Advanced
Ethanol Production from Glycerol Using Immobilized Pachysolen tannophilus During Microaerated Repeated-Batch Fermentor Culture
Ethanol Production from Glycerol Using Immobilized Pachysolen tannophilus During Microaerated Repeated-Batch Fermentor Culture
Journal of Microbiology and Biotechnology. 2015. Mar, 25(3): 366-374
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : September 12, 2014
  • Accepted : October 08, 2014
  • Published : March 28, 2015
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Hye-Geun Cha
Department of Biotechnology, Korea National University of Transportation, Jeungpyung 368-701, Republic of Korea
Yi-Ok Kim
Department of Biotechnology, Korea National University of Transportation, Jeungpyung 368-701, Republic of Korea
Woon Yong Choi
Department of Medical Biomaterials Engineering, Kangwon National University, Chuncheon 200-701, Republic of Korea
Do-Hyung Kang
Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea
Hyeon-Yong Lee
Department of Food Science and Engineering, Seowon University, Cheongju 361-742, Republic of Korea
Kyung-Hwan Jung
Department of Biotechnology, Korea National University of Transportation, Jeungpyung 368-701, Republic of Korea
khjung@cjnu.ac.ckr

Abstract
Herein, we established a repeated-batch process for ethanol production from glycerol by immobilized Pachysolen tannophilus . The aim of this study was to develop a more practical and applicable ethanol production process for biofuel. In particular, using industrial-grade medium ingredients, the microaeration rate was optimized for maximization of the ethanol production, and the relevant metabolic parameters were then analyzed. The microaeration rate of 0.11 vvm, which is far lower than those occurring in a shaking flask culture, was found to be the optimal value for ethanol production from glycerol. In addition, it was found that, among those tested, Celite was a more appropriate carrier for the immobilization of P. tannophilus to induce production of ethanol from glycerol. Finally, through a repeated-batch culture, the ethanol yield (Y e/g ) of 0.126 ± 0.017 g-ethanol/g-glycerol ( n = 4) was obtained, and this value was remarkably comparable with a previous report. In the future, it is expected that the results of this study will be applied for the development of a more practical and profitable long-term ethanol production process, thanks to the industrial-grade medium preparation, simple immobilization method, and easy repeated-batch operation.
Keywords
Introduction
In the biodiesel production process, an abundance of glycerol has recently been encountered as a byproduct of transesterification reactions of oil feedstock [1 , 14] . There have been many studies on utilization of the produced glycerol to attain more valuable products [5 , 13] , in which the ethanol production from glycerol has particularly received much attention. Conversion of the byproduct glycerol to ethanol as a biofuel is regarded as a process for the complete utilization of oil feedstock as fuel [4 , 11 , 12] . In addition, in the economic analysis of glycerol as a substrate for ethanol production, it was shown that the cost for producing ethanol using glycerol was comparatively superior to that of using corn starch [18] . In general, it is known that bacteria and yeast can convert glycerol to ethanol, although they have quite different metabolic pathways for glycerol utilization [11 , 12 , 18] . Among yeasts, Pachysolen tannophilus ( P. tannophilus ) was demonstrated by several studies to have the ability to convert glycerol to ethanol under controlled aeration conditions, particularly under microaeration [11 , 12] . It has been reported that the utilization of glycerol in P. tannophilus is associated with the mitochondrial electron transport chain [6 , 8] . Air supply in low amounts is necessary for the ethanol production in P. tannophilus , while sufficient air supply can activate oxidative metabolism.
In the present study, we investigated the development of a more practical and applicable ethanol production process for biofuel, where the yeast P. tannophilus was used for the production of ethanol from glycerol. In particular, the microaeration rate was optimized for maximization of the ethanol production, with analysis of the relevant metabolic parameters. In addition, an appropriate immobilization method to allow ethanol production by P. tannophilus was also investigated. Finally, to verify the establishment of a practical process applicable to the ethanol production industry, the repeated-batch culture for long-term ethanol production was studied, where the medium was withdrawn and filled repeatedly during the period that batch culturing was conducted.
