Fuel 119 (2014) 328–334
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Stability of the process of simultaneous saccharification and fermentation of corn flour. The effect of structural changes of starch by stillage recycling and scaling up of the process _ Lewandowicz a, Piotr Kubiak a, Wioletta Błaszczak b Daria Szymanowska-Powałowska a,⇑, Grazyna a b
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-627 Poznan, Poland Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Division of Food Science, Tuwima 10, 10-747 Olsztyn, Poland
h i g h l i g h t s Stillage recycling does not affect effectiveness of repeated SSF process. Amount of unhydrolyzed residual starch was similar in every SSF cycles. The repeated SSF process is stable in scaling up and in industrial scale.
a r t i c l e
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Article history: Received 15 December 2011 Received in revised form 22 December 2012 Accepted 18 November 2013 Available online 2 December 2013 Keywords: Ethanol Simultaneous saccharification and fermentation Native corn starch
a b s t r a c t Intensive development of the transport sector and a rise in the prices of fossil fuels boost the demand for fuels from alternative sources of energy, including biofuels. New energy-efficient technologies of fuel production from renewable resources are developed. The aim of the present study was to examine the factors influencing the effectiveness of the process of simultaneous saccharification and fermentation of corn flour with full stillage recycling. The effect of structural changes of starch granules during the long-term repeated SSF process as well as the scale of the process were investigated. Commercially available STARGEN 001 enzymatic preparation and Saccharomyces cerevisiae strain Red Star Ethanol Red were used in the experiment. The results proved that raw material quality is of the utmost importance for the effectiveness of this processes. Bacterial contamination of the raw material caused decreased ethanol productivity despite similar substrate utilization. Process scale turned out to be a second significant factor influencing the SSF outcome. Increase of bioreactor volume resulted in decreased productivity. Repeated stillage recycling and the resulting concentration of broth ingredients has a lesser impact on the process. Ethanol content and the amount of residual starch was independent of the number of operation cycles. Formation of porous granules is predominant as starch undergoes hydrolysis. The affinity of the amylolytic enzyme used towards crystalline and amorphous regions is equal. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The economical competitiveness of different technologies for fuel ethanol production depends on the cost of the first stage of this process – obtainment of a solution of fermentable sugars. Three types of raw material requiring different processing can be used for ethanol manufacturing. Sugar crops (sugarcane or sugar beet) require only the extraction process before fermentation. From starch-containing plants (cereal grains, potato tubers, cassava roots) the polysaccharide first has to be extracted and then hydrolyzed. Lignocellulosic biomass (wood, straw) has to undergo the most complicated pretreatment prior to fermentation that ⇑ Corresponding author. Tel.: +48 61 846 60 10; fax: +48 61 846 6003. E-mail address:
[email protected] (D. Szymanowska-Powałowska). 0016-2361/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fuel.2013.11.034
includes removal of lignin (non-saccharide fraction) followed by hydrolysis of cellulose. The latter process is far more difficult than the saccharification of starch. Therefore, despite the fact that most of agricultural by-products are lignocellulosic materials, production of second generation bioethanol is currently economically not competitive [1]. For this reason, almost all bio-ethanol is produced from grain and sugarcane. Moreover, much effort is made to decrease the cost of the production of this biofuel using starchy raw materials, i.e. to improve the hydrolysis stage. The conventional process of enzyme hydrolysis of starch to produce fermentable sugars involves following steps: gelatinisation, liquefaction with thermostable a-amylase, and saccharification [2]. The energy consumption of theses processes usually amounts to about 30–40% of all energy required for ethanol production [3]. However, recently a new enzyme mix – STARGEN 001 – has
D. Szymanowska-Powałowska et al. / Fuel 119 (2014) 328–334
