利用两级UASB消化处理实现木薯燃料乙醇的水循环型生产_英文_

BIOTECHNOLOGY AND BIOENGINEERING

Chinese Journal of Chemical Engineering , 18(5) 837—842 (2010)

Water-recycled Cassava Bioethanol Production Integrated with Two-stage UASB Treatment *

SUN Fubao (孙付保), MAO Zhonggui (毛忠贵)**, ZHANG Jianhua (张建华), ZHANG Hongjian (张宏建), TANG Lei (唐蕾), ZHANG Chengming (张成明), ZHANG Jing (张静)and ZHAI Fangfang (翟芳芳)

Key Laboratory of Industrial Biotechnology, Ministry of Education, Wuxi 214122, China

Fermentation and Ecological Engineering Laboratory (FEEL), School of Biotechnology, Jiangnan University, Wuxi 214122, China

Abstract Considering limited success in target-hitting discharge from alcohol industry, our attention was directed toward a recycling use of distillery spentwash (DS) in cassava bioethanol production by using a two-stage up-flow

anaerobic sludge blanket bioremediation (TS-UASBB). With the TS-UASBB, 24

SO ?

, COD, N and P in the effluent

from the DS degraded significantly and their concentrations were kept at 0.2 g·L ?1, 2.0 g·L ?1, 1.0 g·L ?1 and 15 mg·L ?1, respectively, in 13 batch processes for water-recycled ethanol fermentation. With the effluent used directly as dilu-tion water, no heat-resistant bacteria were found alive. The thirteen-batch ethanol production individually achieved 10% after 48 h fermentation. The starch utilization ratio and total sugar consumption were 90% and 99.5%, respec-tively. The novel water-recycled bioethanol production process with ethanol fermentation and TS-UASBB has a considerable potential in other starchy and cellulosic ethanol production.

Keywords cassava, bioethanol production, thermophilic and mesophilic, up-flow anaerobic sludge blanket, dis-tillery spentwash, recycle and reuse

1 INTRODUCTION

Cassava (Manihot esculenta), also called yuca or manioc, is a perennial woody shrub of Euphorbiaceae (spurge family), which is a native of South America and grows in tropical and subtropical areas. As the third largest source of carbohydrates for human food in the world, the cassava has become a good starchy material in food and fermentation industry. In China, cassava based bioethanol production reaches almost 1 million tons per year, approximately accounting for 60% of the total bioethanol output.

For production of 1 L ethanol, 8-15 L distillery spentwash (DS), also known as stillage, vinasse or distillery slop, is generally released. The large amount of DS results in a serious disposal or treatment prob-lem because of its high organic content (100 g·L ?1), strong acidity (pH 3.8-4.5) and dark brown color.

Since a large amount of water used in ethanol production is mainly responsible for the DS discharge, a concept for DS recycle was proposed [1-3], which was considered as the first priority for decreasing wa-ter consumption and DS discharge from alcohol fer-mentation [4, 5]. However, it was found that the direct use of DS had an adverse effect on both fermentation time and alcohol yield [2]. Thus a fraction of DS [<(30%-50%)] was reused [6], and some physico-chemical methods, especially the membrane process, were applied [7-9]. Owing to its flux decline and membrane fouling, the membrane filtration is not used extensively in ethanol industries. Therefore, it is nec-essary to consider some novel strategy. As an environmentally friendly and economically acceptable method for waste treatment, the up-flow anaerobic sludge blanket (UASB) bioremediation is considered desirable for treatment of high-strength organic wastewater because of its high biomass con-centration and rich microbial diversity [10-12]. The anaerobic digestion is also suitable for treating the DS, owing to some significant advantages, i .e ., less energy input, low nutrient demand, minimal sludge formation, and surplus biogas cogeneration [13, 14]. However, dis-charge standards are often stringent and beyond the scope of anaerobic digestion [14], so that some post- treatments including physico-chemical and aerobic processes are necessary [15]. As a result, these proc-esses are less cost-competitive.

