Initial Cell Concentration Research Articles

Ultrasonic disruption of yeast cells: Underlying mechanism and effects of processing parameters

During the ultrasonic treatment of yeast cells, the damage to the cell walls and membranes was monitored by assessing the release of cell wall polysaccharides and intracellular proteins, respectively. At a low acoustic intensity (10 W/cm2), the polysaccharides were released faster than the proteins, whereas at higher acoustic intensities (24 and 39 W/cm2), this trend was reversed. At elevated temperatures, additional cell wall polysaccharides were released, whereas fewer intracellular proteins were released. Increasing processed volumes, initial cell concentrations, and salt concentrations led to the decrease of both fractions. However, the total releases per ultrasound treatment remained constant regardless of the processed volumes, and increased with the increase of initial cell concentrations. The results suggest that the ultrasonic disruption of yeast cells begins with the breakdown of the cell wall before continuing to the cell membrane. These findings may offer new avenues for exploring more efficient cell disruption or microbial inactivation processes. Ultrasonic technology has been intensively studied for microbial inactivation or cell disruption. It is believed that the inner cell membrane is the target of the ultrasonic damage. The mechanism of microbial inactivation via ultrasound involves the thinning of the cell membranes. In this study, we monitored the ultrasonic damage to yeast cell walls and membranes by monitoring the release of cell wall polysaccharides and intracellular proteins, respectively. Our results demonstrate that the ultrasonic disruption of yeast cells begins with the breakdown of the cell wall before continuing to the cell membrane. Increasing the temperature weakens the cell wall and thermally coagulates the intracellular proteins. Increasing the processed volumes, initial cell concentrations, and salt concentrations reduces the releases of cell wall polysaccharides and intracellular proteins from the viewpoint of yeast cells. However, the total releases of each fraction per ultrasound treatment remain constant regardless of the processed volumes, and increase with increasing initial cell concentrations. Our results may offer new insights towards the exploration of more efficient industrial processes for microbial inactivation or cell disruption, and in elucidating the effects of processing parameters.

Alcoholic Fermentation with Self‐Flocculating Yeast in a Tower Upflow Reactor

AbstractEthanol production using self‐flocculating yeast in a batch tower upflow reactor system operating with a recirculation loop was examined. Ethanol productivity, yield, and residual sucrose concentration were evaluated experimentally according to a central composite design with initial cell and sucrose concentrations and recirculation flow rate as independent variables. Yeast cell concentration strongly influenced the reactor performance. Alcoholic fermentation was conducted using this strain and reactor configuration which allowed for high productivity and high sucrose conversion. The ethanol yield was comparable with industrial yields. A kinetic study of the fermentation process under optimized conditions was performed using the experimental data and considering inhibition by sucrose and ethanol.

Evaluation of metal biouptake from the analysis of bulk metal depletion kinetics at various cell concentrations: theory and application.

Bioavailability of trace metals is a key parameter for assessment of toxicity on living organisms. Proper evaluation of metal bioavailability requires monitoring the various interfacial processes that control metal partitioning dynamics at the biointerface, which includes metal transport from solution to cell membrane, adsorption at the biosurface, internalization, and possible excretion. In this work, a methodology is proposed to quantitatively describe the dynamics of Cd(II) uptake by Pseudomonas putida. The analysis is based on the kinetic measurement of Cd(II) depletion from bulk solution at various initial cell concentrations using electroanalytical probes. On the basis of a recent formalism on the dynamics of metal uptake by complex biointerphases, the cell concentration-dependent depletion time scales and plateau values reached by metal concentrations at long exposure times (>3 h) are successfully rationalized in terms of limiting metal uptake flux, rate of excretion, and metal affinity to internalization sites. The analysis shows the limits of approximate depletion models valid in the extremes of high and weak metal affinities. The contribution of conductive diffusion transfer of metals from the solution to the cell membrane in governing the rate of Cd(II) uptake is further discussed on the basis of estimated resistances for metal membrane transfer and extracellular mass transport.

