282 Chiang Mai J. Sci. 2015; 42(2)

Chiang Mai J. Sci. 2015; 42(2) : 282-293http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Improvement of Continuous Ethanol Fermentationfrom Sweet Sorghum Juice by Saccharomycescerevisiae using Stirred Tank Bioreactor Couplingwith Plug Flow BioreactorNaulchan Khongsay [a], Lakkana Laopaiboon [b,c] and Pattana Laopaiboon*[b,c][a] Graduate School, Khon Kaen University (KKU), Khon Kaen, 40002, Thailand.[b] Department of Biotechnology, Faculty of Technology, KKU, Khon Kaen, 40002, Thailand.[c] Fermentation Research Center for Value Added Agricultural Products, KKU, Khon Kaen, 40002, Thailand.*Author for correspondence; e-mail: patlao@kku.ac.th

Received: 17 July 2013Accepted: 8 October 2013

ABSTRACTThe continuous ethanol production from sweet sorghum juice in a continuous stirred

tank bioreactor (STR) and a combined bioreactor was compared. The combined bioreactorcomprised an STR and three tubular (plug flow) bioreactors (TB) in series with a total workingvolume of 3320 ml. The fermentation was carried out at 30°C by Saccharomyces cerevisiae NP01. The sweet sorghum juice containing 250 g l-1 of total sugar was introduced into thesystems at total dilution rates (D) of 0.007, 0.02 and 0.04 h-1. Our results clearly show that thedegree of ethanol production depends on the residence time of the fermentation broth in thesystem. In the STR system at D = 0.007 h-1, the ethanol concentration (P), yield (YP/S) andproductivity (QP) were 67.28 ± 0.37 g l-1, 0.52 ± 0.01 g g-1 and 0.47 ± 0.00 g l-1 h-1, respectively.Using the combined bioreactor (CSTR + TB) could improve ethanol production efficiency.The sugar consumption and ethanol production of the combined bioreactor system weremarkedly higher than those of the STR system. The highest P value (106.01 ± 0.47 g l-1) wasobtained when using the combined system at D 0.007 h-1; and under this condition, the YP/S

and QP values were 0.50 ± 0.01 g g-1 and 0.76 ± 0.02 g l-1 h-1, respectively.

Keywords: continuous ethanol fermentation, plug flow bioreactor, stirred tank bioreactor(STR), Saccharomyces cerevisiae, sweet sorghum

1. INTRODUCTIONThe use of biofuel to replace oil is one

of the most viable ways to ensure a sustainableenergy future. Ethanol production as analternative fuel energy resource has been asubject of great interest since the oil crisis ofthe 1970s [1]. Therefore, the development of

a fermentation process using economical rawmaterials is important for production ofthe biofuel ethanol on a commercial scale [1].In Thailand, the main raw materials used forethanol production are sugarcane molassesand cassava. Regarding the energy policy of

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the Thai government, ethanol productionwill be increased to 9,000,000 litres/day inthe year 2022 [2]. Therefore, it is possible thatThailand may face a shortage of sugarcanemolasses and cassava.

Sweet sorghum [Sorghum biocolor (L.)Moench] is a high biomass- and sugar-yieldingcrop. It has been of particular interest as asubstrate for ethanol production becausethe juice from its stalks contains highlyfermentable sugars, primarily sucrose,fructose and glucose, as well as many essentialtrace elements for microbial growth andethanol production [3-7]. The pH of the juiceis also in the optimum pH range (pH 4.0 to5.5) for yeast growth and ethanol production[8]. Moreover, after juice extraction, sweetsorghum bagasse can be hydrolysed intosugars for ethanol production [9]. The sweetsorghum can be cultivated at nearly alltemperatures and tropical climate areas, andit has a growing period of 120-150 days [7,10].In Thailand, the average yield of sweetsorghum cultivar KKU40, 90-100 days old,is 30-50 ton/ha corresponding to about15-25 dry ton/ha [11].