In a previous study [11] , it was reported that controlled aeration had an important role in ethanol production from glycerol by P. tannophilus , wherein microaeration was conducted at 0.05l/min (0.083 vvm) in a chemically defined medium consisting of yeast nitrogen base without amino acids (YNB w/o amino acids) (Becton Dickinson, USA) and glycerol. These experimental conditions were a little different from those implemented in this study in terms of medium composition. However, we compared the results of the previous study [11] with those in the present study.
The immobilization methods for yeast were compared in view of operational durability for cell adsorption and ethanol production, as these variables are necessary for the long-term operation of an ethanol-producing system. In particular, Celite (a trademarked brand name of diatomaceous earth) was investigated as the carrier for the immobilization of P. tannophilus , together with DEAE-corncob [10] and DEAE-cotton [9] . Celite has sometimes been used as an immobilization carrier of yeast cells and enzymes [7 , 17] . However, the immobilization of P. tannophilus on Celite has not yet been reported, and furthermore, there have been no reports on the immobilization of P. tannophilus for the production of ethanol from glycerol. Throughout this study, we established the first platform strategy for long-term ethanol production from glycerol, wherein P. tannophilus was immobilized on Celite and the aeration was finely controlled to maintain microaeration conditions.
Materials and Methods
- Chemicals
Glycerol and Celite 545 (particle size, 38-46 µm; specific gravity, 2.3) were purchased from Daejung Chemicals and Materials Co. (Gyoenggi-Do, Korea) and Yakuri Pure Chemicals Co. (Kyoto, Japan), respectively. Yeast extract, peptone, and YNB w/o amino acids were obtained from Becton Dickinson (Franklin Park, NJ, USA). Specifically, industrial-grade yeast extract was purchased from Choheung Corp. (Gyoenggi-Do, Korea). CSL (corn-steep liquor) was kindly supplied by Samyang Genex Corp. (Seoul, Korea). All other chemicals were of reagent grade.
- Yeast and Medium
The yeast strain used in this study was P. tannophilus ATCC 32691. The following four kinds of media were used: YPD medium (yeast extract, 10 g/l; peptone, 20 g/l; glucose, 20 g/l), YPG medium (yeast extract, 10 g/l; peptone, 20 g/l; glycerol, 20 g/l), YCG medium (glycerol, 20 g/l; CSL, 20 ml/l; industrialgrade yeast extract, 5 g/l; (NH 4 ) 2 SO 4 , 3.0 g/l; KH 2 PO 4 , 2.4 g/l; and MgSO 4 ·7H 2 O, 1.2 g/l), and YNBG medium (glycerol, 20 g/l; YNB w/o amino acids, 6.8 g/l).
- Fermentor Culture with Microaeration
The fermentor culture was conducted in a 2.5 L jar fermentor (KoBiotech, Republic of Korea) with a working volume of 800 ml. The seed culture volume of 80 ml was used. During fermentation, the temperature, pH, and agitation speed were maintained at 30℃, 5.0, and 200 rpm, respectively. The pH was maintained by adding a phosphoric acid solution (10% (v/v)) or ammonia water, as necessary. The air supply rate was finely controlled using a sparger with an air flow meter (Cole-Parmer, USA), which had a scale-range from zero to 800 ml/min.
- Immobilization ofP. tannophilus
Prior to the immobilization of yeast cells using Celite, the immobilization conditions were optimized. To optimize the initial OD 600 (optical density of the cell suspension at 600 nm), yeast cell suspensions with various OD 600 values were prepared in 20 ml of YCG medium in a 50 ml conical tube, after which 2.0 g of Celite was added to the cell suspensions. Next, the mixture of cells and Celite was shaken briefly. After the conical tube was kept static for 30 sec to confirm the formation of a lower Celite-containing layer, the OD 600 of the upper solution was measured. Optimization of the time for cell immobilization was also carried out by varying the time periods in which the conical tubes (OD 600 of yeast cell suspensions = 8.0) were kept static and measuring the resulting OD 600 of the upper solution. Finally, the cell immobilization for flask and fermentor cultures was conducted using Celite based on the optimized conditions obtained.
Immobilization of yeast cells using DEAE (2-(diethylamino)ethyl chloride hydrochloride)-corncob and DEAE-cotton was conducted according to the protocols previously described in detail [9 , 10] .
