Treatment of, and Candida utilis biomass production from shochu wastewater; the effects of maintaining a low pH on DOC removal and feeding cultivation on biomass production
© Watanabe et al.; licensee Springer. 2013
Received: 5 August 2013
Accepted: 1 October 2013
Published: 6 October 2013
Shochu wastewater (SW; alcoholic distillery wastewater) contains large amounts of organic compounds (25,000 – 60,000 COD mg/L), nitrogen (1,000 – 6,000 T-N mg/L), and phosphorus (500 – 1,000 T-P mg/L). Despite its high nutrient content, SW is highly perishable, which limits its utilization for animal feed and fertilizer. Therefore, SW is mainly treated by methane fermentation. On the other hand, a feed yeast, Candida utilis, can utilize various organic compounds and be utilized as a yeast extract source and animal feed. We previously bred a mutant, C. utilis UNA1, that accumulates a large amount of nitrogen. Here, we investigated the use of C. utilis UNA1 to treat highly concentrated SW. With fed-batch cultivation using a 5-L jar fermenter, controlling pH at 5.0 with H2SO4, 62.9% of DOC, 38.4% of DTN, and 44.5% of DTP were stably removed from non-diluted barley shochu wastewater (BSW), and about 16.7 kg of freeze-dried yeast biomass was obtained. The yeast sludge biomass generated from BSW contains about 60% crude protein. Furthermore, using H2SO4 to control pH increased the sulfur content of wastewater, which increased the methionine composition of yeast sludge biomass.
Shochu is a traditional Japanese distilled liquor made from barley, sweet-potato, rice, and other crops. In the south Kyushu region, the annual discharge of shochu waste is about 689,000 ton/year. Although shochu waste contains large amounts of nutrients (Ikeda et al. 2012), its high perishability limits its use for animal feed and fertilizer (Tsuyumu et al. 2003). Shochu waste has high concentrations of suspended solids (30,000 – 65,000 SS mg/L), organic compounds (25,000 - 60,000 COD mg/L), nitrogen (1,000 – 6,000 T-N mg/L), and phosphorus (500 – 1,000 T-P mg/L). This makes it too expensive to treat by the conventional activated sludge method. The main treatment method of shochu waste is solid–liquid separation, and the liquid part (shochu wastewater; SW) is treated by combining a methane fermentation process, a physical phosphorus removing process, and a conventional activated sludge process.
Previously, the National Research Institute of Brewing (NRIB) of Japan developed an aerobic wastewater treatment method using a combination of yeasts and activated sludge (Yoshizawa 1978). This system removes large amounts of organic compounds, requires little space, and discharges little waste sludge. This method is useful for treating food and beverage industry wastewater (Yoshizawa 1978) and has also been used to treat SW in laboratory experiments for over thirty years (Saito et al. 1983; Suzuki et al. 1991; Watanabe et al. 2009; Yoshii et al. 2001).
The wastewater treatment abilities of yeast strains can be improved by non-recombinant techniques. We found that phenotypes of the PHO regulatory system of Saccharomyces cerevisiae (Oshima 1997) are useful for improving the phosphorus accumulation capacity (Watanabe et al. 2008). We also found that positive selection of methylamine-resistant mutants to isolate URE2 mutants (Kamerud & Roon 1986) was useful for finding strains with improved nitrogen accumulation ability (Watanabe et al. 2013b). We also confirmed this method can apply to a wastewater treatment yeast Hansenula anomala (Watanabe et al. 2013c).
However, it is necessary to keep these yeasts as the dominant microorganism in the yeast treatment tank by adding chemicals, such as HClO and HCl (Yoshizawa et al. 1980), and by replacing the yeast sludge with fresh seed yeast sludge at regular intervals (Watanabe et al. 2009). On the other hand, treating wastewater with yeast is also considered an attractive way to produce yeast biomass resource. For example, cheese whey was used to produce a food yeast, Kluyveromyces fragilis (Paul et al. 2002). Yeast biomass productivities of Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis were investigated using brewery’s spent grains hemicellulosic hydrolyzates (Duartc et al. 2008). A feed yeast Candida utilis was used to produce biomass from salad oil manufacturing wastewater (Zheng et al. 2005). Furthermore, the effect of C. utilis on the degradation of forages was investigated (Ando et al. 2006).
