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Effect of fruit pulp supplementation on rapid and enhanced ethanol production in very high gravity (VHG) fermentation

Effect of fruit pulp supplementation on rapid and enhanced ethanol production in very high... Background: The energy crisis and climate change necessitate studying and discovering of new processes involved in the production of alternative and renewable energy sources. Very high gravity (VHG) fermentation is one such process improvement aimed at increasing both the rate of fermentation and ethanol concentration. The technology involves preparation and fermentation of media containing 300 g or more of dissolved solids per liter to get a high amount of ethanol. Findings: Saccharomyces cerevisiae was inoculated to the very high gravity medium containing 30% to 40% w/v glucose with and without supplementation of three selected fruit pulps (mango, banana, and sapota). The fermentation experiments were carried out in batch mode. The effect of supplementation of 4% fruit pulp/puree on the metabolic behavior and viability of yeast was studied. Significant increase in ethanol yields up to 83.1% and dramatic decrease in glycerol up to 35% and trehalose production up to 100% were observed in the presence of fruit pulp. The fermentation rate was increased, and time to produce maximum ethanol was decreased from 5 to 3 days with increased viable cell count. The physical and chemical factors of fruit pulps may aid in reducing the osmotic stress of high gravity fermentation as well as enhanced ethanol yield. Conclusions: It was found that fruit pulp supplementation not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. Production of high ethanol concentration by the supplementation of cheap materials in VHG sugar fermentation will eliminate the expensive steps in the conventional process and save time. Keywords: High gravity fermentation; Osmotic stress; Ethanol; Fruit pulp supplementation Background aimed at increasing both the rate of fermentation and The energy crisis and climate change necessitate study- ethanol concentration. The technology involves prepar- ing and discovering of new processes involved in the ation and fermentation of media containing 300 g or production of alternative and renewable energy sources. more of dissolved solids per liter [4]. VHG fermentation Bioethanol is regarded as a promising alternative energy influences the five basic fermentation assets: (1) plant source, which is both renewable and environmentally and equipment, (2) raw materials, (3) utilities and con- friendly [1,2]. The commonly used ethanol producer in sumables, (4) personnel, and (5) money [5]. High gravity industries is Saccharomyces cerevisiae and the initial fermentation is an accepted method to produce more sugar concentration will not exceed 20% and the con- ethanol in existing fermenters and distil houses (affect- ventional ethanol production process needs high energy, ing item 1), and uses less cooling equipment and pro- high cost and low productivity [3]. Very high gravity duces less effluent (affecting item 3), resulting in higher (VHG) fermentation is one such process improvement yield (affecting item 2) and less staff work (affecting item 4); all these properties decrease the money investment * Correspondence: lvereddy@yahoo.com for ethanol production. Another advantage is an increase Department of Microbiology, Yogi Vemana University, Kadapa, Andhra in opportunities for harvest of high protein spent yeast Pradesh 516003, India [4,6]. Full list of author information is available at the end of the article © 2014 Lebaka et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 2 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 However, the high sugar content of the very high grav- were purchased from the local market of Kadapa, India. ity fermentation medium causes an increase in the os- The fruits were peeled off, and the pulp was separated motic pressure, which has a pessimistic effect on yeast from stones in the case of mango and sapota and pre- cells, and the fermentations are rarely fast and complete. pared as puree with a macerator. The prepared puree The ethanol produced by the yeast also poses negative will have good suspension in the fermentation medium. effects on yeast metabolism like enzyme inhibition and membrane solubility and needs some protectants to Fermentation counteract these effects at the end of the fermentation The very high gravity fermentation medium composition process [3]. S. cerevisiae can ferment an increased is the same as the abovementioned MPYD medium with amount of sugars in the medium when all required nu- high glucose concentration (300 to 400 g/L); 4% fruit trients are provided in adequate amounts [6]. Specific pulp/puree was supplemented to 300 and 400 g/L sugar nutrients, such as nitrogen, trace elements, or vitamins, medium to evaluate the potential effect of fruit pulps in are required to obtain rapid fermentation and high etha- enhancing the ethanol production, and the medium nol levels, which are desirable to minimize capital costs without supplementation of fruit pulp was treated as con- and distillation energy. On a laboratory scale, media are trol. Fermentations were conducted at 30°C in 250-mL often supplemented with peptone, yeast extract, amino Erlenmeyer flasks with 100 mL of fermentation medium. acids, and vitamins [6-8]. However, such addition is not The initial pH was adjusted to 5.5. The progress of feasible in industrial fermentation processes due to the fermentation was monitored by periodical sample analysis. associated high costs. Thus, it is necessary to exploit in- The fermentation was stopped after 5 days, and samples expensive nutrient sources to supply all nutritional re- were kept at −4°C until the analysis. quirements for yeast growth and fermentation. Many investigators studied the effect of inexpensive substances Analytical estimations like soy flour, oils and fatty acids, fungal mycelia, and Sugar concentration was estimated using the Shaffer and fruit pulp [9-12] on the improvement of ethanol produc- Somogyi method [15] as follows: Reducing sugars were esti- tion. In our laboratory, we have tested finger millet and mated using the idometric method of Shaffer and Somogyi horse gram powder supplementation in VHG fermenta- (1933). Sugars containing a free sugar syrup group undergo tion and successfully improved the ethanol production enolization when placed in an alkaline solution. Enediol [13,14]. forms of sugars are highly reactive to acids. The reduced In view of the above, we have screened 15 different copper was quantified by idometric titration using starch commercially