- Flask Culture Using ImmobilizedP. tannophilus
When flask culture was conducted using the immobilized yeast cells on Celite, it was started with YPG medium. During the batch culture, it was confirmed that the residual glycerol concentration was less than 5 g/l, and then Celite was added into the culture broth. Next, the flask was kept static to confirm the formation of a lower Celite-containing layer, and then the upper culture broth was removed by pipetting. Afterward, new YPG medium was added to the flask containing the immobilized yeast cells, and the flask culturing was started for the production of ethanol from glycerol. The flask cultures were performed using 100 ml Erlenmeyer flasks in a shaking incubator at 30℃ and 150 rpm. The protocols for the flask cultures of yeast immobilized on DEAE-corncob and DEAE-cotton were described in detail in previous reports [9 , 10] .
- Detachment of ImmobilizedP. tannophilusfrom Celite
To estimate how strongly the immobilized yeast cells adsorbed on the surface of Celite, detachment of the immobilized P. tannophilus was investigated. The yeast cell suspension (OD 600 = 8.0) was prepared in 20 ml of distilled water in a 250 ml Erlenmeyer flask, and 2.0 g of Celite was added. After the yeast cells were adsorbed onto the Celite using the above-mentioned method, the flask was kept static to confirm the formation of a lower Celite-containing layer, and the upper solution was then decanted. Next, 20 ml of distilled water was added to the flask, and it was shaken vigorously in a shaking incubator for 22 h. During this shaking, the change of OD 600 in the upper solution was monitored intermittently after separation of the upper and lower layers.
- Fermentor Culture with Microaeration Using ImmobilizedP. tannophilus
The basic protocol for the fermentor culture of the immobilized yeast was the same as the above-mentioned conditions for fermentor culture. The culture was started with YCG or YNBG medium. In the first batch culture, Celite was added to the culture broth after confirming that the residual glycerol concentration was less than 5 g/l. Next, the agitation was stopped and the fermentor was kept static to confirm the formation of a lower Celite-containing layer, and then 700-800 ml of the upper culture broth was removed by pumping using a peristaltic pump. Afterward, fresh YCG or YNBG medium was added into the fermentor containing the immobilized yeast cells. The medium replacement methods for ethanol production were conducted repeatedly.
- Analytical Methods
Yeast cell concentration was assessed by measuring OD 600 using a spectrophotometer (Spectronic; Thermo Scientific, Rockford, IL, USA). If necessary, the OD 600 was converted to dry cell weight (DCW, g/l) by the formula DCW = 0.53 × OD 600 . Residual glycerol concentration was measured using a chemical assay method [3] . Ethanol concentration was measured by gas chromatography, as described in detail previously [19] . The oxygen transfer rate coefficient (k L a) was measured by the unsteady-state method [15] , in which the fermentor was equipped with a dissolved oxygen probe (Mettler-Toledo) and filled with 800 ml of distilled water. The specific growth rate (µ), specific ethanol production rate (Q e ), and ethanol yields (Y e/g and Y e/x ) were estimated between time zero to the time when ethanol production was at a maximum value. The method for estimation of these parameters is described in detail in a textbook [16] .
- Electron Microscopy
Scanning electron microscopy (ESEM; FEI Quanta 400, Hillsboro, OR, USA) observations were made after the yeast cell-immobilized Celite was washed with deionized water and dried for 24 h at 60℃ [9 , 10] .
- Statistical Analysis
All experimental data were measured three times using the same sample, and were reported as means with standard deviations, except for the cell growth monitoring of OD 600 . The z-test was conducted using Microsoft Excel.
Results and Discussion
- Ethanol Production from Glycerol byP. tannophilusin Microaerated Fermentor
To investigate the optimal microaeration rate for ethanol production from glycerol by P. tannophilus , fermentor cultures were conducted with various microaeration rates (0.0-1.70 vvm), for which YPG medium was used. As shown in Figs. 1 A and 1 B, as the microaeration rates increased, on the whole, the growth rate and glycerol consumption rate of the yeast cells increased. Ethanol production reached a maximum of about 4.7 g/l at the microaeration rate of 0.05 and 0.11 vvm ( Fig. 1 C). At these rates, the rates of cell growth and glycerol consumption were at medium values among the various microaeration rates. It can be seen in Fig. 1 that as the microaeration rate was increased, the cell growth rate also increased but the ethanol production decreased. However, when no aeration was conducted, cell growth, glycerol consumption, and ethanol production were hardly observed. As shown in Fig. 1 C, the ethanol production rates (from time zero to when ethanol concentration reached the maximum) were almost identical for the various microaeration rates, except with no aeration. It is likely that P. tannophilus had a limited capacity for ethanol production from glycerol, regardless of the microaeration rate. In other words, because P. tannophilus has a limited metabolic flux for the production of ethanol from glycerol, it is probable that the excess of glycerol might be metabolized oxidatively for cell growth.
