reusing a mixture of anaerobic digestion effluent and thin stillage for cassava ethanol production

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Journal of Cleaner Production xxx (2014) 1e7

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Journal of Cleaner Production

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Reusing a mixture of anaerobic digestion effluent and thin stillagefor cassava ethanol production

Ke Wang, Jian-Hua Zhang*, Pei Liu, Hua-Shi Cao, Zhong-Gui Mao*

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

a r t i c l e i n f o

Article history:Received 21 December 2013Received in revised form3 April 2014Accepted 3 April 2014Available online xxx

Keywords:Cassava ethanol fermentationThin stillageAnaerobic digestionSustainable development

* Corresponding authors. Tel./fax: þ86 510 8591829E-mail addresses: [email protected] (J.-H. Zha

(Z.-G. Mao).

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a b s t r a c t

In this study, a novel process was explored for cassava ethanol fermentation. In the process, a portion ofthin stillage was mixed with the effluent from anaerobic digestion of residual thin stillage and was thenreused as process water for the following ethanol fermentation. This process was evaluated at the lab anda pilot scale was performed for anaerobic digestion and ethanol fermentation performance. Ethanolproduction was enhanced in the recycling batch compared with the first batch (freshwater was used).Anaerobic digestion of the thin stillage also showed high efficiency and stability with a chemical oxygendemand (COD) removal rate above 97% and a methane yield of 300 mL/g COD removed at both the laband pilot scale. In addition, the COD and conductivity of the thin stillage remained consistent after the6th and 3rd batch, respectively. In conclusion, in this novel process, energy consumption was reduced,and wastewater was utilized for value-added products, thereby contributing to the sustainable devel-opment in the cassava ethanol industry.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Bioethanol, as a clean and renewable fuel, is considered as agood alternative to replace petroleum oil (Nguyen and Gheewala,2008), and its production has increased considerably in recentdecades. However, bioethanol production is facing someenvironmental-based criticism, which is mainly focused on a smallpositive net energy balance (NEB) achieved (Hill et al., 2006; Lenget al., 2008) and wastewater management. To create a more sus-tainable bioethanol industry, decreasing energy consumption andresolution of the problem of wastewater treatment are crucial.

Ethanol can be fermented from sugar-based (Roukas, 1996) andstarch-based feedstocks (Zafar and Owais, 2006). Among theavailable feedstocks, cassava is of particular interest due to its lowcost, high productivity, and lack of competition for arable land(Sanchez and Cardona, 2008). In the conventional cassava ethanolfermentation process (Fig. 1a), massive freshwater is consumed and8e15 L/L ethanol of distillery waste (with very high COD, sus-pended substance, and low pH) is produced (Saha et al., 2005).Distillery waste is usually treated by solideliquid separation fol-lowed by anaerobic-aerobic digestion, and the effluent is further

6.ng), [email protected]

., et al., Reusing a mixture o014), http://dx.doi.org/10.101

treated using physical or chemical methods to achieve the nationaldischarge standard (Jördening and Winter, 2005; Kim et al., 1997).However, capital investment and operating costs of the aerobicdigestion and further treatment process are high. Furthermore, themassive residual sludge produced should be treated. Consequently,alternative methods were explored to resolve the problem ofwastewater handling. The recycling of wastewater to ethanolfermentation process is one of these alternatives. However, only aportion of the wastewater can be recycled, otherwise the growth ofSaccharomyces cerevisiae will be inhibited due to the accumulationof metabolic byproducts and materials from the feedstocks. Thus,an integrated ethanol-methane fermentation system as anotheralternative was developed. In this system, thin stillage is treated bytwo-stage anaerobic digestion, which produces biogas as fuel(Zhang et al., 2010a, b) and the digestate was recycled as processwater for the following ethanol fermentation. Thus, wastewaterdischarge was eliminated, and freshwater and energy consumptionwas reduced (Fig. 1b). However, sulfuric acid was required to adjustthe slurry pH to an optimum value (6.0) for high-temperatureamylase prior to cooking because the pH of the digestate isapproximately 8.0 with a strong buffer capacity. Thus, when thethin stillage is treated by anaerobic digestion, the sulfate is reducedby sulfate-reducing bacteria (SRB), which out-compete methane-producing bacteria for substrates (H2 and acetate). The reducingproduct, H2S, will cause problems of corrosion, malodor andtoxicity. Sulfide is also involved in the precipitation of non-alkali

f anaerobic digestion effluent and thin stillage for cassava ethanol6/j.jclepro.2014.04.007

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Fig. 1. Process diagram of the conventional process (a), integrated process (b) and improved integrated process (c) for cassava ethanol production.