available fruits to determine their effect on as an indicator. The 1 L reagent contains sodium carbonate very high gravity fermentation in terms of fermentation (25 g), Rochelle salt (25 g), copper sulfate (75 mL from rate and enhancement of ethanol yield. In this paper, we 100 g/L solution), sodium bicarbonate (20 g), potassium presented the results of tropical fruit pulps mango (Man- iodide (5 g), and potassium iodate (3.567 g). A 5 mL solu- gifera indica), banana (Musa paradisiaca), and sapota tion containing 0.5 to 2.5 mg dextrose units was pipetted (Achras sapota), which show a significant effect in enhan- into test tubes and 5 mL of reagent was added; then, the cing the ethanol production during the screening process. solution with the added reagent was mixed well by stirring. Tubes capped with bulbs were placed in a boiling water Materials and methods bath for 15 min and cooled under running water. Next, Organism and cultural conditions 2 mL of idodine-oxalate titrated with 0.005 N sodium Yeast strain S. cerevisiae 3215 was used in all the experi- thiosulfate was added using starch as indicator. Ethanol ments. The yeast strain was obtained from National was determined with the help of gas chromatography Collection of Industrial Microorganisms (NCIM, Pune, [16]. The fermented samples were centrifuged at India). The culture was maintained on MPYD (malt ex- 5,000 rpm for 10 min. The supernatant was used for tract 3 g/L, peptone 5 g/L, yeast extract 3 g/L, and dex- ethanol analysis. An Agilent Systems Gas Chromato- trose 2 g/L) agar (1.5 g/L) slants at 4°C. The inoculum was graph with Flame Ionization Detector (GC-FID) Model prepared by inoculating the slant culture into 25 mL of 6890 Plus instrument (Agilent Technologies Inc., Santa the sterile MPYD liquid medium taken in a 100-mL flask Clara, CA, USA) was used, and conditions were as follows: and growing it on a rotary shaker (100 rpm) for 48 h. The 5% Carbowax 20M glass column (6 ft (2 m), 2-mm inner above produced yeast culture (5%, 1 × 10 cells/mL) was diameter (ID), 1/4 mm). Nitrogen was used as a carrier used as inoculum to initiate the fermentation. gas with a flow of 20 mL/min, and the eluted com- pounds were detected using a flame ionization detector Fruit pulp preparation for supplementation (FID). For this, the fuel gas was hydrogen with a flow The fruits selected for the supplementation, mango (M. rate of 40 mL/min, and the oxidant was air with a flow indiaca), banana (M. praradisiaca), and sapota (A. sapota), rate of 40 mL/min; n-propanol was used as internal Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 3 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 standard. Glycerol on diluted samples was estimated and incompletely fermented by S. cerevisiae.However, using Boehringer kits (Boehringer Mannheim (Roche, the 4% fruit pulp supplementation led to a significant Basel Switzerland); enzymatic test (340 nm) 3 × 11 de- increase in ethanol production, and the final concentra- terminations (code number 10148270035)). Trehalose tion reached 14.5% (w/v) in a shorter time (72 h) with a was estimated in the supernatant by the anthrone productivity of 2.1 g/h/L (Table 1). In the three fruit method as described previously [17]. The pellet ob- pulps selected, mango supplementation gave the high- tained after the trehalose extraction was used for the est yields of ethanol when compared to banana and protein estimation. sapota. In the fruit pulp-supplemented medium, 10% (v/v) of ethanol production was achieved in just 48 h Cell viability after the inoculation. Besides the high fermentation rate Cellular viability was determined by the methylene blue in the supplemented medium, it also decreased the dur- staining technique [18]. A 100 mL sterile solution of ation of fermentation from 5 to 3 days. methylene blue (3.3 mM in 68 mM sodium citrate) was An attempt was made to increase ethanol production mixed with 100 mL of a yeast suspension diluted to up to 18% to 20% (w/v) as in the case of sake fermenta- reach an OD of 0.4 to 0.7 at 620 nm. This mixture was tion, by increasing the sugar concentration from 300 to shaken, and after a 5-min incubation, it was placed in a 400 g/L with supplementation of fruit pulp. In the Thomas counting chamber. The number of stained (in- 400 g/L sugar fermentation with 4% fruit pulp supplemen- active cells) and unstained (active cells) were counted in tation, the ethanol concentration was 12.5% (Table 1). The five different fields with total of at least 200 to 300 cells. sugars were utilized maximally up to 300 g/L. In the three fruit pulps selected, mango supplementation gave the Statistical analysis highest yields of ethanol when compared to banana and All the experiments were carried out three times (tripli- sapota. The ethanol production after 5 days in the control cate), and the mean value with standard deviation and experiments was only 7.5%. significant (P) was determined. SPSS version 11.0 was used for analysis of variance. Effect of fruit pulp supplementation on cell viability Results After 30 to 35 h of fermentation in both supplemented The present study provides potential observation of and control media, cell growth rate was decreased. fruit pulps as supplements in small quantity during fer- After 40 to 50 h, the growth ceased, but glucose fer- mentation stimulating the rate of alcohol production mentation continued slowly until the number of the vi- and final alcohol concentration in very high gravity fer- able cell count decreased and became very low. The mentation. Two sets of batch fermentation experiments viability percentage of yeast cells in the supplemented with two levels of sugar concentrations, 300 and 400 g/ medium was greater than that in the control medium. L (30% and 40%), were carried out with and without The supplementation of fruit pulp led to an increase in fruit pulp supplementation, in order to evaluate the ef- the rate of fermentation and ethanol yield through the fect of fruit pulps. The unsupplemented batch fermen- extended growth phase of cells (Figure 1). In the three tation experiments yielded only 9% (w/v) of alcohol in fruit pulps selected, mango supplementation gave a higher 300 g/L, and a good amount of residual sugars was left cell viability than banana and sapota supplementation. Table 1 Effect of fruit pulp supplementation on ethanol production in 30 and 40% sugar fermentation Serial number Supplement Alcohol concentration (w/v) 24 h % IMP 48 h % IMP 72 h % IMP 30% Sugar 1 Control 2.5 ± 0.3 - 5.5 ± 0.3 - 9.0 ± 0.7 - 2 Mango 4.5 ± 0.5 90 ± 4.5 10 ± 0.8 90.5 ± 8.0 14.5 ± 1.2 80.5 ± 10 3 Banana 4.0 ± 0.5 80 ± 4.0 9.0 ± 1.0 81.5 ± 9.0 13.2 ± 1.0 73.1 ± 8.0 4 Chiku 4.0 ± 0.4 80 ± 4.2 7.5 ± 0.6 68 ± 6.2 12 ± 0.8 66.5 ± 4.8 40% Sugar 1 Control 2.0 ± 0.3 - 4.5 ± 0.5 - 7.5 ± 0.6 - 2 Mango 4.0 ± 0.2 100 ± 2.0 8.4 ± 0.7 93 ± 7.0 12.5 ± 1.0 83.3 ± 10 3 Banana 3.3 ± 0.3 82.5 ± 2.5 8.0 ± 0.6 87.6 ± 6.2 11 ± 0.8 73.3 ± 7.0 4 Chiku 3.2 ± 0.2 77.5 ± 2.0 7.2 ± 0.8 80 ± 6.8 10 ± 1.0 66.5 ± 8.1 % IMP, percentage of improvement. Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 4 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 stimulating the rate of alcohol production and final alcohol concentration in very high gravity fermentation. In unsup- plemented controls of 30% glucose fermentation experi- ments, compared with the supplemented medium, the sugar was not utilized completely. It is evident that at the end of fermentation, yeast requires certain nutrients that aid tolerance to the high concentrations of alcohol it forms. Nearly 70% to 75% of the volume of the final ethanol con- centration was formed within 48 h of fermentation, and al- most all the final concentration of ethanol was formed in 60 h; the remaining 1% or 2% (v/v) took some time for its Figure 1 Effect of fruit pulp supplementation on yeast cell secretion out of the cell. In addition to nutrients, fruit pulps viability in 30% sugar fermentation. Diamond, control; square, also contain good amounts of polyphenols (all flavones, stil- mango; triangle, banana; circle, sapota. benes, flavonones, isoflavones, catechins, chalcones, tan- nins, and anthocyanidins), which are frequently attributed Effect of fruit pulp supplementation on glycerol to antioxidant, metal ion-chelating, and/or free radical scav- production and trehalose enging activity [19]. This may help in keeping the yeast cells Concentrations of glycerol, one of the stress indicators and viable forlongerdurationand producing suchhighconcen- releasers, were decreased in the fruit pulp-supplemented trations of ethanol in 48 h. The supplemented medium had experiments from 954 to 620 mg/L in the 300 g/L fer- higher viable cell count than the control medium. There 7 7 mentation and from 1,266 to 823 mg/L in the 400 g/L was a dramatic drop in cell count from 10 × 10 to 3 × 10 fermentation (Table 2). In the three fruit pulps selected, in the control medium with increase in ethanol concentra- mango supplementation gave less glycerol when com- tion from 5% to 9% (v/v). But in the supplemented medium, pared to banana and sapota supplementation. Trehalose is the cell viability went up even up to 12% (v/v)ethanol. This a disaccharide which is typically produced by yeast when indicates that the threshold concentration of ethanol to it experiences stress conditions. In the present study, yeast inhibition is 9% (v/v). In all cases, the cell viability in- trehalose concentration was in low in the fruit pulp- creased even at high ethanol concentration (12% v/v)inthe supplemented experiments when compared to the unsup- fruit pulp-supplemented medium compared with the con- plemented control experiments (Table 3). In the control trol medium. Alfenore et al. [6] made a similar observation 30% (w/v) fermentation experiments, the trehalose con- in fed-batch fermentation by vitamin feeding strategy that centration was 40 mg/g yeast cells, and in the fruit pulp- enhanced the final ethanol up to 19% (v/v)in45h. supplemented experiments, it was 21 mg/g yeast cells. In The important byproduct formed during ethanol fer- the 40% sugar (w/v) control fermentation experiments, mentation is glycerol. Commonly, its production is high the trehalose concentration was 52 mg/g yeast cells, in high gravity fermentation. Glycerol is the well-known while in the fruit pulp supplementation with aeration compatible solute in S. cerevisiae. Osmophilic yeasts ac- experiments, it was 38 mg/g yeast cells. In the three cumulate glycerol to compensate for high osmotic pres- fruit pulps selected, mango supplementation decreased sure [20,21]. In the present study, the formation of the trehalose to low levels when compared to banana glycerol was found to be high at the growth/logarithmic and sapota supplementation. phase. After cessation of cell growth, glycerol was not present much in the media. The percentage of glycerol Discussion in the supplemented media was low when compared The present study provides potential observation of fruit with that in the control medium. These results con- pulps as supplements in small quantity during fermentation firmed the previous reports that the growth rate of yeast cells is reduced irreversibly in proportion to an increase in external osmolarity [21]. Another important reserve Table 2 Effect of fruit pulp supplementation on glycerol carbohydrate and stress protectant for the yeast is tre- production in 30 and 40% sugar fermentation halose. Trehalose is also considered as one of the most Serial 30% Sugar 40% Sugar effective saccharines in preventing phase transition in number Supplement Glycerol (mg/L) Supplement Glycerol (mg/L) the lipid bilayer and thereby protecting membranes 1 Control 954 ± 62 Control 1,266 ± 75 against damages, and considering the relation of intra- cellular trehalose concentration with the cellular resist- 2 Mango 620 ± 35 Mango 823 ± 54 ance to osmotic stress, trehalose was supposed to act as 3 Banana 757 ± 58 Banana 938 ± 68 an osmoprotectant under osmotic stress [20]. In the 4 Chiku 826 ± 73 Chiku 1,040 ± 47 supplemented medium, trehalose concentration was Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 5 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 Table 3 Effect of fruit pulp supplementation on trehalose accumulation in 30 and 40% sugar fermentation Serial number 30% Sugar 40% Sugar Supplement Trehalose (mg/g yeast cells) Supplement Trehalose (mg/g yeast cells) 1 Control 40 ± 3.4 Control 52 ± 2.5 2 Mango 21 ± 2.8 Mango 38 ± 3.2 3 Banana 29 ± 3.3 Banana 43 ± 4.6 4 Chiku 34 ± 2.6 Chiku 49 ± 3.8 decreased at the end of the fermentation which shows beginning of fermentation and high ethanol stress at the that the cells are not under stress when compared to end of the fermentation. The increased ethanol produc- the control. This could explain the fact that stress induced tion by the fruit pulp supplementation is a significant the genes involved in trehalose synthesis and those in- finding that could also be applied to an industrial fer- volved in degradation, and why the genes responded in a mentation of ethanol utilizing molasses and other raw similar pattern in osmotic and oxidative stress [22]. It has materials as substrates. This may reduce the cost of been reported that the production pattern of protein syn- ethanol production in developing countries like India. thesis is changed dramatically by osmotic and heat stress, The nature of active principles from fruit pulps and their and also depriving amino acids or proteins inhibits trans- mechanism that aids in tolerating high osmotic stress lation initiation through the phosphorylation pathway and enhance ethanol production rate are being investi- [23,24]. gated by the authors. Higher amount of ethanol in the 400 g/L sugar medium Abbreviations was not obtained probably due to the initial high glucose % IMP: percentage of improvement; %: percent; g/l: gram per liter; mg/ concentration that strongly inhibited fermentation. Even l: milligram per liter; mM: millimolar; MPYD: malt extract, peptone, yeast extract, and dextrose (medium); v/v: volume per volume; VHG: very high in such high osmolarity, the supplemented media yield gravity; w/v: weight per volume. 