alternative over its fermentative production.

Sustainable autotrophic production of polyhydroxybutyrate (PHB) from CO2 using a two-stage cultivation system

The technical feasibility of Cupriavidus necator DSM 545 for sustainable autotrophic polyhydroxybutyrate (PHB) production from CO2 using a two-stage cultivation system was evaluated. In this cultivation method, cell mass growth occurred under heterotrophic conditions using two different organic substrates, namely glucose and waste glycerol. In both cases, PHB biosynthesis was triggered by applying nitrogen and oxygen limitation at three different cell mass concentrations under autotrophic conditions using a gas mixture of H2, O2 and CO2. To ensure that the test conditions were relevant for later industrial application, O2 concentration was kept below the safety value during autotrophic PHB production. PHB production from CO2 on waste-glycerol grown cell mass resulted in a PHB concentration of 28g/L, which is the highest reported value in literature for PHB synthesized from CO2 at an O2 concentration below the lower explosion limit of 5vol%. The fermentation performance decreased when nutrient limitation was delayed at higher cell mass concentrations. Furthermore, it was shown that PHB production from CO2 at high cell mass concentration is metabolically feasible, but under the tested conditions the mass transfer of O2 was limiting PHB accumulation. Characterization of the produced polymers showed that the organic carbon source affected the properties of PHB and that the cultivation method developed in this study provided PHB with properties similar to a commercial PHB and PHB typically found in literature. It can be concluded that heterotrophic–autotrophic production of PHB by C. necator is a promising cultivation method to reduce the overall production cost of PHB. In order to compete with the current heterotrophic cultivation system, the oxygen transfer rate must however be enhanced to achieve a higher PHB productivity.