Apart from the development of newsweet sorghum cultivars with high grain andsugar yields [12], fermentation processdevelopment is also important for efficientethanol production. A very high gravity(VHG) technology is one of the methodsthat can enhance ethanol productionefficiency. It is defined as the preparationand fermentation to completion of mediacontaining sugar in excess of 250 g l-1 [13].This technology has several advantages forindustrial applications because it increasesboth the ethanol concentration and the rateof fermentation. In addition, it reducescapital costs, energy costs per litre of alcoholand the risk of bacterial contamination [13].However, substrate inhibition may occurunder initially high sugar levels. To increase

the efficiency of ethanol production, manyprocess improvements have been studiedincluding the continuous fermentation system.

Continuous culture fermentationprovides advantages over batch fermentation,including optimized process conditions formaximum productivity, long-term continuousproductivity, reduced labour costs once ithas reached steady state, reduced vesseldowntime for cleaning, filling and sanitizing,and easy process control and operation[14,15]. Typical bioreactors called stirredtank bioreactors (STRs) have been used forethanol production in both laboratoryresearch and industry applications for along period of time. These bioreactorsare characterized by their well-mixedperformance, but bioreactor engineeringtheories predict that strong productinhibitions can occur because of highproduct concentrations inside thebioreactors [16,17]. Therefore, they are notgood choices for fermentation under VHGconditions, although multi-stage STRs inseries (homogenous continuous culture) canlower product inhibition to some extent.It was reported that tubular bioreactors(heterogeneous continuous culture) could beused in the case of product inhibition asproduct concentration increases graduallyalong their axial directions [18].

Ethanol production from sweet sorghumjuice using STRs combined with plug flowbioreactors or tubular bioreactors has notbeen reported. Therefore, the aim of thisresearch was to investigate methods to increaseethanol production from sweet sorghum juiceusing continuous (chemostat) fermentation bySaccharomyces cerevisiae NP 01. The efficienciesof continuous ethanol production from thesweet sorghum juice by a STR and a combinedbioreactor (STR and plug flow bioreactor)were compared under VHG conditions toaccount for differences in sugar consumption,

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ethanol production and cell viability.To control the bioprocess, the effects ofdilution rate (D) on ethanol production in bothsingle and multi-stage continuous fermenta-tion processes were also investigated.

2. MATERIALS AND METHODS2.1 Microorganism and InoculumPreparation

S. cerevisiae NP 01 isolated fromLoog-pang (Chinese yeast cake) fromNakhon Phanom province, Thailand [19],was inoculated into 150 ml of yeastextract - malt extract (YM) medium and wasincubated on a rotating shaker at 150 rpm,30°C for 15 h. The YM medium contained(g l-1) yeast extract 3, peptone 5, malt extract3 and glucose 10. The yeast cells were thentransferred into 350 ml of the YM mediumto give an initial cell concentration of5×106 cells ml-1 and were further incubatedunder the conditions as previously mentioned.After 15 h, the cells were harvested and usedas an inoculum for ethanol production.

2.2 Raw MaterialSweet sorghum juice extracted from

its stalks (cv. KKU 40) was obtainedfrom Division of Agronomy, Faculty ofAgriculture, Khon Kaen University,Thailand. To avoid storage problem and toprevent bacterial contamination, the juicecontaining total soluble solids of 18°Bxwas concentrated to 75°Bx and stored at4°C before use.

2.3 Ethanol Production MediumThe concentrated juice was diluted with

distilled water to obtain a total sugarconcentration of approximately 250 g l-1 andused as an ethanol production (EP) medium.The medium was transferred into a 2-l STRwith a final working volume of 1 l andautoclaved at 110°C for 40 min, or it was

transferred into a 5-l STR with a finalworking volume of 3320 ml and autoclavedat 110°C for 60 min. A 5-l reservoir wasfilled with the EP medium before sterilization.The sterile EP medium was kept at roomtemperature for use in continuousfermentation.