12.5% (v/v) ethanol with a productivity of 1.73 g/h/L. It is likely that the supplementation of fruit pulp may add Competing interests sugars, thereby contributing to the increased osmotic The authors declare that they have no competing interests. pressure. The old yeast cells ferment slowly when com- Authors' contributions pared with actively growing yeast cells. It is possible to LV participated in the design of the study, carried out the fermentations, produce high ethanol concentrations by extending the analyzed the results, and wrote the manuscript. YJ participated in the experimental procedure and the GC and result analysis. HW conceived the growth phase of yeast to longer periods as in the case of study and participated in analyzing the results and correcting the beer production. It is expected that fruit pulp supplemen- manuscript. All authors read and approved the final manuscript. tation would overcome nutritional deficiencies of yeast and allow them to stay longer in the growth phase and Acknowledgements The author would like to acknowledge the Council of Scientific and that antioxidants protected the yeast cells from osmotic Industrial Research, Government of India and Department of Science and stress and aeration allowed yeast to produce membrane Technology, Government of India for the financial support given in the form lipids to be sustained at higher alcohol concentrations. of research projects entitled ‘Studies on Rapid and Enhanced Production of Ethanol through Very High Gravity (VHG) Fermentation’ (Ref No: 38 (1310)/ During VHG ethanol fermentation, maintaining the redox 11/EMR-II) and ‘Biotechnological production of Acetone-Butanol-Ethanol potential at a constant level is essential, as yeast requires a (ABE) from agricultural biomass using solventogenic bacteria’ (Ref No: SR/FT/ small amount of oxygen to facilitate the synthesis of ste- LS-79/2009). rols and unsaturated fatty acids, which serve as the build- Author details ing blocks for constructing cell membranes [25]. 1 Department of Microbiology, Yogi Vemana University, Kadapa, Andhra Pradesh 516003, India. School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Korea. Department of Food Conclusions Science and Technology, College of Natural Resources, Yeungnam University, It is concluded that fruit pulp supplementation en- Gyeongbuk 712-749, Korea. hanced the rate and yield of ethanol production in a very Received: 22 July 2014 Accepted: 19 October 2014 high gravity medium. It is observed that the selected fruit pulps were not much effective in the 400 g/L sugar fermentation when compared to the 300 g/L sugar fer- References mentation. The decrease in both glycerol and trehalose 1. Cardona C, Sa´nchez O (2007) Fuel ethanol production: process design trends and integration opportunities. Bioresour Technol 98:2415–2457 concentrations by the supplementation would suggest 2. Reddy LVA (2013). Potential bioresources as future sources of biofuels production: that the fruit pulp constituents might be involved in an overview. V. K. Gupta and M. G. Tuohy (eds.), Biofuel technologies, Springer- lowering the osmotic stress induced by high sugar at the Verlag Berlin Heidelberg doi:10.1007/978-3-642-34519-7_9, 2013 Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 6 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 3. Pereira FB, Guimarães PMR, Teixeira JA, Domingues L (2010) Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs. Bioresour Technol 101:7856–7863 4. Thomas KC, Hynes SH, Jones AM, Ingledew WM (1993) Production of fuel alcohol from wheat by VHG technology. Appl Biochem Biotechnol 43:211–226 5. Reeve P (1998) Sweat your fermentation assets. Brewer 12:212–215 6. Bafrncova P, Smogrovicova D, Salvikova I, Patkova J, Domeny Z (1999) Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae. Biotechnol Lett 21:337–341 7. Casey GP, Magnus CA, Ingledew WM (1984) High-gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production. Appl Environ Microbiol 48:639–646 8. Alfenore S, Molina-Jouve C, Guillouet SE, Uribelarrea JL, Goma G, Benbadis L (2002) Improving ethanol production and viability of Saccharomyces cerevisiae by vitamin feeding strategy during fed batch process. Appl Microbiol Biotechnol 60:67–72 9. Damoano D, Wang SS (1985) Improvements in ethanol concentration and fermentor ethanol productivity in yeast fermentations using whole soy flour in batch and continuous recycle systems. Biotechnol Lett 71:35–140 10. Deepak S, Visvanathan L (1984) Effects of oils and fatty acids on the tolerance of distillers yeast to alcohol and temperature. Enzyme Microb Technol 6:78–80 11. Patil SG, Patil BG (1989) Chitin supplement speeds up the ethanol production in cane molasses fermentation. Enzyme Microb Technol 11:38–43 12. Patil SG, Patil BG, Gokhale VD, Bastawde KB, Puntambekar S, Ranjekar PK (2000) Process for the production of alcohol. US Patent no: 6016699. 