Cell Biomass Concentration Research Articles

Microbial lipid production from soybean hulls using Lipomyces starkeyi LPB53 in a circular economy

Soybean hulls are lignocellulosic residuesgeneratedinthe industrial processing of soybean, representing about 5 % of the mass of the whole bean. This by-product isan importantsource of polymers suchas cellulose(34 %) and hemicellulose (11 %),which could bevalorizedvia biotechnology to improvethe economic returnof the oilseed chain. In the present work,soybean hulls were evaluated as a carbon sourcefor biolipid productionbyLipomycesstarkeyi LPB 53. Initially the hulls were treated physicochemically and enzymatically to obtain fermentable sugars. Subsequently, biomass growth was evaluated using different nitrogen sources andthe lipid production was optimized, reaching a maximum cell biomass concentration of 26.5 g/L with 42.5 % of lipids. Around 65 % of the xylose content was consumed.The obtained oil wasmajorlycomposed of oleic, palmitic, palmitoleic, linoleic and stearic fatty acids in a proportion of 54 %, 32 %, 4 %, 3 % and 2 %, respectively.

Modeling of poly-β-hydroxybutyrate production by Bacillus subtilis and its use for feed-forward bioreactor studies.

Successful scale-up of Bacillus subtilis culture for poly-β-hydroxybutyrate (PHB) production was performed in 5-l stirred-tank reactor using batch, fed-batch, and two-stage culture strategies. The kinetics of biomass production, substrate consumption, and PHB production were established in the stirred tank bioreactor in all the studies. A mathematical model was developed to investigate the role of limiting substrate on overall culture metabolism. A fed-batch strategy was predicted on the basis of computer simulations, for maximum PHB production. This was performed by extrapolation of batch model for predicting the feeding rate and suitable time of feeding. Substrate inhibition was studied and the substrate inhibition terms were incorporated in the model. The maximum cell biomass concentration in batch culture (24h) and fed-batch culture (30h) was 1.79 ± 0.03g/l on dry cell weight (DCW) basis and 1.66 ± 0.050g/l on DCW basis and the corresponding PHB content was 68.71% and 85.54% of DCW, respectively. Glucose was found to be the major limiting nutrient during the bioreactor culture. A two-stage culture, where cells were first grown in stage I in LBG media containing excess carbon and thereafter in stage II in OM media, showed biomass production of 1.95 ± 0.045g/l at 4h and PHB production of 93.33% of DCW at 16h. A 9% increase in growth and 25% increase in PHB yield were obtained using two-stage culture with computer-simulated feeding strategy in the 5l reactor. Oxygen limitation was overcome in modified two-stage culture to obtain a PHB production of 98% at 30h. KEY POINTS: • Polyhydroxybutyrate production was studied in a 5-l stirred-tank bioreactor using HPLC • Mathematical model-assisted fed-batch strategy was implemented in bioreactor • Two-stage fed-batch cultivation was implemented and PHB production was 93% of dry weight in Gram-positive bacteria.

Effect of Nitrogen Concentration on the Biosynthesis of Citric Acid, Protein, and Lipids in the Yeast Yarrowia lipolytica.

Yarrowia lipolytica yeast is well known to be able to synthesize citric acid (CA) in large amounts. This study deals with CA biosynthesis, the production of biomass, as well as the accumulation and composition of proteins and lipids in Y. lipolytica VKM Y-2373 grown in media with glucose at different concentrations of ammonium sulfate (from 2 to 10 g/L). It was found that these concentrations of nitrogen source are limiting for the growth of Y. lipolytica and that nitrogen deficiency is the main cause of CA excretion. At the high concentration of (NH4)2SO4 (10 g/L), the accumulation of cell biomass, biomass yield (YX/S), and protein concentration was higher than in the medium with 2 g/L ammonium sulfate by 4.3 times, 143%, and 5.1 times, respectively. CA was accumulated in meaningful quantities only in media containing 3–10 g/L (NH4)2SO4 with the maximum concentration of CA (99.9 g/L) at 4 g/L ammonium sulfate. Also of interest is the technological mode with 6 g/L (NH4)2SO4, which is characterized by high productivity (1.11 g/L × h). It should be noted that biomass contains large amounts of essential amino acids and unsaturated fatty acids and can be used in food biotechnologies and agriculture.