PPT Slide
Lager Image
Effect of microaeration rate on the production of ethanol from glycerol by P. tannophilus in YPG medium. (A) Cell growth; (B) residual glycerol concentration; (C) ethanol production. The box in (B) shows the symbols indicating the microaeration rate.
Unfortunately, it was not possible to adjust the microaeration rate to between 0.0 and 0.05vvm, because the air flow meter did not allow fine control at this range. In addition, the optimal microaeration condition was selected as 0.11 vvm, as this rate could be controlled more easily than 0.05vvm. A previous study reported the optimal aeration rate to be 0.083 vvm when ethanol was produced by P. tannophilus from medium containing YNB w/o amino acids and glycerol [11] . Although the medium compositions were fairly different from each other, the optimal microaeration rate was very similar to that of the previous study.
- Analysis of Metabolic Parameters During Ethanol Production with Microaerated Fermentor
To quantitatively analyze the metabolic parameters of the ethanol production process in Fig. 1 , the oxygen transfer rate coefficient (k L a) and other metabolic parameters (µ, Q e , Y e/g , and Y e/x ) were estimated ( Fig. 2 ). As shown in Fig. 2 A, the microaeration rates conducted in this study (0.05-1.7 vvm) were estimated to have oxygen transfer rate coefficients (k L a) of 0.5-26.2 1/h, which were measured using the unsteady-state method. It is well known that the k L a ranges from 24 to 198 1/h in shaking flask cultures [2] . Therefore, the ethanol production process used in this study was conducted under microaeration conditions that were far lower than those occurring in a shaking flask culture. In particular, the k L a at the optimal microaeration condition in this study (0.11 vvm) was 1.41 1/h, which was far less than that in general aerobic conditions.
PPT Slide
Lager Image
Effects of microaeration rate on the oxygen transfer rate coefficient (kLa) and quantitative metabolic parameters (µ, Qe, Ye/g, and Ye/x). Estimations were conducted using data from Fig. 1. (A) kLa; (B) µ and Qe; (C) Ye/g and Ye/x. The boxes in (B) and (C) show the symbols indicating µ and Qe, and Ye/g and Ye/x, respectively.
As shown in Fig. 2 B, the specific growth rate (µ) increased until about 0.3 1/h with increase of the microaeration rate, after which no more increase was observed. The specific ethanol production rate (Q e ) increased sharply at 0.05vvm until about 0.07 g-ethanol/g-cell/h, where it was maintained until 0.91 vvm, and decreased thereafter. In contrast with µ, the maximal Q e was maintained at about 0.07 g-ethanol/g-cell/h, between 0.05 and 0.91 vvm. Therefore, Q e provided the reason for which microaeration between 0.05 and 0.91 vvm should be used for the production of ethanol from glycerol.
Fig. 2 C shows that Y e/x (ethanol yield based on cell growth) and Y e/g (ethanol yield based on glycerol consumption) reached maximal levels of 0.61 g-ethanol/g-cell and 0.22 g-ethanol/g-glycerol, respectively, when the microaeration rate was 0.05 and 0.11 vvm, respectively. Interestingly, Y e/x and Y e/g also showed the maximum values around 0.05-0.11 vvm, the same as that shown for Q e . It was deduced that the ethanol production at 0.05 and 0.11 vvm in Fig. 1 C likely reached the maximum, since among the metabolic parameters, Q e , Y e/g , and Y e/x all reached maximum values at around 0.05-0.11 vvm. However, it was observed that Q e was about 0.07 g-ethanol/g-cell/h at 0.91 vvm, which may have resulted from the relatively high value of µ.