However, these studies did not investigate the stability of continuous cultivation and the wastewater treatment abilities. Because wastewater is discharged continuously over long periods, the treatment method must be highly stable. In order to continuously treat SW and produce yeast biomass at the same time, we previously bred a mutant C. utilis UNA1 with high nitrogen-accumulating ability (Watanabe et al. 2013a). In that study, we also demonstrated repeated-batch cultivation using barley shochu wastewater (BSW) (two times diluted) and obtained about 10 g dry yeast sludge biomass per liter of wastewater. Because dilution of SW requires large scale reactors and high operational costs, further works to efficiently treat undiluted SW are needed for practical application.
Here, we investigated the use of C. utilis UNA1 to treat undiluted SW. We found that we could do this by controlling the pH and continuously feeding into the reactor. We also estimated the components of yeast sludge biomass.
Materials and methods
Strains and culture conditions
Candida utilis IFO1086 strain and its highly-nitrogen-accumulating mutant UNA1 (Watanabe et al. 2013a) were used in this study. These strains were maintained on YM agar plates (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, and 1% glucose, and 2% agar) at 4°C.
Physico-chemical characteristics of shochu wastewater used in this study
Total sugar (mg/l)
Organic acids (mg/l)
The absorbance at 660 nm (OD660) was measured in order to determine cell density using a spectrometer (Ultrospec 2000, Pharmacia Biotech Co. Ltd., Cambridge, England). Dissolved organic carbon (DOC) was analyzed using a TOC analyzer (TOC-5000A, Shimazu Co. Ltd., Kyoto, Japan). Nitrate (NO3-N), Dissolved total nitrogen (DTN), inorganic phosphate (PO4-P), and total phosphorus were analyzed according standard methods (APHA, AWWA, WEF 1998). pH was measured using a pH meter (HM-30S, TOA DKK Co. Ltd., Tokyo, Japan).
Repeated-batch cultivation using a 1.5-L jar fermenter
The yeast strains were pre-incubated in a test tube containing 5 mL of YM medium with shaking at 30°C for 24 h. The pre-culture was inoculated into 1 L of two-times-diluted sweet-potato shochu wastewater (SSW) in a 1.5-L jar fermenter. The cultivation conditions were agitating at 400 rpm, aerating at 0.7 LPM, and incubating at 30°C. After 24 h cultivation, 0.5 liters of culture was replaced with new 0.5 liters of SSW every 12 h. The replacing cycles were repeated 30 times for C. utilis UNA1 and 12 times for C. utilis IFO1086.
Thus, thy hydraulic retention time (HRT) and the sludge retention time (SRT) were 24 h. The DOC-volume loading of SSW was about 16 kg/m3/d.
pH controlled-batch cultivation using a 5-L jar fermenter
C. utilis UNA1 was pre-incubated in 50 mL YM medium using a 300-mL flask with shaking at 120 rpm, at 30°C for 24 h. The pre-culture was inoculated into 3 L of 5-times-diluted BSW. The cultivation conditions were agitating at 400 rpm, aerating at 0.5 LPM, and incubating at 30°C. When pH exceeded 5.0, H2SO4 solution (0.49%) was automatically added to maintain pH value at 5.0.
Repeated-fed-batch cultivation using a 5-L jar fermenter
C. utilis UNA1 was pre-incubated in 50 mL YM medium using a 300-mL flask with shaking at 120 rpm, at 30°C for 24 h. The pre-culture was inoculated into 2 L of 5-times-diluted BSW. The cultivation conditions were agitating at 400 rpm, aerating at 0.5 LPM, incubating at 30°C, and pH value was maintained at 5.0 using H2SO4 solution (0.49%). After 24 h cultivation, BSW was continuously feeding at 2 L/24 h ratio, and 2 L of culture was removed at every 24 h. After 4 times repeated this cycle, the feeding wastewater was changed to 1:1 mixture of BSW and SSW (mixed shochu wastewater; MSW). Feeding ratio and removing time were also changed to 2 L/20 h and 20 h, respectively. This cycle was also repeated 4 times.