HBsAg Production in Methanol Controlled <i>P. pastoris</i> GS115 Mut<sup>S</sup> Bioreactor Process

When producing recombinant proteins with Pichia pastoris, cultivation parameters, such as induction temperature, dissolved oxygen level and residual methanol concentration play a crucial role in product biosynthesis and subsequent purification, therefore to maximize protein yields, the optimization of these parameters is imperative. Two different Pichia pastoris cultivation strategies for HBsAg VLP production in a 5 L stirred-tank bioreactor and the influence of different cultivation parameters on product yield were investigated. Residual methanol concentrations were controlled at low (>0.01 g/L), medium (1.5-2.0 g/L) and high (5.0-6.0 g/L) levels using a PI-based feed rate control algorithm based on the online methanol sensor signal. Product was purified using a novel and rapid purification method including steps of ammonium sulfate precipitation, size-exclusion chromatography and hydrophobic interaction chromatography. Employing an in-situ methanol sensor probe, the PI-based methanol feed rate control algorithm provided residual methanol concentration control with an average deviation of ±0.4 g/L from set-point value. Employing a cultivation protocol with an increased methanol concentration controlled at 6.0 g/L and a reduced DO level below 10 %, resulting in a final dry cell biomass concentration of 140 g/L and purified HBsAg VLPs yield of 186 mg/L. Developed purification method proved advantageous to other described methods, as it did not include time consuming extraction and centrifugation steps.

Production of β-glucan exopolysaccharide lasiodiplodan by Lasiodiplodia theobromae CCT 3966 from corn bran acid hydrolysate.

The potential utilization of corn bran acid hydrolysate (CBAH) was evaluated as an inexpensive feedstock for the production of a rich carbohydrate and protein medium for lasiodiplodan (LAS) production using the filamentous fungus Lasiodiplodia theobromae CCT 3966. Experiments were performed according to a 22 CCRD experimental design aiming to evaluate the influence of agitation speed (rpm) and temperature (°C) over the production of total cell biomass (TCB) and LAS concentration released to the medium (LAS-M), adhered to biomass (LAS-C), and total (LAS-T). Under the selected conditions (temperature of 28°C and agitation of 200 rpm), 8.73 g·L-1 of LAS-T and 4.47 g·L-1 of TCB were obtained. Recovery of LAS-C with hot water was shown as an alternative to increase the production concentration, although it might require further purification steps. CBAH potential for substitution of synthetic media was demonstrated, indicating that it is an adequate raw material containing all necessary nutrients for LAS production.Key points• Corn bran acid hydrolysate is presented as a suitable substrate for β-glucan production.• Lasiodiplodia theobromae CCT 3966 have the potential for the industrial β-glucan production.• Simple recovering of biomass-adhered lasiodiplodan by hot water extraction.

Effect of light quality on carotenogenic and non-carotenogenic species of the genus Dunaliella under nitrogen deficiency

Changes in growth, pigment content, and photosynthetic response under different light qualities were monitored for two species of the green alga Dunaliella: the carotenogenic D. salina Teodoresco and the non-carotenogenic D. tertiolecta Butcher. Log-phase cultures were exposed to 100% red, 100% blue, and a mix of 50% red and 50% blue at 300 μmol photon m−2 s−1. Cell density, biomass concentration, pigment content and photosynthetic efficiency were recorded during stationary phase, once nitrogen had been depleted from the culture medium. For both species biomass concentration was maximal in red-light, while the lowest biomass concentration was found with blue-light. No significant effect of light quality on cell density was observed for any of the species. The photosynthetic response was completely different between D. salina and D. tertiolecta. In D. tertiolecta nitrate starvation was accompanied by a reduction in photosynthetic efficiency and an increase in non-photochemical quenching processes, regardless of the light condition. This species activated a clear mechanism of energy dissipation, probably related to a xanthophyll cycle. On the other hand, D. salina massively accumulated secondary carotenoids as a response to the same disruptive conditions. Carotenoids protected D. salina cells mainly against blue light, where Fv/Fm remained high. In D. salina, pigment content changed with respect to light quality. Blue light enhanced total carotenoid content, but a mix of red and blue increased total carotenoid concentration in the culture, by stimulating a more balanced increase in dry weight and carotenoid accumulation. The application of specific light spectrum for the production of microalgal biomass with different carotenoid content that has a potentially different market value, is confirmed an important tool for the optimization of artificial-light microalgae cultivation systems.