Filtration methodologies for the clarification and concentration of human mesenchymal stem cells

Currently human mesenchymal stem cells (hMSC) are expanded using microcarrier-based stirred culture systems from one to hundreds of liters of culture volume to guarantee the required cell numbers to be delivered to the clinic. Such culture volumes need to be clarified, ensuring efficient removal of microcarriers, and concentrated without compromising the cells׳ characteristics. The aim of this work was to evaluate the applicability of filtration methodologies, as dead end filtration and tangential flow filtration, for the clarification and concentration of hMSC, respectively.Different process variables and their impact on hMSC quality were evaluated, showing that polypropylene filters with pore sizes higher than 75μm can ensure the removal of microcarriers from the cell suspension bulk, without compromising cells׳ recovery or viability. Furthermore, hMSC could be successfully concentrated up to a factor of ten while maintaining their identity, potency and high cell viability, allowing for the recovery of over 80% of viable cells; an initial cell concentration higher than 2×105 cell/mL, and polysulfone membranes with pore sizes higher than 0.45μm were identified to be key conditions to obtain such concentration factors; shear rate and permeate flux were also shown to impact the cells׳ recovery yields, viability and quality.

Improvement of Sporulation Conditions of a New Strain of <i>Bacillus amyloliquefaciens</i> in Liquid Fermentation

The desirable active ingredients for the development of bioproducts based in Bacillus sp. for the control of soil pathogens are the spores because these structures exhibit more resistance and stability to conditions present during the fermentation, formulation, and storage processes. To improve the sporulation of a native strain of Bacillus amyloliquefaciens (Bs006) using liquid fermentation, some modifications in the concentrations of the components in a previously standardized culture media were made. Subsequently, five sporulation inducers (iron nitrate, mixture of salts, peroxide hydrogen, temperature, and initial cell concentration) were evaluated. The treatment with a mixture of salts in combination with an initial cell concentration of 1 × 108 cells/ml was selected because a final spore concentration of 1.05 × 1010 spores/ml after 66 hours with a fully substrate consumption and sporulation efficiency of 88.7% was obtained. To demonstrate the biological activity of B. amyloliquefaciens Bs006 in Cape gooseberry seedlings, a greenhouse bioassay was conducted, and statistical differences in plant growth-promoting parameters compared with previous media were not found. Additionally, the proposed modified media (coded as JM) presented a benefit-cost ratio 2.65 times higher compared with the baseline media.

好酸性・好熱性古細菌<i>Acidianus Brierleyi</i> による 海底熱水鉱床鉱石のバイオリーチング<b> </b>

This article describes the bioleaching of deep-sea hydrothermal ore deposits by the acidophilic and thermophilic archaeon Acidianus brierleyi at 65℃. Bioleaching experiments with A. brierleyi were used to examine the dependence of leaching rates on three process parameters such as solution pH (pH 1.2–2.0), initial cell concentration (1×1013–1×1014 cells/m3), and initial ore-liquid loading ratio (5–40 kg/m3). The leaching of copper and zinc was markedly accelerated in the presence of A. brierleyi, and greater than 80 % extraction of copper and zinc in the ore sample (38–53 μm) was achieved at 65℃ within 10 days of batch operation. Nearly identical bioleaching rates of zinc and copper were obtained for the deep-sea hydrothermal sulfide (ore sample A) and terrestrial sulfide concentrates (sphalerite and chalcopyrite) by the thermophilic archaeon A. brierleyi. However, the bioleaching of lead was practically negligible because of precipitation of insoluble anglesite. The bioleaching of gallium tended to be suppressed as the initial ore-liquid loading ratio was increased from 5 to 40 kg/m3. The bioleaching of the ore sample (45.4 % SiO2) yielded less than 5 % extraction of gallium in 10 days of batch operation at an initial ore-liquid loading ratio of 40 kg/m3, and gallium in the bioleaching residue was concentrated 1.8-fold of its initial content. Moreover, the concentration of gold in the bioleaching residue was increased by a factor of 1.9 after 10 days of batch operation. It can be concluded that bioleaching is an attractive processing route to recover valuable metals from deep-sea hydrothermal ore deposits.