K. Wang et al. / Journal of Cleaner Production xxx (2014) 1e72

metals in digesters, thus reducing their availability for methane-producing bacteria (Elferink et al., 1994). Furthermore, H2S in thebiogas can increase the cost for methane purification.

To avoid these problems, a new process was proposed (Fig. 1c),in which a portion of the thin stillage (pH 4.2e4.6) was mixed withthe digestate to decrease the pH of the process water, therebyeliminating the consumption of sulfuric acid. In this study, a newprocess was developed and assessed at the lab and on a pilot scale.

2. Methods and materials

2.1. Operation of the new process

In the first batch of ethanol fermentation, tap water was used tomake mash. At the end of the fermentation, the “beer’’ was trans-ferred into a distillation column and distilled. The distillage wascentrifuged (4000� g for 30 min) to obtain the supernatant (thinstillage). Next, 40% of the thin stillage was stored at �20 �C beforeuse. The residual was fed into the anaerobic digester. The digestate

Please cite this article in press as: Wang, K., et al., Reusing a mixture oproduction, Journal of Cleaner Production (2014), http://dx.doi.org/10.101

was then mixed with the thin stillage and recycled to make mashfor the following batch of ethanol fermentation.

2.2. Lab-scale experiments

2.2.1. Preparation of inoculaAngel alcohol yeast (strain AG, a commercial strain of

S. cerevisiae for ethanol production obtained from Hubei AngelYeast Co. Ltd., China) was maintained at 4 �C on yeast extract maltextract glucose (YMG) agar. The yeast was inoculated into a 500-mLErlenmeyer flask, which contained seed medium. The flask wasincubated in a rotating shaker at 100 rpm, 30 �C for 19 h before thecells were used as an inoculum for ethanol fermentation. The seedmedium contained (g/L): glucose 20, yeast extract 8.5, (NH4)2SO41.3, MgSO4$7H2O 0.1, and CaCl2$2H2O 0.06.

2.2.2. Mashing and ethanol fermentationCassava powder (starch content of 65e70% (w/w); size of

approximately 0.45 mm) was mixed with the process water (1 g


Table 1Comparison of the ethanol production in ethanol fermentation using differentcompositions of process water (different letters in the table indicate a significantdifference).

Percentage of thinstillage or tapwater in the mixture

Ethanol production (% v/v)

Fermentation witha mixture of thinstillage and digestate

Fermentation witha mixture of tapwater and digestate

30% 11.45 � 0.09bc 11.22 � 0.03e

40% 11.51 � 0.06ab 11.30 � 0.04de

50% 11.56 � 0.06a 11.37 � 0.06cd

K. Wang et al. / Journal of Cleaner Production xxx (2014) 1e7 3

cassava powder per 3 mL water) in a liquefaction tank. The slurrywas heated with steam to 95 �C after thermotolerant a-amylase(10 IU/g cassava, Genencor China Co. Ltd.) was added and held for2 h and cooled to 60 �C. Glucoamylase (130 IU/g corn, GenencorChina Co. Ltd.) was then added. When the temperature decreasedto 30 �C, the slurry was placed into a 10-L fermentor with aworkingvolume of 8 L (Baoxing Bio-engineering Co. Ltd., China) and 10% (v/v) of the inoculawas added to initiate the fermentation. Next, 0.5 g/L of urea (urea/medium, w/v) was added as the nitrogen source atthe first batch of fermentation (urea was not added in the latterbatches). Temperature was maintained at 30 �C. The total retentiontime in the fermentor was 48 h. The same conditions were alsoused for the experiments performed in the flasks.

2.2.3. Two-stage anaerobic digestion treatmentDetails of the two-stage anaerobic reactors have been previously

described by Wang et al. (2013). The distillage was fed into thethermophilic anaerobic sequencing batch reactor (ASBR) using aperistaltic pump in two days. The effluent was centrifuged (4000�g for 15 min) and pumped into the mesophilic ASBR. The effluent ofthe mesophilic ASBR was centrifuged at 4000� g for 15 min andstored at�20 �C before use. Cycles of both reactors were finished inone day, with 2 h feeding, 10 h reaction, 12 h settling, and 1 mindrainage.

2.3. Pilot-scale studies

The experiments were performed in a pilot plant built in HenanTianguan Fuel Ethanol Co. Ltd., China. The fermentor, thermophilicASBR and mesophilic ASBR had working volume of 1, 5.6, and1.5 m3, respectively. The processing steps and temperature controlswere the same as those described in the lab-scale studies.