been developed by Genencor International that hydrolyzes granular starch. Employing this enzyme makes it possible to effectively perform the process of simultaneous hydrolysis of native starch and ethanol fermentation [4]. During this process sugars liberated by hydrolysis are instantly consumed by yeast. The process of simultaneous saccharification and fermentation (SSF) is energyand water-saving and results in higher ethanol productivity by avoiding the loss of fermenting sugars, which may occur during heating of fermentation broth (i.e. in the Maillard reaction). There are a few important factors determining economic effectiveness of bioethanol production, one of the most important being full utilization of sugars and the related efficient conversion of granular starch into ethanol. The key role in the hydrolysis of granular starch is played by their supramolecular structure, crystallinity and the presence of complexing agents [5,6]. These factors are determined by starch origin. The comparison of the four cheapest commercial starches in their native form in terms of their susceptibility to amylolysis sets them in the following diminishing order: corn P wheat > cassava > potato [7,8]. The second important factor is water management. Firstly, the growing global water shortage leads to an increase in its costs. Secondly, distillery stillage is ranked among especially burdensome industrial effluents, particularly in large biorefineries. One of the methods of water cost reduction applied by distilleries is the reutilization of stillage in the production process which enables not only to reasonably utilize this by-product, but also to significantly reduce the demand for production water. In order to improve the economic outcome of the SSF process, the zero-discharge fermentation system is introduced. This means that the solid-containing whole stillage is first separated using a decanter centrifuge. Afterwards, the solid phase is dried in a drum dryer to produce DDGS (distillers dry grain solids), a valuable co-product used for animal feed, whereas the liquid phase is evaporated in a double-effect evaporator [9]. Another way of improving the zero-discharge fermentation system is the application of the repeated SSF process with complete recycling of stillage liquid fraction. This approach assumes liquid phase recirculation into the simultaneous saccharification and fermentation process [9]. It should be emphasized that coupling hydrolysis to fermentation in time and space through SSF reduces the costs and duration of the process and eliminates the necessity of using two separate vessels [10]. The number of reports on the subject reflects the awareness of the key role played by the efficient hydrolysis of granular starch [11–16]. In spite of much effort of different research groups within the field, a lot of questions remain answered. Still it is not clear which regions of starch granules, crystalline or amorphous, are more susceptible to amylolysis [12,11,17,18]. Moreover, most of the reports utilize experimental systems significantly different from those occurring in industrial practice. The aim of the present study was to examine the factors influencing the effectiveness of simultaneous saccharification and fermentation of corn flour with full stillage recycling. The effects of structural changes of starch granules during the long-term repeated SSF process as well as scaling up of the process were investigated.
2. Materials and methods 2.1. Materials Commercially available corn flour (BIO CORN, Zie˛bice, Poland) was used as raw material for lab scale and pilot plant fermentation experiments. In the industrial scale corn grain ground using 1 mm mesh size was used. The corn grain contained 69% starch in dry mater and was significantly contaminated with soil and sand. Freeze-dried distiller’s yeast – Red Star Ethanol Red (Saccharomyces
329
cerevisiae) obtained from Lesaffre Company (France) was used for the production of ethanol from corn mashes. A mixture of granular-starch hydrolyzing enzymes, containing a-amylase and glucoamylase was used (STARGEN 001, Genencore International, USA) and fungal acid protease GC 106 (Genencore International, USA) were used. 2.2. Simultaneous saccharification and fermentation process The SSF experiments were performed in a 5 L bioreactor BioFlo III (New Brunswick, USA), containing 4 L of the fermentation medium. Corn flour was suspended in unsterilized water to obtain the mash of the concentration of 250 g L 1. The fermentation broth pH was adjusted to 5.0 by the addition of 10% H2SO4. In all cases, the medium was supplemented with acid protease GC 106 (40 lL kg 1 corn flour dry