13. Reddy LVA, Reddy OVS (2005) Improvement of ethanol production in very high gravity fermentation by horse gram (Dolichos biflorus) flour supplementation. Lett Appl Microbiol 41:440–445 14. Reddy LVA, Reddy OVS (2006) Rapid and enhanced production of ethanol in very high gravity (VHG) sugar fermentation by Saccharomyces cerevisiae: role of finger millet (Eleusinae coracana L.) flour. Process Biochem 41:726–729 15. Shaffer PA, Somogyi M (1933) Copper iodometric reagents for sugar determination. J Biol Chem 100:695–713 16. Antony JC (1984) Malt beverages and malt brewing materials: gas chromatographic determination of ethanol in beer. J Assoc Off Annal Chem 67:192–193 17. Aranda JS, Salgado E, Taillandier P (2004) Trehalose accumulation in Saccharomyces cerevisiae cells: experimental data and structured modeling. Biochem Eng J 17:129–140 18. Postgate JP (1967) Viable counts and viability. In: Norris JR, Ribbons DW (eds) Methods in microbiology, vol. 1. Academic Press, New York 19. Ferguson LR (2001) Role of plant polyphenols in genomic stability. Mutat Res 475:89–111 20. Li LL, Ye YR, Pan L, Zhu Y, Zheng S, Lin Y (2009) The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses. BBRC 387:778–783 21. Klipp E, Nordlander B, Krüger R, Gennemark P, Hohmann S (2005) Integrative model of the response of yeast to osmotic shock. Nat Biotechnol 23:975–982 22. Da Costa M, Da Silva C, Mariani D, Fernandes P, Pereira M, Panek A, Eleutherio E (2008) The role of trehalose and its transporter in protection against reactive oxygen species. Biochem Biophys Acta 1780:1408–1411 23. Siderius M, Van Wuytswinkel O, Reijenga K, Kelders M, Mager W (2000) The control of intracellular glycerol in Saccharomyces cerevisiae influences osmotic stress response and resistance to increased temperature. Mol Submit your manuscript to a Microbiol 36:1381–1390 24. Uesono Y, Tohe A (2002) Transient inhibition of translation initiation by journal and benefi t from: osmotic stress. J Biol Chem 277:13848–13855 25. Lin YH, Chien WS, Duan KJ, Chang PR (2011) Effect of aeration timing and 7 Convenient online submission interval during very-high-gravity ethanol fermentation. Process Biochem 7 Rigorous peer review 46:1025–1028 7 Immediate publication on acceptance 7 Open access: articles freely available online doi:10.1186/s40643-014-0022-8 Cite this article as: Lebaka et al.: Effect of fruit pulp supplementation on 7 High visibility within the fi eld rapid and enhanced ethanol production in very high gravity (VHG) 7 Retaining the copyright to your article fermentation. Bioresources and Bioprocessing 2014 1:22. Submit your next manuscript at 7 springeropen.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png "Bioresources and Bioprocessing" Springer Journals

Effect of fruit pulp supplementation on rapid and enhanced ethanol production in very high gravity (VHG) fermentation

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Abstract

Background: The energy crisis and climate change necessitate studying and discovering of new processes involved in the production of alternative and renewable energy sources. Very high gravity (VHG) fermentation is one such process improvement aimed at increasing both the rate of fermentation and ethanol concentration. The technology involves preparation and fermentation of media containing 300 g or more of dissolved solids per liter to get a high amount of ethanol. Findings: Saccharomyces cerevisiae was inoculated to the very high gravity medium containing 30% to 40% w/v glucose with and without supplementation of three selected fruit pulps (mango, banana, and sapota). The fermentation experiments were carried out in batch mode. The effect of supplementation of 4% fruit pulp/puree on the metabolic behavior and viability of yeast was studied. Significant increase in ethanol yields up to 83.1% and dramatic decrease in glycerol up to 35% and trehalose production up to 100% were observed in the presence of fruit pulp. The fermentation rate was increased, and time to produce maximum ethanol was decreased from 5 to 3 days with increased viable cell count. The physical and chemical factors of fruit pulps may aid in reducing the osmotic stress of high gravity fermentation as well as enhanced ethanol yield. Conclusions: It was found that fruit pulp supplementation not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. Production of high ethanol concentration by the supplementation of cheap materials in VHG sugar fermentation will eliminate the expensive steps in the conventional process and save time. Keywords: High gravity fermentation; Osmotic stress; Ethanol; Fruit pulp supplementation Background aimed at increasing both the rate of fermentation and The energy crisis and climate change necessitate study- ethanol concentration. The technology involves prepar- ing and discovering of new processes involved in the ation and fermentation of media containing 300 g or production of alternative and renewable energy sources. more of dissolved solids per liter [4]. VHG fermentation Bioethanol is regarded as a promising alternative energy influences the five basic fermentation assets: (1) plant source, which is both renewable and environmentally and equipment, (2) raw materials, (3) utilities and con- friendly [1,2]. The commonly used ethanol producer in sumables, (4) personnel, and (5) money [5]. High gravity industries is Saccharomyces cerevisiae and the initial fermentation is an accepted method to produce more sugar concentration will not exceed 20% and the con- ethanol in existing fermenters and distil houses (affect- ventional ethanol production process needs high energy, ing item 1), and uses less cooling equipment and pro- high cost and low productivity [3]. Very high gravity duces less effluent (affecting item 3), resulting in higher (VHG) fermentation is one such process improvement yield (affecting item 2) and less staff work (affecting item 4); all these properties decrease the money investment * Correspondence: lvereddy@yahoo.com for ethanol production. Another advantage is an increase Department of Microbiology, Yogi Vemana University, Kadapa, Andhra in opportunities for harvest of high protein spent yeast Pradesh 516003, India [4,6]. Full list of author information is available at the end of the article © 2014 Lebaka et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 2 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 However, the high sugar content of the very high grav- were purchased from the local market of Kadapa, India. ity fermentation medium causes an increase in the os- The fruits were peeled off, and the pulp was separated motic pressure, which has a pessimistic effect on yeast from stones in the case of mango and sapota and pre- cells, and the fermentations are rarely fast and complete. pared as puree with a macerator. The prepared puree The ethanol produced by the yeast also poses negative will have good suspension in the fermentation medium. effects on yeast metabolism like enzyme inhibition and membrane solubility and needs some protectants to Fermentation counteract these effects at the end of the fermentation The very high gravity fermentation medium composition process [3]. S. cerevisiae can ferment an increased is the same as the abovementioned MPYD medium with amount of sugars in the medium when all required nu- high glucose concentration (300 to 400 g/L); 4% fruit trients are provided in adequate amounts [6]. Specific pulp/puree was supplemented to 300 and 400 g/L sugar nutrients, such as nitrogen, trace elements, or vitamins, medium to evaluate the potential effect of fruit pulps in are required to obtain rapid fermentation and high etha- enhancing the