Statistical Modeling of Environmental Factors on Microbial Urea Hydrolysis Process for Biocement Production

Calcium carbonate is a widely used raw material by many industries. It can be precipitated through microbial process within soil pores as cementitious bonding agent between grains for geotechnical applications. It is called microbially induced calcium carbonate precipitation (MICP). Designing an appropriate biogrout material for injection into soil is essential for controlling the amount, type, time, and place of the biocement production within pores. For this purpose, understanding the active reactions and the kinetics of bacterial growth and urea hydrolysis is necessary. A conductometric method and spectrophotometry were used in this study to, respectively, monitor the urea hydrolysis reaction progress and bacterial growth inS. pasteurii-inoculated urea-NB-NH4Cl solution at different level of the environmental factors that are initial cell concentration, urea concentration, and temperature. Variation in conductivity of the solution versus logarithmic scale of time was depicted as microbial ureolysis characteristic curve (MUCC) through which lag duration, specific rate, and potential of urea hydrolysis at each condition were obtained. Central composite face-centered (CCF) design, which is one of the response surface methodologies, was employed to statistically fit polynomial models explaining the bacterial growth and the characteristics obtained from MUCCs in terms of the environmental factors and their interactions. An optimization analysis based on the urea-normalized responses was also carried out.

Critical Assessment of the Time-to-Detection Method for Accurate Estimation of Microbial Growth Parameters

The time-to-detection (TTD) method is a rapid and high throughput approach for the estimation of microbial growth parameters (maximum specific growth rate μmax and lag phase duration λ), which relies on optical density (OD) measurements. The performance of this method depends on several factors that are often selected in an arbitrary way. In this work, a sensitivity analysis was performed to assess the effect of several key factors on the resulting output data of this method with Listeria monocytogenes. The factors showing higher influence on the results include (1) the calibration curve relating viable plate counts and OD data; (2) the approach to estimate TTD values; (3) the detection limit of OD measurements; and (4) the range of the initial cell concentrations considered (Ni). In general, lag phase (λ) estimates were more sensitive than maximum specific growth rate (μmax) estimates. The approach to estimate TTD values and the OD detection limit was the most influential factors for the μmax and λ estimation. This work has illustrated that, despite all the advantages of the TTD method, there are crucial steps in experimental design and data processing that significantly influence its output in terms of lag phase duration and maximum specific growth rate. Practical Applications This study contributes to understanding which are the most influential factors on the output of time-to-detection (TTD) method. These factors can be considered throughout different steps of the method, experimental design, data processing and growth parameter estimation. Guidelines are provided about how the selection of the factors assessed should be made and which variables – i.e., research scope, materials and equipment available for experimental and analytical steps – should be considered. In addition, some limitations of the method regarding the range and quality of the experimental data when implementing an automated global fitting are described. Overall, the performance of the TTD method and the accuracy of microbial growth parameters estimation will be improved.

Control of the Ratio of Inducer to Cell Concentration to Enhance the Phytase Production in Recombinant <i>Pichiapastoris</i>

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.

Investigation of fermentation conditions for teff (Eragrostis tef) malt-wort by Lactobacillus amylolyticus

Teff is nutritious and gluten-free low risk cereal with the potential as an alternative raw material for gluten-free foods and beverages. The aim of the research was to investigate optimal fermentation conditions for teff malt-wort to be fermented by Lactobacillus amylolyticus. The experimental design and statistical analysis were performed using Design Expert statistical software. High lactic acid formation was observed between 42 and 48 °C. An increase and a decrease in temperature beyond the stated range caused a decrease in the formation of lactic acid. Temperature was the dominant factor influencing the three response variables viz. lactic acid to sugar ratio, lactic acid, and pH. However, the main factor influencing extract was time followed by cell concentration. The optimum conditions of the parameters such as temperature, initial pH, initial cell concentration, and fermentation time were 42 °C, 5.4, 1.86 × 105 cells/ml, and 52 h, respectively. With this set of condition, a beverage with pH 3.5, lactic acid concentration 9.5 g/L, and lactic acid to sugar ratio 0.26 was predicted to be produced. The validation experimental runs demonstrated a good agreement between the experimental and predicted values. The sensory evaluation of the product also shows that it was accepted by the assessors.