matter) and STARGEN 001 (2.05 mL kg 1 corn flour dry matter). The fermentation was started with the addition yeast (0.5 g L 1 of the fermentation medium). The batch SSF fermentations were performed in anaerobic, non-sterile conditions at 35 °C with medium agitation rate of 200 rpm for 72 h. After the fermentation period was completed, mash containing ethanol was pumped to a continuous distillation column (UOP3CC, Armfield, UK). At the top of this column, operating at 78.5 °C and reflux ratio of 4:1, carbon dioxide is stripped from the ethanol solution and leads to an ethanol concentration of 93–95% (volume based) in a side stream. The liquid fraction collected at the bottom of the distillation column, was first cooled down to 30 °C and then centrifuged at 4000g for 20 min. Supernatant was used instead of water to prepare the mash using native corn starch in the subsequent SSF batch. The amount of recirculated stillage was kept constant, amounting for 75% replacement of fresh water. The procedure was repeated twenty times over a period of 60 days, using a fresh yeast culture for inoculation during each run. Samples from the 4th, the 7th and the 20th cycles of fermentation were taken for analyses. Studies on the scaling up of fermentation processes comprised fur stages: using laboratory bioreactors of 5 L, then 30 L, next 150 L, and finally 1500 L. The procedure of the simultaneous hydrolysis and fermentation followed the scheme presented above. Industrial scale experiment was made at a agricultural distillery located near Poznan, Poland. The distillery was equipped with a mill coupled to a weighing tank, a mash tub with a stirrer, a jet– cooker, fermentation tanks, a system of pumps, a decantation centrifuge, slop tanks and a distillation column. First, the mash tub of 8000 L was filled with a weighed amount of maize grain taking into consideration moisture content of the raw material. Next, water was added to make up to a final volume of 8000 L. In the produced sweet mash of a concentration of 250 g L 1, pH was adjusted to 5.0 using 10% H2SO4 solution. In the next stage 4 L of STARGEN 001, 0.2 L acid protease GC 106 and 4 kg lyophilised yeasts were added. The entire volume was pumped to a fermentation tank. Fermentation was run for 72 h, with no adjustment of pH or temperature. Mash was stirred every 15 min using screw pumps coupled to fermentation tanks. Fermented mash was distilled on a column by Chemat, Poland. After distillation the eluate was centrifuged using a WD 350 decanter (Spomasz Wronki, Poland). The solid fraction (precipitate) was dried and used as animal feed, while the liquid fraction was used as a substitute of process water for the preparation of another batch of mash. All the experiments were performed in triplicate. The block diagram of the experiment was given at Fig. 1. 2.3. Analytical methods Analysis of sugars, ethanol and glycerol concentration were performed using on HPLC (Merck Hitachi, Germany) equipped with
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CORN + WATER + ENZYMES + YEASTS
SIMULTANEOUS SACCHARIFICATION AND FERMENTATION
SSF WITH RECIRCULATION - INDUSTRIAL SCALE
20 RECIRCULATIONS SCALING - UP
IR, X-ray, HPLC, yeasts enumeration
HPLC, yeasts enumeration HPLC, yeasts enumeration Fig. 1. Flowcharts of the experiment.
refractive index detector and an Aminex HPX-87P column (BioRad, USA) thermostated at 30 °C. 5 mM sulfuric acid solution was used as the mobile phase at a flow rate of 0.6 mL min 1. The starch content was analyzed according to the enzymatic method developed by Holm et al. [19]. The yeast cell populations were determined by a direct microscopic count in a counting chamber after staining with methylene blue. Pure starch was isolated from grain meal using the procedure described by Varatharajan et al. [20]. The purified granules were dried and then deposited on a copper disc, coated with gold using a Jeol JEE-400 vacuum evaporator, and analyzed with a scanning electron microscope (Jeol JSM 5200) under 10 kV accelerating voltage. The X-ray diffractometry was performed with a TUR 62 Carl Zeiss X-ray diffractometer under the following conditions: X-ray tube Cu Ka (Ni filter); voltage 30 kV; current 15 mA; scanning from Q = 2° to 18°. To avoid the influence of relative humidity on relative crystallinity, the starch samples were placed in a desiccator and conditioned in the atmosphere of relative humidity of 92% for 48 h. The FTIR measurements were performed in the solid state with an FTIR Bruker IFS 113v spectrometer under the following conditions: KBr pellet (200 mg 1.5 mg 1), resolution 2 cm 1.