2.4 Continuous Fermentation SystemContinuous fermentation was carried out

into two systems, the STR system and thecombined bioreactor system.2.4.1 Stirred tank bioreactor (STR) system

All continuous fermentations throughoutthis work were conducted in a 5-l STR(Biostat® B, B. Braun Biotech, Germany)with a final working volume of 3320 ml.The yeast cells were inoculated into theSTR to give an initial yeast cell concentrationof 1×108 cells ml-1. The STR was agitated at100 rpm at 30°C. The sterile EP medium inthe feed reservoir was fed into the top of theSTR via a peristaltic pump (Biostat® B, B.Braun Biotech, Germany) when the total sugarof the fermented broth in the STR remainedapproximately one-third of the initial value.The dilution rates or D of the fresh EPmedium into the STR were 0.007, 0.02 and0.04 h-1. Samples were withdrawn at timeintervals for analysis. Steady-state conditionswere indicated by stable viable yeast cells, totalsugar and ethanol levels in the effluent fromthe STR. The continuous cultures wereoperated for at least two times of the residencetime after the system reached steady state.

2.4.2 Combined bioreactor systemThe combined bioreactor system

comprised a 2-l STR (Biostat® B, B. BraunBiotech, Germany) and a three-stage tubular(plug flow) bioreactor in series with a totalworking volume of 3320 ml. The workingvolume of the STR and three plug flowbioreactors were 1000, 770, 770 and 780 ml,

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respectively. Figures 1A and 1B illustratea schematic diagram of the combinedbioreactor system. The yeast cells wereinoculated into each bioreactor to giveinitial yeast cell concentrations of 1×108

cells ml-1. The STR was agitated at100 rpm and the temperature for the wholesystem was controlled at 30°C. The STRwas connected to the 5-l mediumreservoir, the three-stage plug flowbioreactor in series and a waste reservoir.The sterile EP medium in the reservoirwas fed into the top of the STR at total

D 0.007, 0.02 and 0.04 h-1; and the mediumfrom the STR was fed into the bottom ofthe tubular bioreactor 1, 2 and 3, respectively(Figure 1B). The exhaust gas from thethree-stage plug flow bioreactor was washedby bubbling it into a de-ionised water storagetank to recover the entrapped ethanol.Sterile air was supplied to the bottom of thethree plug flow bioreactors at the flow rateof 0.005 vvm to prevent cell settling [18].The continuous fermentation at eachdilution rate was operated for at least tworetention times at steady state.

2.5 Analytical MethodsAfter the system inoculation and

equilibrium of continuous fermentation,samples were taken daily. The viable yeastcell numbers were determined by thedirect counting method with methyleneblue staining using a haemacytometer [20],The fermentation broth was centrifuged at13,000 rpm for 10 min. The supernatantwas detected for total residual sugar byphenol sulfuric acid method [21]. Ethanolconcentration (P, g l-1) was analysed by gas

chromatography (Shimadzu GC-14B,Japan, Solid phase: polyethylene glycol(PEG-20M), carrier gas: nitrogen, 150°Cisothermal packed column, an injectiontemperature of 180°C, flame ionizationdetector temperature 250°C; C-R7 Ae plusChromatopac Data Processor), and 2-propanol was used as an internal standard[5]. Sugar consumption (SC), ethanol yield(Yp/s) and ethanol productivity (Qp) werecalculated by the following equations:

Figure 1 A: The combined bioreactor system composed of STR and three-tubular bioreactorfor ethanol production and B: Schematic diagram of the combined bioreactor system: (1)stirred tank bioreactor; (2) tubular bioreactor; (3) medium reservoir; (4) waste bottle; (5) unitcontrol; (6) burette; (7) sampling ports; (8) peristaltic pump; (9) air filter; (10) exhaust gaswashing tank; (11) stirrer; (12) thermostat water inlet and outlet; (13) air pump; (14) rotameter;(15) agitation motor; (16) condenser; and (17) valves.

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SC (%) = (ITS - RTS)×100/ITSYp/s (g g-1) = P/(ITS - RTS)Qp (g l-1 h-1) = P×D

where ITS is the initial total sugar (g l-1),RTS is the residual total sugar (g l-1), P is theethanol concentration produced (g l-1) andD is the dilution rate (h-1) of a continuousbioreactor system based on the totalworking volume of the bioreactor system.