Through the estimation of the oxygen transfer rate coefficient (k L a), the amount of air supplied under the optimal microaeration rate for ethanol production was confirmed. In addition, through analyses of the metabolic parameters (µ, Q e , Y e/g , and Y e/x ), the reason for the maximal ethanol production at the optimal microaeration rate was also confirmed.
- Ethanol Production from Glycerol Using Industrial-Grade Medium in Microaerated Fermentor
Because we aimed to provide a practical process for ethanol production from glycerol, it was necessary to use YCG medium, wherein the medium consisted of glycerol, CSL, industrial-grade yeast extract, and salts. As shown in Fig. 3 , the fermentor cultures were conducted at 0.11, 0.23, and 0.57 vvm. The microaeration rate was controlled at slightly higher values compared with those in Fig. 1 , though the profiles of cell growth and glycerol consumption were relatively similar to those in Figs. 1 A and 1 B. At the microaeration rate of 0.11 and 0.23 vvm, the maximum ethanol production and ethanol yield (Y e/g ) was about 3.1 g/l and 0.16 g-ethanol/g-glycerol, respectively. These values were better than those achieved at 0.57 vvm, and showed that the range of optimal microaeration rate could be extended to 0.23 vvm. However, the ethanol production and ethanol yield at 0.11 and 0.23 vvm were less than those observed in Fig. 1 . These decreases were likely associated with the nutrient deficiency in YCG medium. In other words, YCG medium was nutritionally insufficient to allow ethanol production using P. tannophilus .
PPT Slide
Lager Image
Effect of microaeration rate on the production of ethanol from glycerol by P. tannophilus in YCG medium. (A) Cell growth; (B) residual glycerol concentration; (C) ethanol production. The box in (B) shows the symbols indicating the microaeration rate.
- Immobilization ofP. tannophilusfor Ethanol Production from Glycerol
Prior to the immobilization of P. tannophilus on Celite, the conditions of cell immobilization were first optimized. As shown in Fig. 4 A, the cell adsorption increased until the initial OD 600 reached 8.0. However, further cell adsorption did not occur at >8.0 of initial OD 600 . In addition, no further cell adsorption was observed when the adsorption time was >1.0 min ( Fig. 4 B). Based on the results in Fig. 4 , it was decided that the conditions of mixing 20 ml of yeast cells (OD 600 = 8.0) with 2.0 g of Celite and then keeping the mixture static for 1.0 min were optimal for adsorption. Under these conditions, about 30% of the yeast cells were immobilized on Celite. The amount of Celite added for immobilization was changed proportionally to the OD 600 of the yeast cells and the culture volume in the flask and fermentor cultures.
PPT Slide
Lager Image
Optimal conditions for the immobilization of P. tannophilus on Celite. Cell adsorption was estimated by subtracting the initial OD600 from the measured OD600, where the medium containing Celite was used as a blank. (A) Effect of initial OD600; (B) effect of adsorption time.
Meanwhile, to choose an appropriate carrier for the immobilization of P. tannophilus , DEAE-corncob, DEAE-cotton, and Celite were compared in terms of the cell growth in culture broth, glycerol consumption, and ethanol production during flask culture of the immobilized cells ( Fig. 5 ). For the DEAE-corncob, although there was no cell growth in the culture broth, glycerol consumption and ethanol production were scarcely observed. A previous report suggested this to be due to the porous structure of the corncob [10] . In other words, because the yeast cells were immobilized in the pore structures in the corncob and it was physically difficult for them to have access to the air supply, the cells could not easily consume glycerol to produce ethanol. In the case of DEAE-cotton, as shown in Fig. 5 A, many cells were detached from the DEAE-cotton and cell growth was subsequently observed in the culture broth. Therefore, it is probable that the glycerol consumption and ethanol production resulted from the detached cells. In contrast with DEAE-corncob and DEAE-cotton, when the yeast cells immobilized on Celite were cultured in flask, glycerol was consumed until about 5.0 g/l, and up to 2.5 g/l ethanol was produced. During this experiment, the cell growth and the detached cells could not be separately investigated because the culture broth contained fine debris of Celite.