Valorization of sugarcane bagasse for bioethanol production through simultaneous saccharification and fermentation: Optimization and kinetic studies

This study focuses on the valorization of sugarcane bagasse (SCB), which is a waste by-product from the sugar industry for ethanol production. A simultaneous saccharification and fermentation process was modelled and optimized. In addition, the kinetics of Saccharomyces cerevisiae cell growth and ethanol formation under the optimized conditions were assessed using the logistic and modified Gompertz models respectively. The response surface methodology (RSM) was used for the optimization with input parameters consisting of enzyme loading (20–100 U/g), temperature (30–50 °C) and yeast titre (1–5 times). A maximum bioethanol concentration of 4.88 g/L was observed under the optimal process conditions of 1 time (yeast titre), 100 U/g (enzyme loading) and 39 °C (temperature). The RSM model revealed that the enzyme loading parameter significantly affected bioethanol production. Kinetics of cell growth gave a maximum specific growth rate (μmax) of 0.15 h−1 and a maximum cell biomass concentration (Xmax) of 2.58 g/L. The modified Gompertz model showed a maximum bioethanol concentration (Pm) and maximum bioethanol production rate (rp, m) of 3.12 g/L and 0.29 g/L/h respectively. These findings demonstrated the potential of sugarcane waste valorization for biofuel production through SSF processes, and provided insights into process design for large scale operations.

Valorisation of potato peel wastes for bioethanol production using simultaneous saccharification and fermentation: Process optimization and kinetic assessment

This study optimized bioethanol production from potato peel wastes on inputs of temperature, pH and solid loading using simultaneous saccharification and fermentation. Subsequently, the kinetics of yeast growth and bioethanol formation under the optimized conditions were assessed using the logistic and modified Gompertz models, respectively. Maximum bioethanol concentration (22.54 g/L) and yield (0.32 g/g) were observed under optimal process conditions of 40 °C (temperature), 5.78 (pH) and 12.25% w/v (solid loading). The logistic model gave a maximum specific growth rate and maximum cell biomass concentration of 0.20 h−1 and 2.43 g/L respectively. Furthermore, the modified Gompertz model displayed a maximum bioethanol production rate, maximum potential bioethanol concentration and a lag time of 1.51 g/L/h, 15.47 g/L and 4.66 h respectively. Optimization and kinetic findings obtained from this study provide significant knowledge for the development of simultaneous saccharification and bioethanol production processes using starch-based agricultural wastes.

Evaluation of surfactant effect on β-poly(L-malic acid) production by Aureobasidium pullulans

β-poly(L-malic acid) (PMLA) is a highly water-soluble biopolymer used in medicine and other industries. PMLA is usually biosynthesized by Aureobasidium pullulans, but in low concentrations, which has restricted its further large-scale production. Here effects of surfactants on PMLA production were investigated. Of various surfactants examined, the addition of Tween 80 at the start of cell growth increased mycelial growth, PMLA production, PMLA productivity and the ratio of PMLA to cell biomass concentration (Yp/x) of A. pullulans. Transmission electron microscopy and flow cytometry showed that cell membrane permeability was increased, improving the secretion of PMLA and uptake of nutrients and oxygen. Proteomic analysis showed that key pathways of PMLA synthesis, along with cellular energy metabolism, were enhanced. Moreover, glutathione (GSH) synthesis and anti-oxidation capacity were also improved. These results indicated that the addition of Tween 80 is an efficient approach for improving the production of PMLA.