3. Results and discussion Our previously reported study on the process of simultaneous saccharification and fermentation of raw corn starch using GSH (granular starch hydrolyzing) enzymes proved that although glycerol, lactic and acetic acid as well as inorganic ions content slightly increases with the increase in the number of recycling cycles, the yield of ethanol production does not decrease [9]. Moreover, the resulting increase in osmotic pressure did not influence yeast condition and enzyme activity. A slight increase in the amount of residual starch, which negatively affects the outcome of the fermentation, was observed. The application of the zero-discharge system, however, allows the solid-containing whole stillage to serve as animal feed after being separated and dried, thus mitigating the aforementioned influence. When the cost-effectiveness of the SSF is considered, the complete hydrolysis of starch to fermentable sugars is a priority. In order to determine the effect of stillage liquid fraction recycling on the effectiveness of starch hydrolysis, structural changes of starch granules were assessed after three selected batches. The 4th, the 7th and 20th cycle of the fermentation process were chosen randomly to this purpose. As shown in Table 1, no significant changes of the fermentation characteristics were observed during the
course of the fermentation between the 4th, the 7th and 20th cycle. Moreover, the results show that the SSF process ran correctly, i.e. starch hydrolysis ran parallel to the fermentation process. Together with the formation of starch hydrolysis products, ethanol concentration in the mash was increasing. After 72 h of the process, ethanol concentration amounted to about 11.5% v/v (90 g L 1). The final alcohol concentration is somewhat lower than than stated by the producer. According to Genencor International Inc. STARGEN 001 used on corn mash of the concentration of 33% dry mass gives the final ethanol concentration of 16–18%. This has also not been confirmed by other authors. Moreover, the results of Wang et al. obtained with STARGEN 001 are similar to ours [21]. These authors compared the process performed according to STARGEN 001 technology with the traditional process of ethanol production with liquefaction at a temperature of 90–120 °C, followed by saccharification with the use of two different combinations of liquefying and saccharifying enzymes (DG1 and DG2; a-amylase and glucoamylase), and completed by fermentation. Using both methods, the authors obtained similar final ethanol concentrations of 14.1–14.2% (v/v) with significantly different sugar profiles in the analyzed systems. It should be emphasized that the data presented in Table 1 proved no accumulation of disaccharides, so lack of glucoamylase activity was not the reason for the lower final concentration of ethanol in this experiment in comparison with the data of Genencor International Inc. However, there was a part of starch which was resistant to enzymatic hydrolysis. The most important factor determining starch resistance to enzymatic attack is its supermolecular structure [22]. Not only the type of crystal structure (A or B) but also the distribution of semi-crystalline and crystalline layers influences the rate of hydrolysis [17]. On the basis of the ‘‘blocklet’’ concept of the supramolecular organization of starch it is postulated that residual, not hydrolyzed material, exhibits a higher degree of crystallinity than native one [17]. However, data reported by Zhang et al. [18] points to even digestion of both crystalline and amorphous regions in native cereal starches during hydrolysis by the mixture of a-amylase and amyloglucosidase. Our