PPT Slide
Lager Image
Flask cultures of immobilized P. tannophilus in YPG medium. DEAE-corncob (●, ■, ▲), DEAE-cotton (○, □, △), and Celite (◇) were used as carriers for yeast cell immobilization. (A) Cell growth in culture broth; (B) residual glycerol concentration; (C) ethanol productio
Through the separate detachment experiment of immobilized yeast cells, it was demonstrated that the yeast cells were not easily detached from the surface of Celite. There was no statistically significant difference in the change of OD 600 in the upper solution after separating the upper and lower layers in the detachment experiment of immobilized P. tannophilus from Celite (6.81 ± 0.91, n = 10, p = 0.5). As a result, Celite was chosen as the immobilization carrier for yeast cells for the long-term process and then keeping the mixture static for ethanol production from glycerol.
- Repeated-Batch Operation for Ethanol Production from Glycerol by ImmobilizedP. tannophilus
Among several operational strategies for an immobilized cell reactor, a repeated-batch operation with immobilized P. tannophilus was chosen for its simplicity and absence of particular equipment requirements except for a fermentor. As shown in Fig. 6 , the yeast cells were immobilized on Celite and the miroaeration rate was controlled at 0.11 vvm. Meanwhile, this microaeration rate was an optimized value through the operation of several batch runs, and was the same as that of Figs. 1 and 2 (data not shown). The first batch run was conducted until 54 h, after which four additional batch runs proceeded until 270 h. It was not possible to monitor the cell growth after Celite was added to the culture broth ( Fig. 6 A). The profiles of glycerol consumption and ethanol production are shown in Figs. 6 B and 6 C through the replacement process of the YCG medium. After Celite addition for cell immobilization, the maximum ethanol productions during the four batch runs ranged from 1.6-1.9 g/l up until 270 h ( Fig. 6 C), with the ethanol yield (Y e/g ) of 0.126 ± 0.017 g-ethanol/g-glycerol ( n = 4). When compared with Figs. 1 C and 3 C, lower ethanol production levels were observed because the glycerol was not consumed completely at each run. In addition, Y e/g was lower than that in Fig. 3 C. This was likely caused by the immobilization of the yeast cells on Celite. The yeast cells immobilized on Celite were not exactly the same as those in Figs. 1 and 3 in terms of chemical and physical conditions.
PPT Slide
Lager Image
Repeated-batch fermentor cultures for the production of ethanol from glycerol using immobilized P. tannophilus in YCG medium. Yeast cells were immobilized on Celite. (A) Cell growth in culture broth; (B) residual glycerol concentration; (C) ethanol production. The arrows indicate the time of Celite addition for immobilization.
Liu et al . [11] previously conducted fed-batch operations to produce ethanol from glycerol using P. tannophilus . The whole medium for ethanol production was prepared using YNB w/o amino acids, which is an expensive and defined medium preparation containing several nutrient ingredients. In this study, the performance of repeated-batch operation for ethanol production using YNBG medium (YNB w/o amino acid-based medium) was also investigated. The operational conditions for the repeated-batch culture were the same as those in Fig. 6 . As shown in Fig. 7 , the first batch run was conducted until 66 h, after which four more batch runs proceeded until 256 h. The profiles of glycerol consumption and ethanol production during the repeatedbatch operation are shown in Figs. 7 B and 7 C. After the addition of Celite for cell immobilization, the maximum ethanol production during the batch runs ranged from 1.9 to 2.4 g/l until 256 h ( Fig. 7 C), with the ethanol yield (Y e/g ) of 0.134 ± 0.025 g-ethanol/g-glycerol ( n = 4). These performances were very similar to those in Fig. 6 . Electron micrographs were obtained from the harvested sample after the culture of Fig. 6 was finished, as shown in Fig. 8 . In particular, the yeast cells immobilized on the surface of the Celite could be observed, where the appearance of the cells was similar to a cluster of eggs.
PPT Slide
Lager Image
Repeated-batch fermentor cultures for the production of ethanol from glycerol using immobilized P. tannophilus in YNBG medium. Yeast cells were immobilized on Celite. (A) Cell growth in culture broth; (B) residual glycerol concentration; (C) ethanol production. The arrows indicate the time of Celite addition for immobilization.