To make the best use of the DS, we proposed a strategy, with the bioethanol production followed by an anaerobic digestion for its DS treatment [16, 17]. We used a thermophilic UASB bioreactor to treat and re-cycle the DS as the dilution water for ethanol fermen-tation [18], but the DS recycle could not be completed by a single thermophilic UASB treatment, as some fermentation-inhibitive volatile fatty acids, such as ace-tic acid, propionic acid, and lactic acid, were produced.

Considering the individual advantage of thermo-philic and mesophilic UASB digestions [10, 19], we carry out the experiment with their combination, namely a two-stage up-flow anaerobic sludge blanket bioremediation (TS-UASBB). To characterize the etha-nol fermentation with recycling DS, some key vari-ables such as concentrations of 24SO ?

, COD, N and P in the TS-UASBB are determined.

Received 2010-03-31, accepted 2010-06-22.

* Supported by the National High Technology Research and Development Program of China (2008AA10Z338) and the National Natural Science Foundation of China (20906041).

** To whom correspondence should be addressed. E-mail: feelingmao@https://www.360docs.net/doc/1518665081.html,

Chin. J. Chem. Eng., Vol. 18, No. 5, October 2010

838 2 MATERIALS AND METHODS 2.1 Materials and apparatus

Cassava was supplied by He’nan Tianguan Group Co., Ltd., He’nan Province, China. It was de-germed and milled drily into powder with an ap-proximately size of 0.38 mm (40 mesh), dried to con-stant mass at 60 °C and then stored in polyethylene plastic containers. The thermophilic and mesophilic anaerobic sludges were provided by Taixing Jinjiang Chemical Industry Co., Ltd., China and Disiman Citric Acid (Wuxi) Co., Ltd., China. They were originally used in the plant to treat the wastewater to meet the desired discharge standard. The ethanol fermentor (10 L) was manufactured by Shanghai Baoxing Bioengineering Equipment Co., Ltd., Shanghai, China. The ethanol distillery system was designed in our laboratory (FEEL). The two UASB bioreactors, also designed by our laboratory (FEEL), were maintained at (60±1) °C and (35±1) °C by a circulator bath, respectively. The former had a reaction region of 12 L and settlement zone of 2 L, and the latter had 10 L and 4 L correspondingly. 2.2 Water-recycled cassava ethanol production integrated with TS-UASBB treatment

The water-recycle process with ethanol fermenta-tion and anaerobic digestion treatment is shown in Fig. 1. The whole process is mainly composed of feedstock dilution, enzymatic saccharification, ethanol fermen-tation, distillation, thermophilic and mesophilic UASB, and effluent reservoir.

2.2.1 Feedstock dilution and enzymatic saccharifi-cation

In the batch run, the cassava powder of 2 kg was diluted at a solid-liquid ratio of 1︰3 with tap water or the effluent (in the reservoir) from TS-UASBB. Then, the broth was adjusted to pH 6.0-6.4 by 30% diluted H 2SO 4 solution. After soaking for 30 min, the broth was heated. At 50-60 °C, it was added with thermo-philic amylase [20 kU·ml ?1, Genencor (Wuxi) Bio- Products Co., Ltd.] at 10 U·g ?1 dry feedstock, heated to 100 °C and then maintained at 100 °C for 1 h, to make the starch hydrolyze completely. After cooled to

60 °C by pumping tap water into the jacket, the mash was calibrated to pH 4.2-4.4 with diluted H 2SO 4 and added with glucoamylase [130 kU·ml ?1, Genencor (Wuxi) Bio-Products Co., Ltd.] at 130 U·g -?1 dry feedstock for saccharifying. After saccharification for 30 min, a cassava sweet mash was obtained.