ethanol production, and the medium nol levels, which are desirable to minimize capital costs without supplementation of fruit pulp was treated as con- and distillation energy. On a laboratory scale, media are trol. Fermentations were conducted at 30°C in 250-mL often supplemented with peptone, yeast extract, amino Erlenmeyer flasks with 100 mL of fermentation medium. acids, and vitamins [6-8]. However, such addition is not The initial pH was adjusted to 5.5. The progress of feasible in industrial fermentation processes due to the fermentation was monitored by periodical sample analysis. associated high costs. Thus, it is necessary to exploit in- The fermentation was stopped after 5 days, and samples expensive nutrient sources to supply all nutritional re- were kept at −4°C until the analysis. quirements for yeast growth and fermentation. Many investigators studied the effect of inexpensive substances Analytical estimations like soy flour, oils and fatty acids, fungal mycelia, and Sugar concentration was estimated using the Shaffer and fruit pulp [9-12] on the improvement of ethanol produc- Somogyi method [15] as follows: Reducing sugars were esti- tion. In our laboratory, we have tested finger millet and mated using the idometric method of Shaffer and Somogyi horse gram powder supplementation in VHG fermenta- (1933). Sugars containing a free sugar syrup group undergo tion and successfully improved the ethanol production enolization when placed in an alkaline solution. Enediol [13,14]. forms of sugars are highly reactive to acids. The reduced In view of the above, we have screened 15 different copper was quantified by idometric titration using starch commercially available fruits to determine their effect on as an indicator. The 1 L reagent contains sodium carbonate very high gravity fermentation in terms of fermentation (25 g), Rochelle salt (25 g), copper sulfate (75 mL from rate and enhancement of ethanol yield. In this paper, we 100 g/L solution), sodium bicarbonate (20 g), potassium presented the results of tropical fruit pulps mango (Man- iodide (5 g), and potassium iodate (3.567 g). A 5 mL solu- gifera indica), banana (Musa paradisiaca), and sapota tion containing 0.5 to 2.5 mg dextrose units was pipetted (Achras sapota), which show a significant effect in enhan- into test tubes and 5 mL of reagent was added; then, the cing the ethanol production during the screening process. solution with the added reagent was mixed well by stirring. Tubes capped with bulbs were placed in a boiling water Materials and methods bath for 15 min and cooled under running water. Next, Organism and cultural conditions 2 mL of idodine-oxalate titrated with 0.005 N sodium Yeast strain S. cerevisiae 3215 was used in all the experi- thiosulfate was added using starch as indicator. Ethanol ments. The yeast strain was obtained from National was determined with the help of gas chromatography Collection of Industrial Microorganisms (NCIM, Pune, [16]. The fermented samples were centrifuged at India). The culture was maintained on MPYD (malt ex- 5,000 rpm for 10 min. The supernatant was used for tract 3 g/L, peptone 5 g/L, yeast extract 3 g/L, and dex- ethanol analysis. An Agilent Systems Gas Chromato- trose 2 g/L) agar (1.5 g/L) slants at 4°C. The inoculum was graph with Flame Ionization Detector (GC-FID) Model prepared by inoculating the slant culture into 25 mL of 6890 Plus instrument (Agilent Technologies Inc., Santa the sterile MPYD liquid medium taken in a 100-mL flask Clara, CA, USA) was used, and conditions were as follows: and growing it on a rotary shaker (100 rpm) for 48 h. The 5% Carbowax 20M glass column (6 ft (2 m), 2-mm inner above produced yeast culture (5%, 1 × 10 cells/mL) was diameter (ID), 1/4 mm). Nitrogen was used as a carrier used as inoculum to initiate the fermentation. gas with a flow of 20 mL/min, and the eluted com- pounds were detected using a flame ionization detector Fruit pulp preparation for supplementation (FID). For this, the fuel gas was hydrogen with a flow The fruits selected for the supplementation, mango (M. rate of 40 mL/min, and the oxidant was air with a flow indiaca), banana (M. praradisiaca), and sapota (A. sapota), rate of 40 mL/min; n-propanol was used as internal Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 3 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 standard. Glycerol on diluted samples was estimated and incompletely fermented by S. cerevisiae.However, using Boehringer kits (Boehringer Mannheim (Roche, the 4% fruit pulp supplementation led to a significant Basel Switzerland); enzymatic test (340 nm) 3 × 11 de- increase in ethanol production, and the final concentra- terminations (code number 10148270035)). Trehalose tion reached 14.5% (w/v) in a shorter time (72 h) with a was estimated in the supernatant by the anthrone productivity of 2.1 g/h/L (Table 1). In the three fruit method as described previously [17]. The pellet ob- pulps selected, mango supplementation gave the high- tained after the trehalose extraction was used for the est yields of ethanol when compared to banana and protein estimation. sapota. In the fruit pulp-supplemented medium, 10% (v/v) of ethanol production was achieved in just 48 h Cell viability after the inoculation. Besides the high fermentation rate Cellular viability was determined by the methylene blue in the supplemented medium, it also decreased the dur- staining technique [18]. A 100 mL sterile solution of ation of fermentation from 5 to 3 days. methylene blue (3.3 mM in 68 mM sodium citrate) was An attempt was made to increase ethanol production mixed with 100 mL of a yeast suspension diluted to up to 18% to 20% (w/v) as in the case of sake fermenta- reach an OD of 0.4 to 0.7 at 620 nm. This mixture was tion, by increasing the sugar concentration from 300 to shaken, and after a 5-min incubation, it was placed in a 400 g/L with supplementation of fruit pulp. In the Thomas counting chamber. The number of stained (in- 400 g/L sugar fermentation with 4% fruit pulp supplemen- active cells) and unstained (active cells) were counted in tation, the ethanol concentration was 12.5% (Table 1). The five different fields with total of at least 200 to 300 cells. sugars were utilized maximally up to 300 g/L. In the three fruit pulps selected, mango supplementation gave the Statistical analysis highest yields of ethanol when compared to banana and All the experiments were carried out three times (tripli- sapota. The ethanol production after 5 days in the control cate), and the mean value with standard deviation and experiments was only 7.5%. significant (P) was determined. SPSS version 11.0 was used for analysis of variance. Effect of fruit pulp