3. RESULTS AND DISCUSSION3.1 Continuous Ethanol Production inStirred Tank Bioreactor

The pH, viable yeast cell, total sugarand ethanol concentrations during thecontinuous fermentation process at the

different dilution rates are illustrated inFigure 2. The amount of sugar consumptionand ethanol production decreased withincreasing the dilution rate. This was dueto the fact that the residence time ofthe medium in the bioreactor decreased.The pH in the broth decreased slightlywithin 30 h and was constant at approximately4.5 throughout the experiment. Similarresults was observed by Narendranathand Power [8] who reported that duringethanol fermentation the pH of the mediumand the ethanol concentrations were inthe range of 4.0 to 4.5 and 36 to 67 g l-1,respectively, which was a possible way tocontrol the bacterial contamination duringthe fermentation.

Figure 2 Fermentation parameters during continuous ethanol production from the sweetsorghum juice in the 5-l STR at the dilution rates of 0.007, 0.02 and 0.04 h-1. pH (×), viableyeast cells (O), total sugar ( ), and ethanol (•). The arrows indicate the start time of continuousmode at the different dilution rates.

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The yeast cell concentrations in thebroth were relatively high, ranging fromlog 7.38 to log 8.43 cells ml-1 at steady state(Figure 2). However, the yeast could notcompletely consume the sugar in the sweetsorghum juice at all D values (Figure 2).The total sugar utilization was approximately49, 32 and 27% of the initial values atD 0.007, 0.02 and 0.04 h-1, respectively.The fermentation parameters of ethanolproduction in the STR are shown inTable 1. The ethanol concentration (P)decreased as the D value increased aspreviously mentioned, but the ethanolproductivity (Qp) increased as the D valueincreased. However, the productivity is indirect proportion to the dilution rate andproduct concentration. Productivity value willincrease to a certain limit with increasing

dilution rate since product concentrationdecreases with increasing dilution rate [22].In this study, the ethanol yields (YP/S) at alldilution rates tested were similar with thevalues of 0.51 to 0.52 g g-1 correspondingto yield efficiencies of approximately94 to 96% of the theoretical yield (0.54) basedon sucrose (the main sugar in the sweetsorghum juice). High YP/S values indicatedthat most sugar in the juice was converted toethanol. At the end of the experiments, thejuice at D 0.007 h-1 was re-introduced to testthe stability of this process. The fermentationparameters of the second feeding were notsignificantly different compared to those atthe first feeding at the same D value (0.007h-1), indicating that the system operationcould be reversed and the results obtainedwere reliable.

Table 1. Fermentation parameters of continuous ethanol production from sweet sorghumjuice in the stirred tank bioreactor (STR) at different dilution rates.

D (h-1)

0.007a

0.020.04

0.007b

Parameters (mean ± SD)SC (%)

49.29 ± 0.8531.64 ± 0.4027.31 ± 0.4448.97 ± 1.06

RTS (g l-1)130.93 ± 1.90176.51 ± 0.83187.68 ± 0.93129.62 ± 2.88

P (g l-1)67.28 ± 0.3742.81 ± 1.2736.12 ± 0.2468.83 ± 1.92

Qp (g l-1 h-1)0.47 ± 0.000.86 ± 0.031.44 ± 0.010.48 ± 0.01

Yp/s (g g-1)0.52 ± 0.010.52 ± 0.010.51 ± 0.010.52 ± 0.00

a First feeding at the beginning of the experiment.b Second feeding at the end of the experiment.D = dilution rates, SC = sugar consumption, RTS = residual total sugar, P = ethanol concentration,Qp = ethanol productivity and Yp/s = ethanol yield.

3.2 Continuous Ethanol ProductionUsing The Combined Bioreactor

In the STR system, high sugar levelsremaining in the juice even at the lowest Dvalue (0.007 h-1) might have been due tosubstrate inhibition. This was supportedby Ingledew [23] who reviewed that sugarconcentrations ≥ 25% (w/v) affected osmoticpressure which was one of the mainfactors leading to the reduction of yeastviability and ethanol yield. Using the plugflow bioreactors in series, the substrate

inhibition might be alleviated to someextent [24].