studies using X-ray diffractometry, IR spectroscopy and scanning electron microscopy did not confirm the thesis that hydrolysis of starch preferentially takes place in amorphous regions. X-ray diffraction patterns of native corn starch as well as starch isolated after 4th, the 7th and the 20th cycle of the fermentation process (data not shown) did not reveal any changes neither in the type of crystal structure nor in the degree of crystallinity. However, different mechanism of enzymatic hydrolysis catalyzed by enzymes of different biological origin could results in different affinity to crystal and amorphous regions of starch [23]. Likewise, IR spectroscopy did not show any changes caused by the simultaneous saccharification and fermentation process, both in the functional groups as well as in the finger-print range (data not shown). Even the bands 1047 cm 1 and 1022 cm 1, considered as sensitive to the amount of crystalline and amorphous starch fractions did not change in the result of the hydrolysis process [24]. However, the FTIR technique does not enable differentiation between the A- and B-type of crystallinity and is less a powerful tool in crystal structure research than X-ray diffraction [25,26]. As observed in the SEM images of starch samples isolated after the 7th cycle of the fermentation (Fig. 2) the predominant part consisted of granules with the pores. This is a typical observation, reported by different research groups [7,27–29]. However, pyramid-shaped residuals, also described by Zhang et al., are present at the very last stage of the hydrolysis [18]. The latter as well as undigested starch granules were less prevalent (Fig. 2). The above observation suggests that the limits of the SSF were not reached and the degree of hydrolysis can be enhanced. Before commercialization, developed technologies usually undergo a scale-up procedure. Our research was was performed in
91.1 ± 1.0 40.1 ± 0.2 1.0 ± 0.1 9.2 ± 0.1 27.4 ± 0.3 12.0 ± 0.9 3.0 ± 0.2 3.7 18.0 ± 1.2 1.2
20th 7th 4th
87.4 ± 0.9 43.2 ± 0.2 4.0 ± 0.3 2.1 ± 0.1 23.0 ± 0.3 10.0 ± 0.5 3.0 ± 0.2 4.0 18.0 ± 1.5 1.2
20th
78.1 ± 0.9 87.5 ± 0.9 1.0 ± 0.1 10.8 ± 0.1 36.7 ± 0.3 10.0 ± 0.5 3.0 ± 0.2 3.6 17.0 ± 1.3 1.6
7th
76.2 ± 0.8 91.4 ± 0.8 1.0 ± 0.1 11.4 ± 0.1 26.5 ± 0.3 11.0 ± 0.4 3.0 ± 0.2 3.5 17.0 ± 1.4 1.6
4th
77.3 ± 0.8 77.1 ± 0.9 5.0 ± 0.2 9.7 ± 0.1 20.1 ± 0.3 9.0 ± 0.3 2.0 ± 0.2 3.7 19.0 ± 1.7 1.6 55.2 ± 0.5 146.3 ± 1.6 1.0 ± 0.1 21.4 ± 0.2 35.2 ± 0.3 10.0 ± 0.6 4.0 ± 0.3 3.7 14.0 ± 1.2 2.3
20th 7th
53.1 ± 0.4 157.6 ± 1.7 1.0 ± 0.1 23.6 ± 0.2 23.6 ± 0.2 11.0 ± 0.9 3.0 ± 0.1 3.6 14.0 ± 1.1 2.2
4th
56.2 ± 0.3 130.4 ± 1.6 1.0 ± 0.1 17.8 ± 0.1 18.0 ± 0.2 9.0 ± 0.9 2.0 ± 0.1 3.7 13.0 ± 0.9 2.3 0.0 210.2 ± 2.1 3.0 ± 0.2 11.3 ± 0.1 33.0 ± 0.2 8.0 ± 0.8 1.0 ± 0.1 5.0 9.0 ± 0.8 –
20th
Ethanol (g L 1) Starch (g L 1) Disaccharides [(g/L*)10 1] Glucose (g L 1) Glycerol (g L 1) Lactic acid [(g L 1) 10 1] Acetic acid [(g L 1) 10 1] pH Yeast [(cfu 108 mL 1) 10 1] Volumetric productivity (g EtOH L
1
h
1
)
7th
0.0 210.2 ± 2.2 5.0 ± 0.2 12.9 ± 0.1 22.0 ± 0.2 9.0 ± 0.7 1.0 ± 0.1 5.0 9.0 ± 0.7 –
4th
0.0 210.1 ± 2.1 2.0 ± 0.1 15.1 ± 0.1 15.4 ± 0.1 7.0 ± 0.6 1.0 ± 0.1 5.0 9.0 ± 0.8 –
72 Operational cycle 48 Operational cycle 24 Operational cycle 0 Operational cycle
Fermentation time (h) Process parameter
Table 1 Time course of SSF processes with stillage recycling after hydrolyzed in the 4th , the 7th and the 20th cycle of the SSF process.
331
90.2 ± 1.1 41.3 ± 0.2 3.0 ± 0.3 1.9 ± 0.1 37.3 ± 0.3 11.0 ± 0.7 4.0 ± 0.3 3.8 18.0 ± 1.4 1.2
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Fig. 2. Scanning electron micrographs (SEM) of corn starch in the 7th cycle of the SSF process.