supplementation on cell viability Results After 30 to 35 h of fermentation in both supplemented The present study provides potential observation of and control media, cell growth rate was decreased. fruit pulps as supplements in small quantity during fer- After 40 to 50 h, the growth ceased, but glucose fer- mentation stimulating the rate of alcohol production mentation continued slowly until the number of the vi- and final alcohol concentration in very high gravity fer- able cell count decreased and became very low. The mentation. Two sets of batch fermentation experiments viability percentage of yeast cells in the supplemented with two levels of sugar concentrations, 300 and 400 g/ medium was greater than that in the control medium. L (30% and 40%), were carried out with and without The supplementation of fruit pulp led to an increase in fruit pulp supplementation, in order to evaluate the ef- the rate of fermentation and ethanol yield through the fect of fruit pulps. The unsupplemented batch fermen- extended growth phase of cells (Figure 1). In the three tation experiments yielded only 9% (w/v) of alcohol in fruit pulps selected, mango supplementation gave a higher 300 g/L, and a good amount of residual sugars was left cell viability than banana and sapota supplementation. Table 1 Effect of fruit pulp supplementation on ethanol production in 30 and 40% sugar fermentation Serial number Supplement Alcohol concentration (w/v) 24 h % IMP 48 h % IMP 72 h % IMP 30% Sugar 1 Control 2.5 ± 0.3 - 5.5 ± 0.3 - 9.0 ± 0.7 - 2 Mango 4.5 ± 0.5 90 ± 4.5 10 ± 0.8 90.5 ± 8.0 14.5 ± 1.2 80.5 ± 10 3 Banana 4.0 ± 0.5 80 ± 4.0 9.0 ± 1.0 81.5 ± 9.0 13.2 ± 1.0 73.1 ± 8.0 4 Chiku 4.0 ± 0.4 80 ± 4.2 7.5 ± 0.6 68 ± 6.2 12 ± 0.8 66.5 ± 4.8 40% Sugar 1 Control 2.0 ± 0.3 - 4.5 ± 0.5 - 7.5 ± 0.6 - 2 Mango 4.0 ± 0.2 100 ± 2.0 8.4 ± 0.7 93 ± 7.0 12.5 ± 1.0 83.3 ± 10 3 Banana 3.3 ± 0.3 82.5 ± 2.5 8.0 ± 0.6 87.6 ± 6.2 11 ± 0.8 73.3 ± 7.0 4 Chiku 3.2 ± 0.2 77.5 ± 2.0 7.2 ± 0.8 80 ± 6.8 10 ± 1.0 66.5 ± 8.1 % IMP, percentage of improvement. Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 4 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 stimulating the rate of alcohol production and final alcohol concentration in very high gravity fermentation. In unsup- plemented controls of 30% glucose fermentation experi- ments, compared with the supplemented medium, the sugar was not utilized completely. It is evident that at the end of fermentation, yeast requires certain nutrients that aid tolerance to the high concentrations of alcohol it forms. Nearly 70% to 75% of the volume of the final ethanol con- centration was formed within 48 h of fermentation, and al- most all the final concentration of ethanol was formed in 60 h; the remaining 1% or 2% (v/v) took some time for its Figure 1 Effect of fruit pulp supplementation on yeast cell secretion out of the cell. In addition to nutrients, fruit pulps viability in 30% sugar fermentation. Diamond, control; square, also contain good amounts of polyphenols (all flavones, stil- mango; triangle, banana; circle, sapota. benes, flavonones, isoflavones, catechins, chalcones, tan- nins, and anthocyanidins), which are frequently attributed Effect of fruit pulp supplementation on glycerol to antioxidant, metal ion-chelating, and/or free radical scav- production and trehalose enging activity [19]. This may help in keeping the yeast cells Concentrations of glycerol, one of the stress indicators and viable forlongerdurationand producing suchhighconcen- releasers, were decreased in the fruit pulp-supplemented trations of ethanol in 48 h. The supplemented medium had experiments from 954 to 620 mg/L in the 300 g/L fer- higher viable cell count than the control medium. There 7 7 mentation and from 1,266 to 823 mg/L in the 400 g/L was a dramatic drop in cell count from 10 × 10 to 3 × 10 fermentation (Table 2). In the three fruit pulps selected, in the control medium with increase in ethanol concentra- mango supplementation gave less glycerol when com- tion from 5% to 9% (v/v). But in the supplemented medium, pared to banana and sapota supplementation. Trehalose is the cell viability went up even up to 12% (v/v)ethanol. This a disaccharide which is typically produced by yeast when indicates that the threshold concentration of ethanol to it experiences stress conditions. In the present study, yeast inhibition is 9% (v/v). In all cases, the cell viability in- trehalose concentration was in low in the fruit pulp- creased even at high ethanol concentration (12% v/v)inthe supplemented experiments when compared to the unsup- fruit pulp-supplemented medium compared with the con- plemented control experiments (Table 3). In the control trol medium. Alfenore et al. [6] made a similar observation 30% (w/v) fermentation experiments, the trehalose con- in fed-batch fermentation by vitamin feeding strategy that centration was 40 mg/g yeast cells, and in the fruit pulp- enhanced the final ethanol up to 19% (v/v)in45h. supplemented experiments, it was 21 mg/g yeast cells. In The important byproduct formed during ethanol fer- the 40% sugar (w/v) control fermentation experiments, mentation is glycerol. Commonly, its production is high the trehalose concentration was 52 mg/g yeast cells, in high gravity fermentation. Glycerol is the well-known while in the fruit pulp supplementation with aeration compatible solute in S. cerevisiae. Osmophilic yeasts ac- experiments, it was 38 mg/g yeast cells. In the three cumulate glycerol to compensate for high osmotic pres- fruit pulps selected, mango supplementation decreased sure [20,21]. In the present study, the formation of the trehalose to low levels when compared to banana glycerol was found to be high at the growth/logarithmic and sapota supplementation. phase. After cessation of cell growth, glycerol was not present much in the media. The percentage of glycerol Discussion in the supplemented media was low when compared The present study provides potential observation of fruit with that in the control medium. These results con- pulps as supplements in small quantity during fermentation firmed the previous reports that the growth rate of yeast cells is reduced irreversibly in proportion to an increase in external osmolarity [21]. Another important reserve Table 2 Effect of fruit pulp supplementation on glycerol carbohydrate and stress protectant for the yeast is tre- production in 30 and 40% sugar fermentation halose. Trehalose is also considered as one of the most Serial 30% Sugar 40% Sugar effective saccharines in preventing phase transition in number Supplement Glycerol (mg/L) Supplement Glycerol (mg/L) the lipid bilayer and thereby protecting membranes 1 Control 954 ± 62 Control 