During the continuous ethanolfermentation in the combined bioreactorsystem, the pH value was relatively constantat approximately 4.0 to 4.5 at all conditionstested (Figure 3A) and no contaminationwas observed (data not shown). Xu et al. [25]reported that lower pH and anaerobicenvironments within fermenters wereunfavourable for the growth of contaminatedmicroorganisms. Therefore, contamination

288 Chiang Mai J. Sci. 2015; 42(2)

could be effectively prevented, and conversionyields of ethanol to sugar could reach ashigh as 90 to 92% of its theoretical value.

At D 0.007 h-1, the steady state wasevident after approximately 158 h of thecontinuous system as indicated by relativelystable levels of viable cell, total sugar andethanol concentrations (Figure 3B, C and D).When D value was increased to 0.02 h-1,the sugar consumption and ethanolproduction by S. cerevisiae NP 01 was lower.The steady state was observed after 53 h ofchanging D value. At D 0.04 h-1, the steadystate was achieved faster than that at

D 0.02 and 0.007 h-1, respectively. The resultsstrongly showed that D value affected sugarutilization in the fermentation brothbecause it related to the residence time ofthe broth in the system. At the end ofthe experiments, the juice at D 0.007 h-1 wasre-introduced to the combined system.The results showed that all fermentationparameters measured were similar to thoseof the first feeding at the same D value(0.007 h-1), indicating that the combinedsystem operation could be reversed.In addition, the measured ethanol loss in theexhaust gas washing tank was not detected.

Figure 3. Fermentation parameters during continuous ethanol production from the sweetsorghum juice in the combined bioreactor at different dilution rates. A: pH, B: log viable cell,C: total sugar and D: ethanol. •: stirred tank, : column 1, : column 2, and O: column 3. Thearrows indicate the start time of continuous mode at each dilution rate.

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The average fermentation parameters inthe combined bioreactor are summarized inTable 2. The results clearly demonstrate thatthe residence time of the fermentation brothin the system had a significant effect onthe main fermentation parameters. At D 0.007h-1, the average residual sugar levels in thestirred tank and the plug flow bioreactors 1,2 and 3 were 176.10 ± 0.73, 130.17 ± 0.71,67.21 ± 2.42 and 43.70 ± 1.13 g l-1, respectively(Figure 3). Under these conditions, the totalsugar was consumed at approximately 30,48, 73 and 83% when the broth passed theSTR and the plug flow bioreactors 1, 2 and3, respectively. The maximum ethanolconcentration of 106.01 ± 0.47 g l-1 or13.44% (v/v) was obtained at the lasttubular bioreactor indicating that the plugflow bioreactor could improve productconcentration. The value of ethanol

concentration is higher than 11.5% (v/v),which is the acceptable level for industrialproduction [25].

When the D value of the system wasincreased to 0.02 and 0.04 h-1, the amount ofsugar consumed and the ethanol producedincreased with increasing residence timeof the broth in the system as found atD 0.007 h-1 (Table 2). However, the ethanolconcentrations decreased dramatically whencompared to those at 0.007 h-1. The maximumethanol concentrations at D 0.02 and 0.04 h-1

were 67.25 ± 1.89 g l-1 (8.52%, v/v) and54.55 ± 1.32 g l-1 (6.91%, v/v), respectively,in the last tubular bioreactor. However,the Yp/s values in each bioreactor at all Dvalues were similar, ranging from 0.50 to0.52 g g-1. The high Yp/s values implied thatby-products of the fermentation wererarely occurred.

Table 2. Continuous ethanol fermentation from sweet sorghum juice at different total dilutionrates in the combined bioreactor.