Thus, the hydraulic retention time (HRT) and the sludge retention time (SRT) of BSW and MSW 48 h and 40 h, respectively. The DOC-volume loadings of BSW and MSW were 20.9 kg/m3/d and 18.9 kg/m3/d, respectively.
Component analysis of yeast sludge biomass
Culture from the jar fermenter cultivation was centrifuged at 7,000 rpm for 10 min to obtain yeast sludge biomass. The yeast sludge biomass was freeze-dried using a BenchTop 4 K Freeze dryers (model 4KBTZL, SP Industries, NY, USA). The freeze-dried yeast sludge biomass was analyzed for protein, fat, fiber, soluble non-nitrogen, and ash according to the manual of Association of Official Analytical Chemists (AOAC 1980). Amino acid analysis was performed by hydrolyzing the freeze-dried yeast sludge biomass with 6 N HCl at 110°C for 22 h and analyzing the hydrolysate with an amino acid auto-analyzer.
Results and discussion
Repeated-batch cultivation of sweet-potato shochu wastewater
BSW contains more organics and total nitrogen than other kinds of SW. Its discharge volume per year is also the largest. We previously confirmed that C. utilis UNA1 could efficiently and stably treat BSW by repeated-batch cultivation using a 1.5-L jar fermenter (Watanabe et al. 2013a). The discharge volume per year of SSW is almost the same as that of BSW. We first investigated the efficiency of SSW using a 1.5-L jar fermenter.
Summary performances of repeated-batch cultivation by C. utilis UNA1 and C. utilis IFO1086
Sweet-potato shochu wastewater
Barley shochu wastewatera
15877 ± 740
15621 ± 1201
19731 ± 311
18444 ± 557
7691 ± 545
8179 ± 927
8075 ± 314
8217 ± 495
Removal ratio (%)
51.6 ± 3.4
47.6 ± 5.9
59.1 ± 1.6
55.5 ± 2.7
1013.3 ± 46.7
985.2 ± 71.5
2736.5 ± 67.0
2674.6 ± 59.7
477.9 ± 18.8
489.6 ± 38.3
1748.4 ± 44.4
1792.9 ± 87.9
Removal ratio (%)
52.8 ± 1.9
50.3 ± 3.9
36.1 ± 1.6
33.0 ± 3.3
289.2 ± 13.9
264.7 ± 17.5
383.1 ± 7.4
378.7 ± 6.3
116.3 ± 10.4
131.5 ± 16.4
195.4 ± 10.9
228.4 ± 20.6
Removal ratio (%)
59.8 ± 3.6
50.3 ± 6.2
49.0 ± 2.8
39.7 ± 5.4
4.08 ± 0.01
3.98 ± 0.01
4.07 ± 0.03
4.18 ± 0.00
7.28 ± 0.27
6.17 ± 0.65
8.00 ± 0.15
8.49 ± 0.07
32.7 ± 2.1
31.4 ± 3.4
34.9 ± 1.2
Effect of pH control on barley shochu wastewater treatment
When SSW and BSW were treated by C. utilis UNA1, the pH increased from about 4.0 to 7.28 – 8.0 (Table 2). The preferred pH of C. utilis cultures is around 4.5 to 6.0. Previously it was shown that the consumption of organic acids by fungi and yeasts caused the pH to change from mildly acidic to mildly alkaline (Tsuyumu et al. 2003; Watanabe et al. 2009). We also thought that the remaining cations (K+ and Ca2+) and ammonium ion generation by deaminization would raise the pH to mildly alkaline. To improve the treatment efficiency and cell growth, we investigated the effect of lowering the pH to the value preferred by C. utilis by acid addition.