four stages, in bioreactors of the following volumes, 5 L, through 30 and 150 up to 1500 L. The most important parameters determining the effectiveness of the technology investigated into were ethanol and starch concentration in the fermentation broth. The data presented in the Table 2 proved that the process performed very stable independently of the volume of the bioreactors. Only during the last 24 h a slight decrease in process kinetics took place. However, the decrease of final concentration of ethanol with the increase of bioreactor volume, by about 7%, 14% and 22% in case of bioreactors of the volume of 30, 150 and 1500 L, respectively, was observed (Table 2). Nevertheless, obtained results are satisfying. They are similar to those obtained by Siquiera et al. [30] in the study of scaling-up the process of ethanol fermentation using soya molasses. Higher values of final concentration of ethanol (19% v/v, which corresponds to 155 g L 1), obtained in the SSF process, was reported by Lamsal et al. [4]. However, that experiment was performed in significantly different conditions: lower scale of the process (100 ml of suspension), higher concentration of corn (30% of d.m), and longer fermentation time (96 h). Our previous experience shows that prolonging the fermentation over 72 h also results in the increase of ethanol concentration in the fermentation broth, however, it is accompanied by decreasing fermentation rate which makes the process ineffective, therefore, it was not included in this study. In contrast to ethanol concentration, utilization of starch was not dependent on the scale of the process and varied in a very narrow range from 89% to 90%. It corresponded to the residual starch concentration in the fermentation broth of 21–23 g L 1 (Table 2). The concentration of low molecular weight sugars was also low and did not exceed 3.5%. The permanent presence of glucose and maltose proved that there were no lack of the carbon source and that the fermentation could be continued. The most important parameter – the ethanol volumetric productivity decreased with the increase of the scale of the process from 1.62 g L 1 h 1 to 1.29 g L 1 h 1. The plateau of this parameter usually was observed after 48th hour of the fermentation process, in case of the biggest bioreactor even after 24th. Similar values of productivity were reported by Devantier et al. [31] for barley mashes, and Mojovic´ et al. [32] for 20% corn mashes. Higher values of productivity were obtained by Nicolic et al. [33], but using gelatinised corn starch as raw material. Concluding, the simultaneous saccharification and fermentation undergoes the scale-up stably. Noteworthy, despite the lack of high temperature processing, bacterial contamination was very low as suggested by the insignificant acetic and lactic
332
Table 2 Time course of SSF processes with stillage recycling after hydrolyzed in the scaling up of the process. Process parameter
Fermentation time (h) 0 Bioreactor volume (L)
Ethanol (g L 1) Starch (g L 1) Disaccharides [(g L 1) 10 1] Glucose (g L 1) Glycerol [(g L 1) 10 1] Lactic acid [(g L 1) 10 1] Acetic acid [(g L 1) 10 1] Yeast cell number [(cfu 108 mL 1) 10 1] Volumetric productivity (g EtOH L 1 h 1)
24 Bioreactor volume (L)
48 Bioreactor volume (L)
72 Bioreactor volume [L]
5
30
150
1500
5
30
150
1500
5
30
150
1500
5
30
150
1500