Total D(h-1)0.007

0.02

0.04

ParametersD (h-1)Vol. (ml)SC (%)P (g l-1)Qp (g l-1 h-1)Yp/s (g g-1)D (h-1)Vol. (ml)SC (%)P (g l-1)Qp (g l-1 h-1)Yp/s (g g-1)D (h-1)Vol. (ml)SC (%)P (g l-1)Qp (g l-1 h-1)Yp/s (g g-1)

The combined bioreactorSTR0.023100029.56 ± 0.7342.62 ± 1.250.98 ± 0.030.52 ± 0.020.07100022.69 ± 1.0931.59 ± 1.842.21 ± 0.130.52 ± 0.000.1310008.67 ± 1.4413.99 ± 1.241.83 ± 0.180.50 ± 0.02

STR to TB10.013177047.93 ± 0.7164.85 ± 2.360.86 ± 0.030.52 ± 0.000.04177033.93 ± 0.2646.76 ± 2.041.82 ± 0.010.50 ± 0.020.08177014.21 ± 3.7122.08 ± 1.111.77 ± 0.110.51 ± 0.00

STR to TB20.009254073.12 ± 1.21100.97 ± 1.000.91 ± 0.010.52 ± 0.010.03254040.99 ± 0.6455.64 ± 0.951.68 ± 0.030.51 ± 0.010.05254025.97 ± 2.1637.73 ± 0.461.89 ± 0.020.52 ± 0.03

STR to TB30.007332082.52 ± 0.56106.01 ± 0.470.76 ± 0.020.50 ± 0.010.02332052.02 ± 0.5467.25 ± 1.891.34 ± 0.020.50 ± 0.010.04332040.28 ± 1.7154.55 ± 1.322.18 ± 0.070.50 ± 0.00

STR = stirred tank bioreactor; TB1, TB2 and TB3 = First, second and third tubular bioreactors, respectively.D = dilution rate, Vol. = working volume, SC = sugar consumption, P = ethanol concentration, Qp = ethanolproductivity and Yp/s = ethanol yield.

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3.3 Comparison of Continuous EthanolProduction Between The STR and TheCombined Bioreactor Systems

The initial total sugar concentrationsfed into the two systems were equal at 257to 258 g l-1 (Table 3). The results showedthat the combined system promoted sugarconsumption and/or reduced substrateinhibition. In addition, it also promoted cellgrowth of S. cerevisiae NP 01 (Figures 2and 3). At the same D values, the sugar

consumptions by the combined bioreactorwere approximately 33, 20 and 13% higherthan those by the STR at D 0.007, 0.02 and0.04 h-1 respectively. The percentage of sugarconsumption by the combined bioreactorat the lowest D value (0.007 h-1) was similarto that by the batch system [26]. Therefore,the continuous operation at D lower than0.007 h-1 was not necessary. In addition,the lower D value, the lower ethanolproductivity.

Table 3. Sugar consumption and fermentation parameters in batch and continuous ethanolfermentation by the STR and the combined bioreactor systems.

D = the dilution rate, STR = stirred tank bioreactor, CR = combined bioreactor, TS = total sugar, RTS = residual totalsugar, SC = sugar consumption, P = ethanol concentration, Qp = ethanol productivity and Yp/s = ethanol yield.

Yp/s

(g g-1)0.52±0.010.50±0.010.52±0.010.50±0.010.51±0.010.50±0.00

D(h-1)0.007

0.02

0.04

System

STRCRSTRCRSTRCR

Initial TS(g l-1)

258.21±0.68257.00±1.34258.21±0.68257.00±1.34258.21±0.68257.00±1.34

RTS(g l-1)

130.93 ± 1.9043.70±1.13176.51±0.83119.96±0.54187.68±0.93149.29±3.42

SC(%)

49.29±0.8582.52±0.5631.64±0.4052.02±0.5427.31±0.4440.28±1.71

P(g l-1)

67.28±0.37106.01±0.4742.81±1.2767.25±1.8936.12±0.2454.55±1.32

Qp

(g l-1 h-1)0.47±0.000.76±0.020.86±0.031.34±0.021.44±0.012.18±0.07

The advantages of serial bioreactorshave been used to reduce the residual sugarconcentration at different D values. Tzengand Fan [27] succeeded with an assimilationof 82% of glucose in cultivation of 200 g l-1

of glucose, in a 8-stage fluidized-bedbioreactor with a total volume of 410 ml atD 0.23 h-1, while Bai et al. [18] achieved 92%sugar utilization in the continuous ethanolproduction from the enriched syntheticmedium containing 280 g l-1 of glucose usinga cascade bioreactor at D 0.012 h-1.