2.4. Statistical analysis

Statistical analyses were performed using Excel and Origin 8.5(Microsoft Corp., USA). Data were analyzed using one-way analysisof variance (ANOVA) with Student’s t-test. Readings were consid-ered significant when the p-value <0.05. Standard errors and errorbars presented in the tables, and figures were calculated usinguntransformed data in ANOVA.

2.5. Analytical methods

Ethanol, glycerol, acetic acid, and lactic acid were determinedusing Dionex U3000 high-performance liquid chromatography(HPLC). The collected samples were centrifuged (10,000� g for10 min) and the supernatant was filtered (0.20-mm filter) prior toanalysis. A 20-mL sample or standard solution was injected into aBio-Rad HPX-87H Aminex ion exclusion column. The column wasoperated at 65 �C and 0.005 mol/L sulfuric acid was used as themobile phase at a flow rate of 0.6 mL/min. A refractive index de-tector (Shodex RI-101, Japan) was used for detection. The COD,conductivity, pH, NH3eN, volatile fatty acid (VFA), alkalinity weredetermined according to standard methods (APHA, 1995).

3. Results and discussion

3.1. Determination of the recycling ratio of thin stillage in the newprocess

In the ethanol fermentation process, actively growing yeastacidifies the culture medium via a combination of differential ionuptake, proton secretion during nutrient transport, direct secretionof organic acids, and CO2 evolution (Walker, 1998). For example,


when ammonium is used as a sole nitrogen source, the medium isacidified rapidly in the first hours of fermentation. Transport ofammonium is directly driven by the electrochemical plasmamembrane potential, which is generated by the plasma membraneATPase (Peña et al., 1987; Van der Rest et al., 1995), inwhich the ionis taken up and the proton is subsequently excreted into the me-dium to maintain a constant intracellular pH, thereby causingacidification of the extracellular medium. Acetic acid, lactic acidand other organic acid produced are in equilibriumwith their salts,thus the pH of the culture medium is maintained in the range from3.5 to 4.5. Thus, the thin stillage obtained after the distillationprocess was used to mix with the digestate to reduce the pH of theprocess water. However, the recycling ratio should be controlled toa suitable level. In conventional corn ethanol fermentation, thinstillage is partially recycled as the fermentation broth for ethanolproduction. Usually, less than a 50% recycling ratio for thin stillageas the fermentation broth (called backset in corn ethanol industry)can be utilized due to solid build-up and toxicity in response toyeast by lactic acid, acetic acid, and/or sodium (Egg et al., 1985;Ingledew, 2003; Shojaosadati et al., 1996). Considering this condi-tion and the pH of the process water (mixture of the thin stillageand digestate), three recycling ratios of thin stillage in processwater, 30%, 40% and 50%, were tested to obtain the optimum value.Mixtures consisting of digestate and 30%, 40% and 50% tap waterwere tested for the control, respectively. These results showed thatthe ethanol production and fermentation rate in fermentationwiththe thin stillage of each percentage were higher compared to thecontrol (Table 1, Fig. 2aec). However, compared with the 50% thinstillage, the 30% and 40% thin stillage showed a higher fermentationrate (Fig. 2d). In addition, the pH of the mixture with 40% thinstillage was close to the optimum pH of thermotolerant a-amylase(Table 2). Thus, 40% was considered to be the most optimal recy-cling ratio of the thin stillage.

3.2. Lab-scale experiments using the new process

3.2.1. Performance of anaerobic digestionIn the new process, 40% of the thin stillage was recycled as the

process water while the remaining portion was treated by anaer-obic digestion. Anaerobic digestion is a complex and multi-stepprocess involving many different microorganisms, which convertthe complex substances to their own cellular material and biogas.Thus, the digestate can be recycled to the fermentation process,which avoids the toxicity to S. cerevisiae caused by an accumulationof some inhibitory materials contained in the thin stillage. How-ever, it has been reported that ethanol fermentation can be affectedby the digestate from the deteriorated anaerobic digester. Zhanget al. (2010a) found that deterioration of anaerobic digestion per-formance causes increased concentrations of organic acids, namelyacetic, propionic, and butyric acids in the digestate, which can haveinhibitory effects on the growth of S. cerevisiae and ethanolfermentation (Zhang et al., 2011, 2010). Thus, stable operation andhigh efficiency of the anaerobic digestion are crucial to the


Fig. 2. Comparison of the fermentation rate in ethanol fermentation using different compositions of process water.