2.2.2 Ethanol fermentation process

Pre-culture of the yeast strain, Saccharomyces cerevisiae (Angle Stock Co., Ltd., Hubei Province, China), was carried out in 250 ml Erlenmeyer flasks containing 100 ml sterilized medium composed of (g·L ?1): glucose, 20; yeast extract, 8.5; NH 4Cl, 1.3; MgSO 4·7H 2O, 0.1 and CaCl 2, 0.06. After a loop of seed from the slant culture was inoculated into the culture, the pre-cultivation was initiated in a rotary shaker at 28 °C and 100 r·min ?1 for 12 h. Then, 10% (volume) of liquid inoculum was inoculated in the sweet mash complemented with urea at 3 g·L ?1. A static fermentation comprising of three stages, namely a prior fermentation at 28 °C for 6-8 h, a main fer-mentation at 32 °C for 16-18 h and a late fermentation at 30 °C for 24 h or 26 h, was carried out. Samples were withdrawn at regular intervals for measurement of pH, reducing sugar, total sugar, and ethanol. The fermentation finished when the residual sugar was approximate 0.5%. After the ethanol distillery, the hot DS (approximate 6 L) was discharged to a reservoir and cooled to 60 °C for the TS-UASBB, whose phys-ico-chemical property is shown in Table 1.

Table 1 Physico-chemical characteristics of the influent

DS from batch ethanol fermentation

Parameter Value

pH 5.04

total suspended solids/mg·L ?1 60500

total COD/mg·L ?1 119000

soluble COD/mg·L ?1 47000 gross protein/mg·L ?1 63

crude fat/mg·L ?1 2623 cellulose/mg·L ?1 18162

hemicelluloses/mg·L ?1 10897

lignin/mg·L ?1 2421

ash content/mg·L ?1

8072

Figure 1 Process of water-recycled bioethanol production with ethanol fermentation and two-stage UASB bioreactor (TS-UASBB)

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2.2.3 TS-UASBB process

To start the TS-UASBB process, thermophilic and mesophilic UASB bioreactors were loaded with anaerobic granular sludge of 4 L and 3 L, respectively, with the rest of their effective volumes complemented with tap water, which was circulated by a peristaltic pump overnight for acclimation. Before the spentwash (COD>100 g·L ?1) was fed to the thermophilic UASB bioreactor (60 °C), it was mixed with FeCl 2 at 2.5 g·L ?1 DS in the reservoir. Then the slurry was pumped at 2 L·d ?1 from the bottom to the bioreactor without dilution or residue removal, and its effluent was dis-charged at 2 L·d ?1. The high-temperature effluent was centrifuged at 4000 r·min ?1 for 30 min. The supernatant (approximate 1.5 L·d ?1) was pumped into the meso-philic UASB bioreactor (35 °C) and the effluent was discharged with the same volume every day. The ef-fluent was collected and kept at 5 °C to be used as an alternative to tap water to dilute the cassava feedstock. 2.3 Analytical methods

Indirect counting method, i .e . pour plate tech-nique, was used to determine the number of infected viable cells in the effluent from the TS-UASBB. The samples were diluted serially, and after the incubation at 30 °C, colonies grown in Petri dishes were used to count the number of viable cells. The fermentation broth was centrifuged at 13000 r·min ?1 for 10 min. The supernatant was then determined for total sugar by a phenol-sulfuric acid method [20]. Ethanol was analyzed by gas chromatography (Agilent 6890A, USA. Solid phase: cross-linked polyethylene glycol; carrier gas: nitrogen; isothermal capillary column at 180 °C; injection temperature 200 °C; flame ionization detec-tor temperature 250 °C; Agilent ChemStation Data Analysis System) and n -butanol was used as an inter-nal standard. The 24SO ?

content, total and soluble chemical oxygen demand (COD and sCOD), total ni-trogen (TN) and soluble TN, and total phosphorus (TP) and soluble TP were determined according to the Stan-dard Methods [21]. Each sample was analyzed in du-plicate, with the average value reported. The standard deviation was less than 6.4%. 3 RESULTS AND DISCUSSION 3.1 Performance of the TS-UASBB

Since the DS recycle in ethanol fermentation de-pends on the performance of the TS-UASBB, the an-aerobic digestion process was first characterized with

some important parameters (24SO ?

, COD, N and P).