0.0 210.2 ± 2.3 2.0 ± 0.1 25.1 ± 02 9.0 ± 0.2 0.0 0.0 8.0 ± 0.5
0.0 208.2 ± 2.1 3.0 ± 0.1 20.2 ± 0.2 8.0 ± 0.3 0.0 0.0 7.0 ± 0.4
0.0 211.1 ± 2.2 3.0 ± 0.2 21.1 ± 0.2 9.0 ± 0.3 0.0 0.0 9.0 ± 0.8
0.0 210.2 ± 2.0 4.0 ± 0.3 20.5 ± 0.2 13.0 ± 0.2 0.0 0.0 8.0 ± 0.9
45.3 ± 0.3 155.1 ± 2.1 5.0 ± 0.2 16.6 ± 0.2 22.0 ± 0.2 8.0 ± 0.4 2.0 ± 0.1 19.0 ± 0.9
41.2 ± 0.3 167.1 ± 1.9 5.0 ± 0.2 15.7 ± 0.1 18.0 ± 0.9 8.0 ± 0.3 3.0 ± 0.1 12.0 ± 0.6
40.2 ± 0.3 165.2 ± 1.5 5.0 ± 0.2 14.3 ± 0.1 19.0 ± 0.9 9.0 ± 0.3 2.0 ± 0.1 15.0 ± 0.5
38.1 ± 0.3 170.1 ± 1.6 6.0 ± 0.3 12.9 ± 0.1 2.5 ± 1.1 10.0 ± 0.3 2.0 ± 0.1 15.0 ± 0.6
82.2 ± 0.3 55.3 ± 0.9 8.0 ± 0.4 5.2 ± 0.1 44.0 ± 1.2 10.0 ± 0.2 2.0 ± 0.1 20.0 ± 0.7
77.2 ± 0.3 61.2 ± 0.3 9.0 ± 0.4 3.3 ± 0.1 33.0 ± 1.2 10.0 ± 0.4 3.0 ± 0.1 17.0 ± 0.8
75.3 ± 0.3 66.3 ± 0.3 7.0 ± 0.4 4.1 ± 0.1 36.0 ± 0.9 10.0 ± 0.3 2.0 ± 0.1 17.0 ± 0.8
71.2 ± 0.3 69.3 ± 0.4 7.0 ± 0.4 6.3 ± 0.1 51.0 ± 0.9 11.0 ± 0.4 2.0 ± 0.1 16.0 ± 0.9
117.0 ± 0.3 22.2 ± 0.2 3.0 ± 0.3 1.2 ± 0.1 75.0 ± 1.1 11.0 ± 0.3 3.0 ± 0.2 24.0 ± 1.2
109.0 ± 0.3 23.3 ± 0.2 4.0 ± 0.3 1.0 ± 0.2 63.0 ± 1.1 11.0 ± 0.3 3.0 ± 0.2 18.0 ± 1.2
101.0 ± 0.3 21.1 ± 0.1 5.0 ± 0.4 1.4 ± 0.4 62.0 ± 1.2 12.0 ± 0.2 3.0 ± 0.2 18.0 ± 1.3
93.0 ± 0.3 23.2 ± 0.2 5.0 ± 0.4 0.8 ± 0.2 72.0 ± 1.2 14.0 ± 0.4 3.0 ± 0.3 19.0 ± 0.9
–
–
–
–
1.9
1.7
1.7
1.6
1.7
1.6
1.6
1.5
1.6
1.5
1.4
1.3
Process parameter
Fermentation time (h) 0 Operational cycle
1
Ethanol (g L ) Starch (g L 1) Disaccharides [(g L 1) 10 1] Glucose (g L 1) Glycerol [(g L 1) 10 1] Lactic acid [(g L 1) 10 1] Acetic acid [(g L 1) 10 1] pH Yeast [(cfu 108 mL 1) 10 1] Bacteria (cfu mL 1) Anaerobic (cfu mL 1) Lactic acid bacteria (cfu mL 1) Volumetric productivity (g EtOH L
1
h
1
)
24 Operational cycle
48 Operational cycle
72 Operational cycle
1th
2th
3th
1th
2th
3th
1th
2th
3th
1th
2th
3th
0.0 172.2 ± 0.5 5.0 ± 0.2 10.1 ± 0.1 11.0 ± 0.1 7.0 ± 0.2 4.0 ± 0.1 5.2 8.6 ± 0.8 4.0 106 5.0 103 0.7 102 –
0.0 174.3 ± 2.7 4.0 ± 0.3 22.1 ± 0.2 42.0 ± 1.2 72.0 ± 0.9 19.0 ± 0.3 3.9 7.6 ± 0.7 3.7 106 4.4 103 1.1 102 –
0.0 173.5 ± 2.3 3.0 ± 0.2 29.1 ± 0.2 64.0 ± 0.8 225.0 ± 1.8 43.0 ± 0.9 3.8 8.5 ± 0.8 4.1 106 3.8 103 1.0 102 –
50.0 ± 0.3 100.3 ± 2.0 1.0 ± 0.1 0.6 ± 0.0 36.0 ± 1.1 57.0 ± 0.9 4.0 ± 0.1 3.9 18.2 ± 1.1 5.8 107 1.8 104 1.8 103 2.1
63.5 ± 0.4 94.7 ± 1.1 1.0 ± 0.1 0.6 ± 0.0 71.0 ± 0.8 115.0 ± 0.9 15.0 ± 0.3 4.1 16.4 ± 1.1 6.6 107 2.6 104 2.1 103 2.6
40.3 ± 0.2 114.5 ± 2.0 2.0 ± 0.1 3.3 ± 0.2 83.0 ± 0.9 237.0 ± 1.9 47.0 ± 0.9 3.7 12.1 ± 1.1 5.1 107 1.9 104 1.7 103 1.8
67.3 ± 0.4 65.1 ± 0.5 1.0 ± 0.1 0.6 ± 0.0 45.0 ± 1.2 64.0 ± 0.7 5.0 ± 0.1 3.7 19.8 ± 1.1 6.5 108 0.6 105 1.5 104 1.4
80.2 ± 0.7 56.3 ± 0.3 1.0 ± 0.1 7.1 ± 0.2 88.0 ± 0.9 153.0 ± 1.6 20.0 ± 0.9 4.9 15.9 ± 1.1 2.5 108 1.6 105 1.1 104 1.7
53.6 ± 0.4 63.1 ± 0.8 1.0 ± 0.1 5.6 ± 0.1 91.0 ± 0.9 292.0 ± 2.1 102.0 ± 1.1 3.4 14.0 ± 1.2 6.0 108 3.8 105 2.1 104 1.1
76.4 ± 0.6 22.8 ± 0.7 10.0 ± 0.1 3.6 ± 0.1 59.0 ± 0.8 82.0 ± 1.1 6.0 ± 0.2 3.7 14.5 ± 1.2 2.0 109 4.0 105 2.1 104 1.0
85.5 ± 0.6 19.7 ± 0.3 34.0 ± 0.6 4.1 ± 0.2 91.0 ± 1.1 215.0 ± 2.1 24.0 ± 0.9 3.7 11.8 ± 1.1 3.0 109 3.2 105 0.9 104 1.2
66.3 ± 0.4 45.1 ± 0.2 30.0 ± 0.6 6.2 ± 0.2 105.0 ± 1.2 325.0 ± 3.1 127.0 ± 1.5 3.3 11.6 ± 1.2 3.3 109 3.6 105 1.5 104 0.9
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Table 3 Time course of SSF processes with stillage recycling after hydrolyzed in the enhancement of the industrial scale.