At the same D values, ethanol efficienciesin terms of P and Qp values by the combinedsystem were higher than those by the STRsystem (Table 3). The P and Qp values in thecombined system increased approximately51 to 58% and 51 to 61%, respectively,when compared with those in the STRsystem. However, the Yp/s values in the two

systems were similar ranging from 0.50 to0.52 g g-1 at all conditions, indicating thatmost sugar in the juice was converted toethanol. In addition, the fermentation processdid not affect Yp/s or ethanol yield implyingthat metabolic pathway of ethanol productionby S. cerevisiae NP 01 under all conditions werenot changed.

The SC, P and Qp values in our study werelower than those (92%, 124.6 g l-1 and 1.50 gl-1 h-1, respectively) reported by Bai et al. [18].One of the main reasons might be due to thedifference in the EP medium. The enrichedsynthetic medium containing 280 g l-1 ofglucose was used in Bai et al. [18], whilethe sweet sorghum juice (~260 g l-1 of totalsugar) without nutrient supplementationwas used as the EP medium in this study.

It was reported that under no aeration inthe STR system, the product inhibition was

Chiang Mai J. Sci. 2015; 42(2) 291

occurred at ~80 g l-1 of ethanol in thefermented medium [28]. Therefore, lower Pvalues in the STR system in our study mightbe mainly due to substrate inhibition as thehighest ethanol concentration produced wasonly ~68 g l-1. However, in the combinedbioreactor, severe product inhibition wasrarely observed. The viable yeast cellconcentrations were still relatively higheven in the plug flow bioreactor 3 whichcontained 106 g l-1 of ethanol (Figure 3).The product inhibition in the combinedbioreactor in series was less because theproduct concentration increased graduallyalong the height of the plug flow reactor [18].In contrast, well-mixed performance of theSTR system caused severe product inhibitioninside the bioreactor.

In this study, the sweet sorghum juice withno nutrient supplement was used as the EPmedium, and the sugar consumption underthe continuous system with the combinedbioreactor was 83% sugar consumption.Complete sugar utilization resulting in higherethanol production may occur if someessential nutrients for ethanol production aresupplemented in the sweet sorghum juice.This was supported by Nuanpeng et al. [29]who found that 9 g l-1 of yeast extract wasrequired for completion of sugar utilizationin batch ethanol production from sweetsorghum juice containing 280 g l-1 of totalsugar.

4. CONCLUSIONSThe results obtained from this study

clearly demonstrated that the combinedbioreactor (STR combined with threeplug flow bioreactors) was successfully usedfor improvement of continuous ethanolproduction from sweet sorghum juiceunder VHG fermentation. The ethanolproduction efficiencies in terms of the ethanolconcentration and its productivity increased

51 to 58% and 51 to 61%, respectively,when compared with those of the typicalsystem (STR). Complete sugar utilization bythe system may be achieved by nutrientsupplementation in the sweet sorghum juice.

ACKNOWLEDGMENTSThe authors would like to thank the

Higher Education Research Promotion andNational Research University Project ofThailand through Biofuels Research Clusterof Khon Kaen University (KKU), Office ofthe Higher Commission Education andCenter for Alternative Energy Research andDevelopment (AERD), KKU, Thailand forfinancial support. We would like to thankAssistant Prof. Dr. Paiboon Danviruthai,Faculty of Technology, KKU for providingthe NP 01 strain; Associate Prof. Dr. PrasitJaisil, Faculty of Agriculture, KKU forproviding sweet sorghum juice andAssociate Prof. Dr. Aroonwadee Chanawong,Faculty of Associated Medical Sciences, KKUand Dr. Preekamol Klanrit, Faculty ofTechnology, KKU for their internal reviewsof this paper.

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