3.1.1 24

SO ?

content of DS before and after TS-UASBB process

When the 24SO ?

concentration of substrate is high, anaerobic reduction of sulfate frequently inhibits the growth of some microorganisms, leading to ab-normal performance in reactors [22, 23]. Thus it is

necessary to detect the 24SO ?

content change in the TS-UASBB. In the ethanol fermentation, diluted sul-furic acid is frequently used to adjust the broth pH. In this study, a FeCl 2 solution was added at 2.5 g·L ?1 DS into the influent reservoir before thermophilic UASB

treatment to avoid the adverse effect of 24SO ?

[23, 24]. As shown in Fig. 2, the 24SO ?

content of the DS influent was low, 1.0 g·L ?1

, at the initial stage of TS-UASBB because tap water was used in the ethanol fermentation. With the effluent from TS-UASBB used,

the 24SO ?

content of DS influent almost maintained at 1.15 g·L ?1 regardless of the addition of diluted H 2SO 4 for pH adjustment in the enzymatic saccharification

process. In the TS-UASBB treatment, the 24SO ?

con-tent of first- and second-stage effluent decreased to approximate 0.3 g·L ?1 and 0.2 g·L ?1, respectively.

Moreover, the 24SO ?

content of effluent was main-tained at such a low level for a long time (thir-teen-batch ethanol fermentation). The result indicated that some anaerobic sulfate reducing bacteria exhib-ited a positive activity during the TS-UASBB process,

probably because large amount of 24SO ?

reduced to sulfur and sulfide [24]. Consequently, the TS-UASBB displayed a good, stable operation performance in re-ducing the high 24SO ?

content of DS and preventing the accumulation of 24SO ? in the effluent.

Figure 2 Change of 24

SO ?

content in the TS-UASBB with

water recycled in bioethanol production

(Fermentation: prior stage at 28 °C for 6-8 h, main stage at 32 °C for 16-18 h and late stage at 30 °C for 24-26 h; TS-UASBB treatment: the first stage at 60 °C; the second stage at 35 °C) ● DS influent; ■ first effluent; ▲ second effluent

3.1.2 COD change in the TS-UASBB

The experiment was carried out to determine COD content in the TS-UASBB to evaluate its ability to cope with the high-strength DS. As shown in Fig. 3, after the first-stage UASB bioreactor became stable, the COD and sCOD contents of influent increased slowly during 102 days, from 110 to 130 g·L ?1 and from 85 to 100 g·L ?1, respectively. Fig. 3 also illumi-nates the COD/sCOD change in the mesophilic (sec-ond-stage) UASB bioreactor. The influent COD and sCOD increased from 12 to 17 g·L ?1 and from 10 to 15 g·L ?1, respectively. The COD/sCOD of influent increased significant (by 5 g·L ?1), while that of effluent increased slightly, from 2 to 3 g·L ?1. With the meso-philic UASB digestion, the COD removal achieved 80%.

Chin. J. Chem. Eng., Vol. 18, No. 5, October 2010

840

Figure 3 Change of COD and sCOD in the TS-UASBB COD 0, sCOD 0—in the influent DS to the first-stage bioreactor (60 °C); COD 1, sCOD 1—in the influent to the second-stage bioreactor (35 °C); COD 2, sCOD 2—in the effluent from the second-stage bioreactor

○ COD 0; △ COD 1; □ COD 2; ● sCOD 0; ▲ sCOD 1; ■ sCOD 2

Additionally, for the DS the ratio of sCOD to to-tal COD was less than 80%, but it improved to 85%-90% after the thermophilic UASB digestion and declined to approximate 50% after the mesophilic UASB digestion. It could be postulated that the first-stage treatment contributed more to the hydroly-sis of large insoluble organic substances of DS to some small soluble substances. The second-stage treatment preferentially converted the soluble matter to biogas. These data evidenced that the two TS-UASBB proc-esses exhibited a good ability to remove COD from the DS. The effluent was at a low COD level, imply-ing that it was possible to use the effluent as an alter-native to tap water to dilute the feedstock of batch ethanol fermentation.