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acids concentrations (Table 2). However, it was probably due to high quality raw material (corn flour) used in the pilot plant experiment. Significantly different conditions were employed during the last experiment performed in a distillery. Firstly, corn grains were of rather low quality. They were partially underdeveloped and contaminated with soil. The concentration of starch in grains was of near 69%, whereas in the case of lab-scale experiments it was near 86%. Moreover, the moisture content was similar to critical value for microbial growth, thus indicating partial decomposition. However, such quality of the raw material is typical for distillery practise. It results in a relatively low pH value of the fermentation mash (5.1–5.2) at the beginning of the process with no need of its adjustment. The pH lowered to 3.3–3.6 after three days of the fermentation (Table 3). While it is typical of ethanol fermentation to observe a pH drop, the increased microbial contamination and the resulting acid formation added caused increased acidification [34]. The reports of other authors concerning industrial scale experiments pointed out the very low efficiency of the process. For example, Suresh et al. [35], using 25% mashes containing damaged wheat or sorgo grains, reached the final ethanol concentration of 4.4% and 3.5% (v/v) respectively. Higher concentration of ethanol (6.7% w/v) was reported by Montesinos and Navarro [36]. However that process was preceded by liquefaction at the temperature of 60 °C. Summarizing, the distillery experiment shows the potential of the SSF process, which could be competitive to the traditional technology. Stillage recycling appears to be economically justified as it minimizes water consumption and waste emission, thus contributing greatly to the overall production costs. Keeping appropriate technological standards could make it possible to obtain a final ethanol concentration of 12% (w/v). As the high temperature substrate pretreatment is omitted, the process becomes less energy demanding but bacterial contamination monitoring and stricter hygiene regimes become critical [37,38]. Contamination problems can be avoided through the use of antibiotics [39,40], it is, however, banned in Europe (in contrast to America) due to the utilization of stillage for fodder. 4. Conclusions The described repeated batch SSF process with stillage recycling conducted with corn flour and the use of STARGEN 001 enzyme preparation runs efficiently in spite of the accumulation of glycerol, organic acids and inorganic ions in the fermentation broth. The most important factor influencing effectiveness of this processes is the quality of the raw material. Bacterial contamination of the raw material caused lower ethanol productivity despite similar utilization of starch. Instead of ethanol, bacterial metabolites i.e. lactic and acetic acids were produced. The scale of the process proved to be another important factor influencing the effectiveness of the simultaneous saccharification and fermentation. The increase of bioreactor volume resulted in decrease of productivity accompanied by similar level of starch utilization. The effect of the number of recirculation cycles is a less important factor. Ethanol content as well as the amount of residual starch is independent of the number of operation cycles. Hydrolysis of starch undergoes with predominant formation of porous granules and an additional small amount of undigested granules and pyramid-shaped residuals. Crystalline and amorphous regions are evenly digested. References [1] Mussato SI, Dragone G, Guimarães PMR, Silva JPA, Carneiro LM, Roberto IC, et al. Technological trends, global market, and challenges of bio-ethanol production. Biotechnol Adv 2010;2010(28):817–30.
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