Microalgal cell disruption via ultrasonic nozzle spraying.

The objective of this study was to understand the effect of operating parameters, including ultrasound amplitude, spraying pressure, nozzle orifice diameter, and initial cell concentration on microalgal cell disruption and lipid extraction in an ultrasonic nozzle spraying system (UNSS). Two algal species including Scenedesmus dimorphus and Nannochloropsis oculata were evaluated. Experimental results demonstrated that the UNSS was effective in the disruption of microalgal cells indicated by significant changes in cell concentration and Nile red-stained lipid fluorescence density between all treatments and the control. It was found that increasing ultrasound amplitude generally enhanced cell disruption and lipid recovery although excessive input energy was not necessary for best results. The effect of spraying pressure and nozzle orifice diameter on cell disruption and lipid recovery was believed to be dependent on the competition between ultrasound-induced cavitation and spraying-generated shear forces. Optimal cell disruption was not always achieved at the highest spraying pressure or biggest nozzle orifice diameter; instead, they appeared at moderate levels depending on the algal strain and specific settings. Increasing initial algal cell concentration significantly reduced cell disruption efficiency. In all UNSS treatments, the effectiveness of cell disruption and lipid recovery was found to be dependent on the algal species treated.

Optimizing cultivation strategies for robust algal growth and consequent removal of inorganic nutrients in pretreated livestock effluent.

Dilution was employed as a pretreatment strategy to increase light transmittance and decrease ammonia toxicity in piggery effluent prior to the cultivation of microalgae. The dilution effect was quantitatively determined based on both the maximum specific nutrient consumption rate and the maximum growth coefficient to minimize the usage of diluent. The biomass productivity of microalgae was also evaluated to select the best species among the five different candidates examined. A 20-fold dilution of piggery wastewater resulted in decreased chromaticity (584mgPt-CoL(-1)) and total nitrogen (76mgL(-1)), on which the microalgae cultivation was more effective for an algal growth compared to the other dilution factors. If the initial cell concentration of Scenedesmus quadricauda increased, the production of biomass tended to improve. Robust growth and harvesting of S. quadricauda were achieved, and the associated consistent removal of inorganic nutrients was accomplished during the semi-continuous cultivation of the best species.

Novel Filtration Photobioreactor for Efficient Biomass Production

A filtration photobioreactor (FPBR) has been designed in this study. It requires a much shorter time to start up than existing biofilm systems, while providing easy biomass harvest. Microalgae are retained on a membrane through filtration so that microalgae growth is spatially separated from nutrient discharge and recharge. Therefore, additional processes for removing viable cells from spent medium are not required, and CO2 transfer efficiency into the medium can be improved without affecting cell growth. Performance of the FPBR for Chlorella sp. cultivation has been studied under various operating conditions, and an operating time and initial cell concentration for achieving optimum productivity has been selected. With a medium recycle rate of 5 mL min–1 and a feed gas containing 7.5% CO2, the biomass productivity was 13.56 g m–2 d–1 as the cell concentration increased from 2.54 to 20 g m–2 at 35 °C, which was higher than that achieved in a biofilm system for Chlorella sp. cultivation.

Biocapture of CO2 from biogas by oleaginous microalgae for improving methane content and simultaneously producing lipid

This study aimed to use oleaginous microalgae to capture CO2 from biogas for improving methane content and simultaneously producing lipid. Several microalgae were screened for their ability to grow and produce lipid using CO2 in biogas. A marine Chlorella sp. was the most suitable strain for capturing CO2 and producing lipid using biogas (50% v/v CO2 in methane) as well as using 50% v/v CO2 in air. The medium and operating conditions were optimized through response surface methodology (RSM). The optimal concentrations of KNO3 and K2HPO4 were 0.80gL−1 and 0.06gL−1, respectively. The optimal operating conditions were: initial pH of 7.8, initial cell concentration of 107.5cellsmL−1, light intensity of 4500lux and gas flow rate of 0.03Lmin−1. After optimization, 89.3% of CO2 was removed from biogas and the methane content was increased up to 94.7%. The lipid productivity was 94.7mgL−1day−1.