1,266 ± 75 against damages, and considering the relation of intra- cellular trehalose concentration with the cellular resist- 2 Mango 620 ± 35 Mango 823 ± 54 ance to osmotic stress, trehalose was supposed to act as 3 Banana 757 ± 58 Banana 938 ± 68 an osmoprotectant under osmotic stress [20]. In the 4 Chiku 826 ± 73 Chiku 1,040 ± 47 supplemented medium, trehalose concentration was Lebaka et al. Bioresources and Bioprocessing 2014, 1:22 Page 5 of 6 http://www.bioresourcesbioprocessing.com/content/1/1/22 Table 3 Effect of fruit pulp supplementation on trehalose accumulation in 30 and 40% sugar fermentation Serial number 30% Sugar 40% Sugar Supplement Trehalose (mg/g yeast cells) Supplement Trehalose (mg/g yeast cells) 1 Control 40 ± 3.4 Control 52 ± 2.5 2 Mango 21 ± 2.8 Mango 38 ± 3.2 3 Banana 29 ± 3.3 Banana 43 ± 4.6 4 Chiku 34 ± 2.6 Chiku 49 ± 3.8 decreased at the end of the fermentation which shows beginning of fermentation and high ethanol stress at the that the cells are not under stress when compared to end of the fermentation. The increased ethanol produc- the control. This could explain the fact that stress induced tion by the fruit pulp supplementation is a significant the genes involved in trehalose synthesis and those in- finding that could also be applied to an industrial fer- volved in degradation, and why the genes responded in a mentation of ethanol utilizing molasses and other raw similar pattern in osmotic and oxidative stress [22]. It has materials as substrates. This may reduce the cost of been reported that the production pattern of protein syn- ethanol production in developing countries like India. thesis is changed dramatically by osmotic and heat stress, The nature of active principles from fruit pulps and their and also depriving amino acids or proteins inhibits trans- mechanism that aids in tolerating high osmotic stress lation initiation through the phosphorylation pathway and enhance ethanol production rate are being investi- [23,24]. gated by the authors. Higher amount of ethanol in the 400 g/L sugar medium Abbreviations was not obtained probably due to the initial high glucose % IMP: percentage of improvement; %: percent; g/l: gram per liter; mg/ concentration that strongly inhibited fermentation. Even l: milligram per liter; mM: millimolar; MPYD: malt extract, peptone, yeast extract, and dextrose (medium); v/v: volume per volume; VHG: very high in such high osmolarity, the supplemented media yield gravity; w/v: weight per volume. 12.5% (v/v) ethanol with a productivity of 1.73 g/h/L. It is likely that the supplementation of fruit pulp may add Competing interests sugars, thereby contributing to the increased osmotic The authors declare that they have no competing interests. pressure. The old yeast cells ferment slowly when com- Authors' contributions pared with actively growing yeast cells. It is possible to LV participated in the design of the study, carried out the fermentations, produce high ethanol concentrations by extending the analyzed the results, and wrote the manuscript. YJ participated in the experimental procedure and the GC and result analysis. HW conceived the growth phase of yeast to longer periods as in the case of study and participated in analyzing the results and correcting the beer production. It is expected that fruit pulp supplemen- manuscript. All authors read and approved the final manuscript. tation would overcome nutritional deficiencies of yeast and allow them to stay longer in the growth phase and Acknowledgements The author would like to acknowledge the Council of Scientific and that antioxidants protected the yeast cells from osmotic Industrial Research, Government of India and Department of Science and stress and aeration allowed yeast to produce membrane Technology, Government of India for the financial support given in the form lipids to be sustained at higher alcohol concentrations. of research projects entitled ‘Studies on Rapid and Enhanced Production of Ethanol through Very High Gravity (VHG) Fermentation’ (Ref No: 38 (1310)/ During VHG ethanol fermentation, maintaining the redox 11/EMR-II) and ‘Biotechnological production of Acetone-Butanol-Ethanol potential at a constant level is essential, as yeast requires a (ABE) from agricultural biomass using solventogenic bacteria’ (Ref No: SR/FT/ small amount of oxygen to facilitate the synthesis of ste- LS-79/2009). rols and unsaturated fatty acids, which serve as the build- Author details ing blocks for constructing cell membranes [25]. 1 Department of Microbiology, Yogi Vemana University, Kadapa, Andhra Pradesh 516003, India. School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Korea. Department of Food Conclusions Science and Technology, College of Natural Resources, Yeungnam University, It is concluded that fruit pulp supplementation en- Gyeongbuk 712-749, Korea. hanced the rate and yield of ethanol production in a very Received: 22 July 2014 Accepted: 19 October 2014 high gravity medium. It is observed that the selected fruit pulps were not much effective in the 400 g/L sugar fermentation when compared to the 300 g/L sugar fer- References mentation. The decrease in both glycerol and trehalose 1. Cardona C, Sa´nchez O (2007) Fuel ethanol production: process design trends and integration opportunities. Bioresour Technol 98:2415–2457 concentrations by the supplementation would suggest 2. Reddy LVA (2013). Potential bioresources as future sources of biofuels production: that the fruit pulp constituents might be involved in an overview. V. K. Gupta and M. G. 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Mol Submit your manuscript to a Microbiol 36:1381–1390 24. Uesono Y, Tohe A (2002) Transient inhibition of translation initiation by journal and benefi t from: osmotic stress. J Biol Chem 277:13848–13855 25. Lin YH, Chien WS, Duan KJ, Chang PR (2011) Effect of aeration timing and 7 Convenient online submission interval during very-high-gravity ethanol fermentation. Process Biochem 7 Rigorous peer review 46:1025–1028 7 Immediate publication on acceptance 7 Open access: articles freely available online doi:10.1186/s40643-014-0022-8 Cite this article as: Lebaka et al.: Effect of fruit pulp supplementation on 7 High visibility within the fi eld rapid and enhanced ethanol production in very high gravity (VHG) 7 Retaining the copyright to your article fermentation. Bioresources and Bioprocessing 2014 1:22. Submit your next manuscript at 7 springeropen.com

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"Bioresources and Bioprocessing"Springer Journals

Published: Dec 1, 2014

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

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