PPT Slide
Lager Image
Electron microscopy images of P. tannophilus immobilized on Celite (from Fig. 6). Samples for observation were collected at the time when the cultures were finished. (A) Celite and (B, C, D) yeast cells immobilized on Celite. Magnification of the electron micrographs: (A) 400×, (B) 5,000×, (C) 10,000×, and (D) 12,000×.
The ethanol yield based on glycerol consumption (Y e/g ) had first been shown to be 0.22 g-ethanol/g-glycerol in YPG medium ( Figs. 2 B and 2 C) and 0.16 g-ethanol/g-glycerol in YCG medium ( Figs. 3 B and 3 C), respectively, at the optimal microaeration rate. These results were obtained from the batch operation, whereas the Y e/g values in Figs. 6 and 7 were obtained from repeated-batch operations with yeast cells immobilized on Celite. Although the Y e/g in Figs. 6 and 7 could not be directly compared with those in Figs. 1 and 3 , it is necessary to improve the YCG medium for increasing Y e/g , as YPG medium cannot be used in the industrial production of ethanol from glycerol. However, the process for medium improvement might lead to an increase in the total cost of ethanol production. In future study, a strategic approach will be needed in view of profitability.
Liu et al . [11] showed Y e/g values ranging from 0.16 to 0.28 g-ethanol/g-glycerol during batch culture using YNB w/o amino-acid-based medium. In addition, Y e/g in the fedbatch culture was about 0.13 g-ethanol/g-glycerol during operation for about 250 h, where the culture was fed twice with the concentrated medium after the initial batch culture. In the study of Liu et al . [11] , the initial glycerol concentrations of 2-10% (v/v) in batch culture and 5% (v/v) in fed-batch culture were used for the ethanol production from glycerol. Therefore, the final concentrations and volumetric productivity of ethanol were not directly compared with those in this study. As mentioned above, we observed Y e/g values of 0.22 g-ethanol/g-glycerol in YPG medium and 0.16 g-ethanol/g-glycerol in YCG medium, respectively, during batch culture. Therefore, the Y e/g of the batch culture in this study was remarkably comparable with those of the previous study [11] . In addition, the Y e/g values of the repeated-batch cultures in this study ( Figs. 6 and 7 ) were also remarkably comparable with that of the previous study [11] . However, Liu et al . [11] showed ethanol production from glycerol conducted in the YNB w/o amino-acids-based medium, which should be substituted for a more cost-effective medium ingredient for industrial ethanol production. In addition, the Y e/g values decreased gradually during the fed-batch culture. Liu et al . [11] described that there was extreme decrease in the ethanol production due to ethanol inhibition, where little ethanol production in the last batch culture occurred.
In this study, we have provided the first investigation of the operational feasibility of repeated-batch culture for ethanol production with the use of P. tannophilus immobilized on Celite, particularly with the use of YCG medium. Because the YCG medium consisted of glycerol, CSL, industrial-grade yeast extract, and salts, the operation of a more practical ethanol production process from glycerol is expected in terms of process profitability. In particular, it was shown herein that Celite was an appropriate carrier for the immobilization of P. tannophilus , as Celite is an easily available and cheap material. In addition, the immobilization procedure was very simple without requiring any pretreatment, where Celite was simply added into the culture broth for immobilization. As a tool of long-term operation for ethanol production, the repeated-batch culture is known to be attractive. More long-term operation of this repeated-batch culture will be able to closely resemble the continuous process for ethanol production, whereas more equipment and instrumentation are not needed for continuous operation.
In conclusion, we established a repeated-batch process for the production of ethanol from glycerol by P. tannophilus immobilized on Celite for the first time, for which the microaeration rate was also optimized. In addition, a more practical medium ingredient was used herein in consideration of the process profitability. The ethanol yield (Y e/g ) was 0.126 ± 0.017 g-ethanol/g-glycerol ( n = 4), which was remarkably comparable with a previous report. In the future, it is expected that the results of this study will be used for the development of a more practical and profitable long-term ethanol production process.
Acknowledgements
This research was supported by a grant from the Marine Biotechnology Program Funded by the Ministry of Oceans and Fisheries, Korea.