Modelling and predicting the simultaneous growth of Listeria monocytogenes and psychrotolerant lactic acid bacteria in processed seafood and mayonnaise-based seafood salads

A new combined model for Listeria monocytogenes and psychrotolerant Lactobacillus spp. was constructed and evaluated for processed seafood and mayonnaise-based seafood salads. The new model was constructed by combining existing cardinal parameter models for L. monocytogenes and Lactobacillus spp. using the classical Jameson effect to model microbial interaction. Maximum population density (MPD) values of L. monocytogenes were accurately predicted in processed seafood with a known initial cell concentration of Lactobacillus spp. For these experiments, average MPD values of 4.5 and 4.3 log (cfu/g) were observed and predicted, respectively for L. monocytogenes. In seafood salads, growth of L. monocytogenes continued at a reduced growth rate after Lactobacillus sakei had reached their MPD. This growth pattern was successfully described by an expanded version of the classical Jameson effect model, but only for products with pH of 6.0 or higher. For seafood salads with pH below 6.0 the performance of the new model was unacceptable, primarily due to prediction of no-growth by L. monocytogenes when growth was actually observed. Within its range of applicability the new model can be valuable for risk assessment and risk management of processed seafood as well as for evaluating the compliance of products with the EU regulation for ready-to-eat foods.

Microalgae Harvest through Fungal Pelletization—Co-Culture of Chlorella vulgaris and Aspergillus niger

Microalgae harvesting is a labor- and energy-intensive process and new approaches to harvesting microalgae need to be developed in order to decrease the costs. In this study; co-cultivatation of filamentous fungus (Aspergillus niger) and microalgae (Chlorella vulgaris) to form cell pellets was evaluated under different conditions, including organic carbon source (glucose; glycerol; and sodium acetate) concentration; initial concentration of fungal spores and microalgal cells and light. Results showed that 2 g/L of glucose with a 1:300 ratio of fungi to microalgae provided the best culturing conditions for the process to reach >90% of cell harvest efficiency. The results also showed that an organic carbon source was required to sustain the growth of fungi and form the cell pellets. The microalgae/fungi co-cultures at mixotrophic conditions obtained much higher total biomass than pure cultures of each individual strains; indicating the symbiotic relationship between two strains. This can benefit the microbial biofuel production in terms of cell harvest and biomass production.

Quick detection of cell to cell communication in gram negative bacteria by colour change of polymer matrix entrapping reporter bacteria

Quick and easy bioassay system for detection of N-acyl-L-homoserine lactones (AHLs) became possible by determining colour change of polymer matrix surfaces entrapping Chromobacterium violaceum CV026 cells, which accumulates purple pigment violacein inside cells through cell–cell communication system termed as quorum sensing (QS). The AHL is popularly produced as one of the QS signals to regulate various cell functions, and its concentration increase can lead to activate the QS. Sensitive detection of producing AHLs could be realised on the polymer surfaces as the gradual changing purple because the AHL synthase defective strain CV026 could respond to produce purple violacein in the presence of AHLs derived from other bacteria. The b* coordinate representing the difference between yellow and blue chromas in the CIE-L*a*b* colour space was obviously negatively correlated with absorbance at 585 nm of butanol extracted violacein from cells irrespective of culture time and initial concentration of cells at inoculation. Time evolution of the chroma at the matrix surface expressed in a*, b* plane was investigated to generate a master curve even when the QS was activated with different concentrations of N-hexanoyl-L-homoserine lactone or N-octanoyl-L-homoserine lactone. As compared with the master curve for the culture broth in a*, b* plane, the matrix surface meaningfully expands since the refractive measurement can give intensive signals at the matrix surface during the initial period of the sequential QS activation. The proposed biosensor can detect the AHL within 3–5 h when the AHL mediated QS system of unknown bacteria is about to be activated, where the conventional agar plate assay needs >16 h.