Table 3


integrated system. Data on the performance of the anaerobicdigestion is summarized in Table 3. The COD removal rate was98.0% and the methane yield was 317 mL/g COD removed onaverage, indicating a high efficiency of anaerobic digestion. The VFAconcentration of the digestate was 132e214 mg/L, whereas thealkalinity concentration was 2350e4335 mg/L for CaCO3. The VFAto alkalinity ratio, which is recognized as a key indicator of stableanaerobic digestion, was lower than the recommended levels,indicating a stable operation of anaerobic digestion (Sung andSantha, 2003). A stable operation can also be proven by theconsistent pH value. Furthermore, the NH3eN concentration of thedigestate remained at a high level (759mg/L on average). Moreover,a high concentration of ammonium in the digestate (>200 mg/L)could slightly reduce ethanol production (data not shown). Thus,the NH3eN removal process was added as further treatment in thisstudy.

3.2.2. Performance of ethanol fermentationEthanol fermentation was successfully performed for 10

batches. Tap water was used as the process water in the first batch,and the mixture of the thin stillage and digestate was used insubsequent batches (2e10), which were termed as recyclingbatches of fermentation. Although the error of measurement and aslight change of total sugar in each batch of fermentation can causea fluctuation in ethanol production, the average ethanol concen-tration in recycling batches was relatively higher compared to thefirst batch, which indicated that ethanol production was promoted

Table 2Comparison of different process streams in terms of pH, COD and conductivity.

Thin stillage Digestate Tap water 30%a 40%a 50%a

pH 3.90 7.83 7.45 6.31 5.91 5.40COD (mg/L) 37,600 2440 n.d.b e e e

Conductivity (mS/cm) 5310 7250 504 e e e

a Mixture of digestate and thin stillage with a different percentage of thin stillage.b Not detectable.


in the system (Fig. 3a). The glycerol and lactic acid concentrations atthe end of ethanol fermentation showed similar trends with theCOD in the thin stillage, while the acetic acid concentration fluc-tuated as the recycling process proceeded (Fig. 4, Fig. 5a).

3.2.3. Thin stillage characteristicsThe COD, conductivity and pH of the thin stillage were investi-

gated in this process. As shown in Fig. 5a and b, both the COD andconductivity increased as the recycling proceeded. However, withan increase in recycling batches, the COD and conductivity wereultimately balanced after the 6th and 3rd batch, respectively.Conductivity represents the strength of inorganic ions. Thecontinuous transfer of ions from the rawmaterial and nutrient saltsadded caused an increase in the concentration of inorganic ions(i.e., conductivity) in thin stillage. However, as the recycling processprogressed, a portion of the inorganic ions was removed from thesystem with the residues at the centrifugation step. Consequently,conductivity of the thin stillage increased at an early stage of theprocess, but ultimately remained consistent. The COD of the thinstillage represents the concentration of easily degraded organicmatter during anaerobic digestion. In the process, 40% of the thinstillage was recycled to the next batch of fermentation, while theother portion was treated by anaerobic digestion. Thus, someorganic materials will recycle and accumulate in the thin stillageduring the process. In addition, with the recycling of thin stillage,

Performance of two-stage anaerobic digestion treatment of thin stillage in lab-scaleand pilot-scale experiments (average value).

Parameters Lab-scale Pilot-scale

COD removal rate (%) 98.0 � 1.1 97.4 � 3.5VFAs (mg/L)a 160 � 31 212 � 24Alkalinity (mg CaCO3/L)a 3431 � 697 3579 � 738NH3eN (mg/L)a 759 � 90 698 � 77pHa 7.85 � 0.13 7.94 � 0.15Methane yield (mL/g COD removed) 317 � 19 303 � 21

a Parameters in the effluent from anaerobic digestion.


Fig. 3. Absolute difference in ethanol production (ethanol concentration in recycling batch minus ethanol concentration in the first batch) in different batches at the lab-scale (a)and pilot-scale (b).

Fig. 4. By-products (a. glycerol, b. lactic acid, c. acetic acid) of the ethanol fermentation in different batches in lab-scale experiments.


the organic materials will remarkably accumulate in the firstseveral batch of thin stillage. However, further accumulation willoccur at a lower level or no accumulation will be detected in thesubsequent batch of thin stillage. Data indicated that the accumu-lated material will no longer increase after the 6th batch offermentation. This finding allowed us to conclude each experimentafter seven recycles. The pH values of the thin stillage varied from4.25 to 4.62 (Fig. 5c).