Bioprocess and downstream optimization of recombinant human growth hormone in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is a well-established expression host, which is often used in the production of protein pharmaceuticals. This work aimed to evaluate the effect of various concentrations of ascorbic acid in mixed feeding strategy with sorbitol/methanol on productivity of recombinant human growth hormone (r-hGH). The relevant concentration of ascorbic acid (5, 10, or 20 mmol) and 50 g/L sorbitol were added in batch-wise mode to the medium at the beginning of induction phase. The rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. Total protein and r-hGH concentrations were analyzed and the results compared with sorbitol/methanol feeding using one-way analysis of variance. Moreover, an effective clarification process using activated carbon was developed to remove process contaminants like pigments and endotoxins. Finally, a three-step chromatographic process was applied to purify the product. According to the obtained results, addition of 10 mmol ascorbic acid to sorbitol/methanol co-feeding could significantly increase cell biomass (1.7 fold), total protein (1.14 fold), and r-hGH concentration (1.43 fold). One percent activated carbon could significantly decrease pigments and endotoxins without any significant changes in r-hGH assay. The result of the study concluded that ascorbic acid in combination with sorbitol could effectively enhance the productivity of r-hGH. This study also demonstrated that activated carbon clarification is a simple method for efficient removal of endotoxin and pigment in production of recombinant protein in the yeast expression system.

Metabolic phenotyping of CHO cells varying in cellular biomass accumulation and maintenance during fed-batch culture.

CHO cell lines capable of high-level recombinant protein product biosynthesis during fed-batch culture are still generally obtained by intensive empirical screening of transfected cells rather than knowledge-guided cellular engineering. In this study, we investigate how CHO cell lines create and maintain cellular biosynthetic capacity during fed-batch culture to achieve the optimal combination of rapid exponential proliferation and extended maintenance of high cell biomass concentration. We perform a comparative meta-analysis of mitochondrial and glycolytic functions of 22 discrete parental CHO cell lineages varying in fed-batch culture performance to test the hypotheses that (i) "biomass-intensive" CHO cells exhibit conserved differences in metabolic programming and (ii) it is possible to isolate parental CHO cell lines with a biomass-intensive phenotype to support fed-batch bioproduction processes. We show that for most parental CHO cell lines, rapid proliferation and high late-stage culture performance are mutually exclusive objectives. However, quantitative dissection of mitochondrial and glycolytic functions revealed that a small proportion of clones utilize a conserved metabolic program that significantly enhances cellular glycolytic and mitochondrial oxidative capacity at the onset of late-stage culture. We reveal the central importance of dynamic metabolic re-programming to activate oxidative mitochondrial function as a necessary mechanism to support CHO cell biosynthetic performance during culture.

Autoflocculation of Scenedesmus Quadricauda: Effects of Aeration Rate, Aeration Gas CO2 Concentration and Medium Nitrogen Concentration

After microalgal cultivation step, a recent method to harvest microalgae is adding flocculant into microalgal liquid culture to cause flocculation. Flocculated cells are easy to concentrate since their sedimentation time and successive filtering time are expected to be shorter. In this sense, autoflocculation of microalgae may be a beneficial phenomenon for easing the harvesting process of microalgae. In this study, autoflocculation was investigated for the growth of Scenedesmus quadricauda, a type of fresh water microalgae, at 25 ºC in a 5-L column photobioreactor. The BG11 medium was used. The effects of aeration rate, CO2 concentration in aeration gas and nitrogen concentration in culture medium on auto flocculating behavior were investigated. Higher aeration rate, higher CO2 concentration and higher nitrogen concentration caused more flocculation. In other words, autoflocculation became prominent at the condition range where the final cell biomass concentration was lower, in general.
 Keywords: Autoflocculation, Scenedesmus quadricauda, aeration rate, aeration gas CO2 concentration, medium nitrogen concentration

Flow cytometry to estimate the cell disruption yield and biomass release of Chlorella sp. during bead milling

A number of visual, chemical and fluorescence-based methods are generally employed for monitoring of algae cell growth, culture health and biomass concentration. These methods are often time-consuming, demand destructive and high volume sampling. Rapid, efficient, cost-effective and automated methods which facilitate high-throughput and non-destructive sampling would highly benefit microalgae biotechnology. It is known in literature that with flow cytometry it is possible to monitor microalgae growth and microalgae culture health. Flow cytometry, however, has not been used to estimate biomass release and cell disruption yield. In this study with representative cultures of Chlorella sp., flow cytometry data were generated by a long pass filter (>670nm) and proved to be a promising technique to rapidly evaluate these parameters during cell disruption by bead milling. Both a laboratory and a commercial culture of Chlorella were bead milled. Prior to and during bead milling, biomass release was evaluated gravimetrically and the cell count was determined via hemocytometer manual counting and 3 flow cytometry methods: 1) direct event count of a certain population, 2) quadrant-upper right event count of a long pass filter (>670nm) and 3) a calculation based on the long pass filter data. The data of all methods were compared and correlated to gravimetric biomass release data. Manual counting resulted in an underestimation of the cell disruption yield as one of the Chlorella cultures manual counts did not agree with biomass release data. Good correlations for both cultures were found for cell disruption yield calculations based on flow cytometry data and gravimetric biomass release data. Flow cytometry is therefore an efficient analytical method to rapidly screen disruption yields during cell disruption in microalgae and can be a substitute for the time-consuming direct methods (e.g. gravimetric, manual counting) for estimating biomass release and cell disruption yield.