Modeling of Clostridium tyrobutyricum for Butyric Acid Selectivity in Continuous Fermentation

A mathematical model was developed to describe batch and continuous fermentation of glucose to organic acids with Clostridium tyrobutyricum. A modified Monod equation was used to describe cell growth, and a Luedeking-Piret equation was used to describe the production of butyric and acetic acids. Using the batch fermentation equations, models predicting butyric acid selectivity for continuous fermentation were also developed. The model showed that butyric acid production was a strong function of cell mass, while acetic acid production was a function of cell growth rate. Further, it was found that at high acetic acid concentrations, acetic acid was metabolized to butyric acid and that this conversion could be modeled. In batch fermentation, high butyric acid selectivity occurred at high initial cell or glucose concentrations. In continuous fermentation, decreased dilution rate improved selectivity; at a dilution rate of 0.028 h−1, the selectivity reached 95.8%. The model and experimental data showed that at total cell recycle, the butyric acid selectivity could reach 97.3%. This model could be used to optimize butyric acid production using C. tyrobutyricum in a continuous fermentation scheme. This is the first study that mathematically describes batch, steady state, and dynamic behavior of C. tyrobutyricum for butyric acid production.

English

Using differential scanning calorimetry (DSC) and plate count (PC), growth of Escherichia coli was measured in four initial E. coli cell concentrations cultured in tryptic soy broth (TSB) at 35°C. The calorimeter measured the energy released and/or absorbed by E. coli cells during growth and expressed it as thermograms. The resulting thermograms accurately reflected cell growth and activity, and exhibited a close correlation with growth values recorded by PC. Heat release decreased notably in the final stage of the logarithmic growth phase, and no signal was generated once the stationary phase had begun. DSC accurately quantified growth in E. coli at different initial cell concentrations grown in TSB at 35°C. Depending on initial E. coli concentration, DSC detected growth starting after just a few minutes and up to five hours. The detection limit of calorimeter was 4.3 ± 0.4 log10 CFU. DSC is an effective alternative method for measuring bacterial growth in TSB in real time. This is the first report of DSC use for measuring bacterial growth.   Key words: Differential scanning calorimetry (DSC), Escherichia coli, thermograms

Role of cultural variables in tailoring poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer synthesis in the diazotrophic cyanobacterium Aulosira fertilissima CCC 444

An important objective in the field of biopolymer research is to study the broad range of cultural conditions; by suitable choice of composition and processing conditions, polymer can often be tailor-made to have specific and desirable properties. Aulosira fertilissima CCC 444 when cultivated in a mixture of fructose and valerate produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer up to 537.5 mg L−1 containing 25 mol% 3HV monomers. Manipulation of the culture conditions such as varying pH, temperature, initial cell concentrations and dark incubation significantly affected the accumulation as well as composition of P(3HB-co-3HV) copolymer; therefore, the polymer finds a wider application. The molar fraction of 3HV tended to increase up to 35 mol% in the early phase of A. fertlissima cultivation, i.e. on the 7th day. An initial inoculum size of 80 mg dcw L−1 was found optimum for P(3HB-co-3HV) copolymer accumulation. Lower temperature conditions favoured the accumulation of copolymers containing higher molar fractions of 3HV monomers as compared to high temperature conditions. It was also observed that 3HV molar fractions in the copolymer can tailor up to 50 mol% by maintaining the pH of culture medium at 9.5, without significantly affecting the copolymer content (72 % dry cell weight). This opens up new possibilities of using A. fertilissima CCC 444 as a suitable feedstock for production of P(3HB-co-3HV) copolymer with varied 3HB and 3HV fractions for various industrial and pharmaceutical applications.