3.1.3 Change of nitrogen content in the TS-UASBB

Figure 4 describes the profile of nitrogen content in the TS-UASBB. In the early period, total nitrogen (TN 0) and soluble TN 0 of the DS (influent) were low, at 2.64 and 1.75 g·L ?1, respectively, owing to the utilization of tap water in the batch ethanol fermenta-tion. Then the TN 0 (soluble TN 0) of influent DS was almost stable at a level of 2.85 g·L ?1 (1.70 g·L ?1). For

the effluent from TS-UASBB, the total nitrogen (TN 1) and soluble TN 1 was lower, close to 1.00 and 0.70 g·L ?1, respectively, with a slight fluctuation. Experi-mental results showed that the nitrogen concentration of effluent after the TS-UASBB reduced greatly and maintained at a low level. Additionally, most nitrogen contained in the effluent was soluble, which would be a good nitrogen source, favorable to yeast growth dur-ing ethanol fermentation. Moreover, the nitrogen con-tent of the effluent treated by TS-UASBB stayed at a low level for a long time in thirteen-batch ethanol fer-mentation, without significant accumulation, which is desirable for the water recycle in ethanol fermentation. 3.1.4 Phosphorus content in the TS-UASBB

Under anaerobic conditions, the microorganism first liberates phosphate to the liquid phase, resulting in eutrophication when it is in excess [25]. Accordingly, the phosphorus concentration is also a key parameter for the TS-UASBB. To characterize the treatment performance, it is reasonable to detect the phosphorus content in the TS-UASBB. As shown in Fig. 5, the total phosphorus (TP 0) of the influent increases from 200 to 250 mg·L ?1 approximately, with the effluent used instead of tap water in ethanol fermentation. The soluble TP0 of the influent DS is from 175 to 150 mg·L ?1. The total phosphorus (TP 1) and soluble TP 1 contents of the effluent degrade to around 15 and 5 mg·L ?1, respectively. It indicates that phosphorus does not accumulate and tends to be consumed in the

TS-UASBB.

Figure 5 Phosphorus content in the TS-UASBB

[TP 0—total phosphorus content of the DS influent to the first-stage UASB bioreactor (60 °C); TP 1—total phosphorus content of the effluent from the second-stage UASB bioreactor (35 °C)]

spentwash: ○ TP 0; ● soluble TP 0 discharge: △ TP 1; ▲ soluble TP 1

The above experimental results show that 24SO ?

, COD, N and P do not accumulate significantly with the water recycle. These substances are at low levels in the TS-UASBB, which is helpful for the ethanol fermentation because they probably act as nutrients for yeast growth and ethanol production [10]. There-fore, it is of interest to further characterize the effect

of water recycle on batch ethanol fermentation.

Figure 4 Nitrogen content in the TS-UASBB

[TN 0—total nitrogen of DS influent to the first-stage UASB bioreactor (60 °C); TN 1—total nitrogen of effluent from the second-stage UASB bioreactor (35 °C)]

○ TN 0; ● soluble TN 0; △ TN 1; ▲ soluble TN 1

Chin. J. Chem. Eng., Vol. 18, No. 5, October 2010 841

3.2 Sterilization performance of the effluent from TS-UASBB

The bioremediation for treating the DS com-monly involves a complex microbial consortium. It is indispensable to evaluate the sterilization of effluent from TS-UASBB since ethanol fermentation is a pure breed cultivation process. Considering the subsequent high-temperature enzymatic saccharification process, the sterilizing test was carried out under the scope of thermophilic α-amylase activity, as shown in Table 2. Under the mild sterilization condition of 100 °C and 10 min, the viable population in sterilized effluent was up to 340 cfu·ml ?1. With the long sterilization time of 40 min, however, the microbial number decreased remarkably to 95 cfu·ml ?1. No obvious microbes were observed in the sterilized effluent after the sterilization time was longer than 50 min. No microbes were found alive in the effluent sterilized at 115 and 121 °C for a short time (10 min). Thus the mild condition of 100 °C and 50 min was sufficient for sterilizing the effluent.