Fermentative Production of Xylitol: A First Trial on Xylose Bifurcation

Background/Objectives: Xylitol production through chemical processes pathway involves high energy usage and production cost. Alternative method via microbial biotransformation and biocatalyst offer more sustainable and environmental friendly feedstock to be used for xylitol production. Methods: Production of xylitol by Aspergillus niger PY11 using different conditions on 2 carbon source, glucose and xylose, were done for the development of this research. Batch fermentation of A. niger PY11 was conducted for 4 days or 96 hours in temperature set at 30oC and agitation speed of 200 rpm. Samples were taken at 12 hours interval, filtered and analyzed for cell biomass, remaining sugar and D-xylitol concentration. The production of biomass and xylitol was monitored through dry-mass weight of mycelium and by HPLC, respectively. Findings: From the results of the utilization of single carbon source, fermentation of D-xylose produced the highest xylitol yield, which was 0.101 g xylitol/g D-xylose consumed, with the xylitol titre of 1.139 g/l was obtained (equivalent to 0.482 g xylitol/g biomass). However, the highest cell growth was observed when fermentation were conducted using a mixture of D-xylose and D-glucose at the ratio of 3:1, which resulted the biomass yield of 0.239 g biomass/g D-xylose (equivalent to 0.211 g xylitol/g biomass). Total amount of 44.94% of added D-xylose was consumed during the fermentation. Applications/Improvements: This paper shown that the addition of glucose had resulted higher biomass growth of A.niger PY11, thus subsequently increased the bioconversion of xylose to xylitol.

Kinetics of Synthesis of Gold Nanoparticles by Acinetobacter sp. SW30 Isolated from Environment.

Cell biomass and metal salt concentration have great influence on morphology of biosynthesized nanoparticle. The aim of present study was to evaluate the effect of varying cell density and gold salt concentrations on synthesis of nanoparticles and its morphology, which has not been studied in bacteria till now. When cells of Acinetobacter sp. SW30 were incubated with different cell density and gold chloride concentrations, tremendous variation in color of colloidal solution containing gold nanoparticles (AuNP) was observed indicating variation in their size and shapes. Surprisingly, monodispersed spherical AuNP of size ~19nm were observed at lowest cell density and HAuCl4 salt concentration while increase in cell number resulted in formation of polyhedral AuNP (~39nm). Significance of this study lays in the fact that the shape and dispersity of AuNP can be customized depending up on the requirement. FTIR spectrum revealed shift from 3221 to 3196cm-1 indicating the presence and role of amino acids in Au3+ reduction while possible involvement of amide I and II groups in stabilization of AuNP. The rate constant was calculated for cell suspension of 2.1×109cfu/ml challenged with 1.0mM HAuCl4, incubated at 30°C and pH 7 using the slopes of initial part of the plot log (Aα-At) versus time as 1.99×10-8M. Also, this is the first study to report the kinetics of gold nanoparticle synthesis by Acinetobacter sp. SW30.

High-rate fermentative hydrogen production from beverage wastewater

Hydrogen production from beverage industry wastewater (20 g/Lhexose equivalent) using an immobilized cell reactor with a continuous mode of operation was studied at various hydraulic retention times (HRT, 8–1.5 h). Maximum hydrogen production rate (HPR) of 55 L/L-d was obtained at HRT 1.5 h (an organic loading of 320 g/L-dhexose equivalent). This HPR value is much higher than those of other industrial wastewaters employed in fermentative hydrogen production. The cell biomass concentration peaked at 3 h HRT with a volatile suspended solids (VSS) concentration of 6.31 g/L (with presence of self-flocculating Selenomonas sp.), but it dropped to 3.54 gVSS/L at 1.5 h HRT. With the shortening of HRT, lactate concentration increased but the concentration of the dominant metabolite butyrate did not vary significantly. The Clostridium species dynamics was not significantly affected, but total microbial community structure changed with respect to HRT variation as evident from PCR–DGGE analyses. Analysis of energy production rate suggests that beverage wastewater is a high energy yielding feedstock, and can replace 24% of electricity consumption in a model beverage industry.