References
Abbaszaadeh A , Ghobadian B , Omidkhah MR , Najafi G 2012 Current biodiesel production technologies: a comparative review. Energy Convers. Manage. 63 138 - 148    DOI : 10.1016/j.enconman.2012.02.027
Atkinson B , Mavituna F 1983 Biochemical Engineering and Biotechnology Handbook The Nature Press London, UK 772 - 773
Bondioli P , Bella LD 2005 An alternative spectrophotometric method for the determination of free glycerol in biodiesel. Eur. J. Lipid Sci. Technol. 107 153 - 157    DOI : 10.1002/ejlt.200401054
Clomburg JM , Gonzalez R 2013 Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol. 31 20 - 28    DOI : 10.1016/j.tibtech.2012.10.006
Dobson R , Gary V , Rumbold K 2012 Microbial utilization of crude glycerol for the production of value-added products. J. Ind. Microbiol. Biotechnol. 39 217 - 226    DOI : 10.1007/s10295-011-1038-0
Gancedo C , Gancedo JM , Sols A 1968 Glycerol metabolism in yeasts; pathways of utilization and production. Eur. J. Biochem. 5 165 - 172    DOI : 10.1111/j.1432-1033.1968.tb00353.x
Khare SK , Nakajima M 2000 Immobilization ofRhizopus japonicuslipase on celite and its application for enrichment of docosahexaenoic acid in soybean oil. Food Chem. 68 153 - 157    DOI : 10.1016/S0308-8146(99)00166-1
Klingenberg M 1970 Localization of the glycerol-phosphate dehydrogenase in the outer phase. Eur. J. Biochem. 13 247 - 252    DOI : 10.1111/j.1432-1033.1970.tb00924.x
Lee S-E , Kim Y-O , Choi W-Y , Kang D-H , Lee H-Y , Jung K-H 2013 Two-step process using the immobilizedSaccharomyces cerevisiaeandPichia stipitisfor ethanol production from the hydrolysate ofUlva pertusaKjellman. J. Microbiol. Biotechnol. 23 1434 - 1444    DOI : 10.4014/jmb.1304.04014
Lee S-E , Lee CG , Kang D-H , Lee H-Y , Jung K-H 2012 Preparation of corncob grits as a carrier for immobilizing yeast cells for ethanol production. J. Microbiol. Biotechnol. 22 1673 - 1680    DOI : 10.4014/jmb.1202.02049
Liu X , Jensen PR , Workman M 2012 Bioconversion of crude glycerol feedstocks into ethanol byPachysolen tannophilus. Bioresour. Technol. 104 579 - 586    DOI : 10.1016/j.biortech.2011.10.065
Maleszka R , Wang PY , Schneider H 1982 Ethanol production from D-galactose and glycerol byPachysolen tannophilus. Enzyme Microb. Technol. 4 349 - 352    DOI : 10.1016/0141-0229(82)90059-X
Rywiñska A , Juszczyk P , Wojtatowicz M , Robak M , Lazar Z , Tomaszewska L , Rymowicz W 2013 Glycerol as a promising substrate forYarrowia lipolyticabiotechnological applications. Biomass Bioenergy 48 148 - 166    DOI : 10.1016/j.biombioe.2012.11.021
Salvi BL , Panwar NL 2012 Biodiesel resources and production technologies - a review. Renew. Sustain. Energy Rev. 16 3680 - 3689    DOI : 10.1016/j.rser.2012.03.050
Shuler ML , Kargi F 2002 Bioprocess Engineering: Basic Concepts 2nd Ed. Prentice-Hall New Jersey 294 -
Shuler ML , Kargi F 2002 Bioprocess Engineering: Basic Concepts 2nd Ed. Prentice-Hall New Jersey 165 -
Vieira AM , Sá-Correia I , Novais JM , Cabral JMS 1989 Could the improvements in the alcoholic fermentation of high glucose concentrations by yeast immobilization be explained by media supplementation? Biotechnol. Lett. 11 137 - 140    DOI : 10.1007/BF01192190
Yazdani SS , Gonzalez R 2007 Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr. Opin. Biotechnol. 18 213 - 219    DOI : 10.1016/j.copbio.2007.05.002
Yeon J-H , Lee S-E , Choi WY , Kang D-H , Lee H-Y , Jung K-H 2011 Repeated-batch operation of surface-aerated fermentor for bioethanol production from the hydrolysate of seaweedSargassum sagamianum. J. Microbiol. Biotechnol. 21 323 - 331