3.3. Pilot-scale experiments

Pilot-scale experiments were performed under more realisticconditions to support results obtained from lab-scale experiments.The entire process was operated for a total of 48 days and 12


batches of ethanol fermentation were performed. Fig. 3b andTable 3 showed the results of ethanol fermentation and anaerobicdigestion performance. The average ethanol production for therecycling batches of fermentation was 13.55% (v/v), which wasslightly higher than that of the first batch of fermentation. The CODremoval efficiency and methane yield for the anaerobic digestionwas 97.4% and 303 mL/g COD removed, respectively, which weresimilar to the lab-scale experiment. The VFA concentrations in thedigestate were relatively higher compared to the lab-scale experi-ment. However, the VFA to alkalinity ratios for the digestateremained within the recommended level (<0.1). Low VFA to alka-linity ratio and stable pH value indicated a stable operation ofanaerobic digestion treatment of the thin stillage. The ammoniumconcentration was comparable with the lab-scale findings.


Fig. 5. COD (a), conductivity (b), and pH (c) of thin stillage in different batches in lab-scale experiments.


Currently, the bioethanol industry is faced with four challenges:1) Developing technologies to produce bioethanol from lignocel-lulosic biomass (Mussatto et al., 2010). The main technologiesinclude lignocellulose biomass pre-treatment, such as the appli-cation of new, engineered enzyme systems for cellulose hydrolysisand developing microorganisms, which are able to metabolize allpentose and hexose sugars and simultaneously withstand thestress imposed by the process inhibitors; 2) Exploration of veryhigh gravity (VHG) fermentation technology (Bai et al., 2008); 3)Providing biomass feedstocks with the nature of presenting a highnet energy gain, with ecological benefits and is economicallycompetitive and feasible for large-scale production withoutaffecting food provision (Sims et al., 2010); and 4) The achievementof an ideal balance between the energy consumed for bioethanolproduction and management of waste for other value-addedproducts (Hickey and Motylewski, 2007). Previous studies havebeen performed to identify solutions for this challenge.Shojaosadati et al. (1996) recycled 15%e70% (v/v) of de-alcoholizedstillage to molasses ethanol fermentation and found that the use ofup to 50% stillage did not greatly affect the yield of ethanol of up to10 repeated experiments, but the ethanol yield was adverselyaffected when the recycling ratio was greater than 50%. Egg et al.(1985) studied the recycling of stillage in grain sorghum ethanolfermentation and obtained similar results with Shojaosadati. Therecycling portion of stillage reduced the energy required forwastewater treatment and water consumption; however, theremaining wastewater still retained some contaminants andrequired further treatment or land disposal. Agler et al. (2008) andLee et al. (2011) adopted anaerobic digestion to treat corn thinstillage, and the produced biogas and decreased energy consump-tion in thin stillage evaporation and drying enhanced the NEB ratioin the corn ethanol production process. Furthermore, Gao and Li(2011) explored the possibility of using the digestate to replacefreshwater and nutrients for bioethanol production. These resultsshowed that the digestate can enhance bioethanol concentrationby up to 18% compared to freshwater-derived bioethanol and thesynergistic effect of nutrients, anaerobes, biochemical processingand enzymes in the digestate are contributing factors to thisenhanced ethanol production. However, reusing the digestate forethanol production requires the usage of sulfuric acid as previouslydescribed. In this study, a portion of thin stillage was mixed with


the digestate of the residual thin stillage and the mixture was thenreused as the process water for the following cassava ethanolfermentation. This technology resolved the problems faced bysolely recycling the thin stillage or the digestate. In addition,ethanol productionwas promoted in this process. However, furtherinvestigation is required to explain the increase in ethanolproduction.

4. Conclusions

A novel process was developed to resolve the problem of stillagemanagement in cassava ethanol fermentation. In the process, aportion of the thin stillage was treated by anaerobic digestion toproduce biogas, and the effluent was then mixed with the residualthin stillage and used as the process water for the next batch offermentation. The system reduced the energy and water con-sumption and achieved the goal of “zerowastewater” in the cassavaethanol production process. In addition, ethanol production wasenhanced.

Acknowledgments

This work was supported by the Science & Technology Programof Jiangsu Province (No. BE2011623) and Scientific Research Projectof Provincial Environmental Protection Bureau of Jiangsu Province(No. 2012047). We thank the Tianguan Fuel Ethanol Co. Ltd. fortheir support, and anonymous reviewers for critically evaluatingthis manuscript.

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reusing a mixture of anaerobic digestion effluent and thin stillage for cassava ethanol production

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