The effluent from TS-UASBB contained no heat- resisting bacteria. Since the enzymatic saccharification of cassava starch was processed at 100 °C for 1 h, it was feasible to adopt the effluent for the dilution of feedstock without extra sterilization. Consequently, the microbiologic characteristic of the effluent was favorable to its reuse in ethanol fermentation. 3.3 Batch ethanol fermentation with recycling the DS in TS-UASBB

In order to assess the water recycle comprising batch ethanol fermentation and TS-UASBB, a thir-teen-batch fermentation using the effluent from mesophilic UASB treatment as dilution water was developed. The sugar consumption, starch utilization ratio and ethanol production of batch ethanol fermen-tation were examined (Figs. 6 and 7). For ethanol fermentation using tap water, the residual total sugar content reduced to 0.5% after 48 h. The starch utiliza-tion ratio and ethanol production reached 90% and 10.9%, respectively. When the effluent from TS-UASBB

was used as dilute water, the sugar was consumed fast in the thirteen-batch fermentation. After 48 h fermen-tation, the starch utilization ratio was above 90% and

the residual total sugar content was low at 0.5%. The ethanol production in 13 batches maintained at 10.5%.

The result indicates that there is no obvious difference

with tap water and effluent from TS-UASBB. The

effluent from TS-UASBB can be used to dilute the cassava feedstock. More importantly, the DS is reus-able for over ten times as dilution water in cas-sava-based ethanol fermentation after the TS-UASBB. In short, the combination of the thermophilic and mesophilic UASB bioreactors exhibited good behav-ior in treating the high-strength DS. The superior

treatment performance is responsible for recycling the

Figure 6 Total sugar consumption in the thirteen-batch ethanol fermentation with water recycle

(Fermentation: prior stage at 28 °C for 6-8 h, main stage at 32 °C for 16-18 h and late stage at 30 °C for 24 h or 26 h)

Table 2 Viable microbial population (cfu·ml ?1

) contained

in the effluent from second-stage UASB (35 °C) after sterilization at different temperatures and time Viable microbial population/cfu·ml ?1

Sterilization time/min 100 °C 115 °C 121 °C

10 340 0 0 15 322 0 0

20 210 0 0

30 200 0 0 40 95 0 0 50 0 0 0 60 0 0 0

Chin. J. Chem. Eng., Vol. 18, No. 5, October 2010

842 water successfully in ethanol production for a long period. This explains why the thirteen-batch ethanol fermentation using the effluent as dilution water pre-sents similar performance with that using tap water. The novel strategy of water-recycled ethanol produc-tion integrated with TS-UASBB realized satisfactory water recycle (80%-85%, by volume) in batch ethanol fermentation, while a small amount of water was con-sumed (15%-20%). Besides, rich biogas was also generated. In this study, the biogas yielded was ap-proximate 120 ml·g ?1 COD (data not shown), which was unsatisfactory. If the residue from solid-liquid separation after the first-stage treatment was exploited, there would be more biogas output. 4 CONCLUSIONS

A bioethanol production process integrating a TS-UASB

B was achieved with the complete DS recy-cle for 13 batches. The TS-UASBB effectively re-duced the content of 2

4SO ?, COD, N and P in the DS and kept them relatively stable at 0.2 g·L ?1, 2 g·L ?1, 1.0 g·L ?1 and 15 mg·L ?1, respectively. With the efflu-ent from the TS-UASBB used directly as dilution wa-ter, the thirteen-batch ethanol fermentation exhibited a good performance comparable to that using tap water. The ethanol production individually achieved 10% and the starch utilization ratio was 90% after 48 h. The novel process has a potential application in other starchy and cellulosic bioethanol industry. REFERENCES

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Figure 7 Starch utilization ratio and ethanol production of batch ethanol fermentation with process water recycle (Fermentation: prior stage at 28 °C for 6-8 h, main stage at 32 °C for 16-18 h and late stage at 30

°C for 24 h or 26 h)

starch utilization; ethanol production