Membrane applications for microalgae cultivation and harvesting: a review

With renewed interest in microalgae due to their potential for biofuel and bioproducts production, efficient cultivation and harvesting mechanisms are needed to increase the economic competitiveness of microalgal products against traditional sources. With pore sizes ranging from microns to angstroms, membranes provide tailored functions for solid/liquid separation (cell retention, biomass concentration and dewatering), gas/liquid separation (gas delivery and removal), and solute/liquid separation (bioproduct recovery, feedstock preparation and effluent recycling) that are problematic or not possible with other technologies. Existing knowledge on membrane systems used in other disciplines, such as environmental engineering, marine science, and biomedicine, can be applied to algae production. Though membranes have great potential to facilitate cultivation and harvesting, challenges in energy reduction and fouling mitigation need to be overcome for long-term, cost-effective application.

Research on Biological Materials with Uranium Biosorption by Microalgae: A Review

With the development of nuclear science, uranium-contained wastewater was heavily generated. If not properly handled, it is bound to bring environmental and human health hazards. Conventional uranium-contained wastewater treatment technologies are limited in certain aspects, such as high reagent and energy consumption, secondary pollution and so on. The microalgae-based biosorption technique for treating uranium-contained wastewater is an economical, simple, effective and feasible approach. This paper summarized the basic mechanism of the technology, and discussed the effects of different pH, algal cell biomass concentration, initial uranium ion concentration and the growth state of algal cells on the biological treatment process of uranium-contained wastewater. Finally, the study explored the future prospects of the technology.

Effects of nitrogen on growth and carbohydrate formation in Porphyridium cruentum

Abstract The microalga Porphyridium cruentum (Rhodophyta) has several industrial and pharmaceutical uses, especially for its polysaccharide production. This study aimed to investigate the influence of nitrogen levels as reflected by altered N:P ratios on the production and content of biomass and carbohydrate. N:P molar ratios were altered in batch cultures to range from 1.6 to 50 using the Redfield ratio of 1:16 as reference. Algal growth (estimated as final cell number, biomass concentration and maximum specific growth rate) was negatively affected at low N:P ratios. The optimal N:P ratio for growth was identified at 35–50, with specific growth rates of 0.19 day−1 and maximum cell concentrations of 59·108 cells L−1 and 1.2 g dry weight of biomass L−1. In addition, variation in cell size was seen. Cells with larger diameters were at higher N:P ratios and smaller cells at lower ratios. The cellular carbohydrate content increased under reduced nitrogen availability. However, because accumulation was moderate at the lowest N:P ratio, 0.4 g per g dry weight biomass compared to 0.24 at the Redfield ratio of 16:1, conditions for increased total carbohydrate formation were identified at the N:P ratios optimal for growth. Additionally, carbohydrates were largely accumulated in late exponential to stationary phase.

Optimizing and validating the production of ethanol from cheese whey permeate by Kluyveromyces marxianus UFV-3

The purpose of this study was to optimize the production of ethanol from cheese whey permeate using Kluyveromyces marxianus UFV-3. We used the response surface methodology (RSM) with a central composite rotational design (CCRD) to evaluate the effects of pH (4.5–6.5), temperature (30–45 1C), lactose concentration (50–250 g l � 1 ), and cell biomass concentration (A 600 2–4). We performed 29 fermentations under hypoxia in cheese whey permeate and seven fermentations for the validation of the equation obtained via RSM. Temperature was the most significant factor in optimizing ethanol production, followed by pH, cell biomass concentration and lactose concentration. The conditions for producing ethanol at yields above 90% were as follows: temperature between 33.3 and 38.5 1C, pH between 4.7 and 5.7, cell biomass concentration between A600 2.4 and 3.3, and lactose concentration between 50 and 108 g l � 1 .T he equation generated from the optimization process was validated and exhibited excellent bias and accuracy values for the future use of this model in scaling up the fermentation process.