The effect of pH control on process performance of batch cultivation with C. utilis UNA1 using 5-times diluted barley shochu wastewater
20.1 ± 0.2
18.4 ± 0.1
7.58 ± 0.53
5.00 ± 0.05
DOC removal amount (mg/l)
5337 ± 4
6597 ± 49
DOC removal ratio (%)
61.1 ± 0.0
75.5a ± 0.6
DTN removal amount (mg/l)
596.5 ± 2.9
563.0 ± 3.5
DTN removal ratio (%)
44.2 ± 0.2
41.4 ± 0.3
DTP removal amount (mg/l)
137.9 ± 1.3
142.9 ± 0.9
DTP removal ratio (%)
75.1 ± 0.7
75.8 ± 0.5
Effect of fed-batch cultivation on shochu wastewater treatment
C. utilis UNA1 (and C. utilis IFO1086) cannot grow in non-diluted BSW in batch cultivation, and thus dilution with water by 4 – 5 times is needed. However, dilution of BSW requires that the treatment tank be larger, which increases the operational costs. On the other hand, the culture could be kept stable and efficiently treated by repeated-batch cultivation by replacing half the volume of treated wastewater with new 2-times-diluted BSW (Watanabe et al. 2013a). Therefore, we investigated the effect of continuous feeding into large amounts of culture on SW treatment by C. utilis UNA1.
Treatment of barley shochu wastewater
Summary performances of fed-batch cultivation by C. utilis UNA1
41969 ± 91
31598 ± 359
15573 ± 1315
10830 ± 617
Removal ratio (%)
62.9 ± 3.1
65.7 ± 2.0
6113.8 ± 83.5
3660.1 ± 84.8
3764.7 ± 224.7
1894.1 ± 19.7
Removal ratio (%)
38.4 ± 3.7
48.3 ± 0.5
787.0 ± 6.2
542.0 ± 2.2
436.7 ± 34.7
229.2 ± 2.6
Removal ratio (%)
44.5 ± 4.4
57.7 ± 0.5
79.1 ± 1.5
68.4 ± 0.7
Treatment of mixed shochu wastewater
In order to concentrate SSW and to dilute BSW, we treated a 1:1 mixture of SSW and BSW (MSW). With repeated-batch cultivation, cell growth in MSW was about twice that in non-diluted SSW (Tables 2, 4). About 14.4 kg freeze-dried yeast biomass was obtained from 1 ton of MSW cultivation. Furthermore, removal ratios of DOC, DTN, and DTP of MSW treatment were higher than those of BSW (Table 4). Therefore, these results indicate that feeding cultivation of MSW with controlled pH is useful for both wastewater treatment and cell biomass production.
A marine thraustochytrid, Schizochytrium sp. strain KH105, was used to produce polyunsaturated fatty acids and xanthophylls from SW (v2006). However, it did not utilize and remove organic compounds from SW, and thus glucose addition was needed. In contrast, C. utilis UNA1 can utilize and removed over 60% of DOC, resulting in a large amount of yeast sludge biomass (Table 4).
Regarding wastewater treatment, over 30% of DOC, DTN, and DTP were still remained in yeast treated SW (Table 4). We previously confirmed that effluent of treated BSW was efficiently treated by a combination of nitrification/denitrification cycle treatment and activated sludge process (Watanabe et al. 2013a; Watanabe et al. 2009). In a laboratory-scale demonstration, 50 cycles (25 days) removed 98.9% of DOC, 95.7% of DTN, and 94.1% of DTP from BSW (Watanabe et al. 2013a). Thus, the remaining DOC, DTN, and DTP of yeast treated SW could be easily removed by conventional treatment methods.
Chemical composition of C. utilis in each treatment process
General compositions of yeast sludge biomass generated in each treatment process with shochu wastewater
(% of dry matter)
Amino acid compositions of yeast sludge biomass generated in each treatment process with shochu wastewater
(% of crude protein)
In the present study, we investigated the cultivation process of highly concentrated SW in order to effectively treat wastewater and to produce large amounts of yeast sludge biomass to reduce operational costs. With fed-batch cultivation using a 5-L jar fermenter, controlling pH value at 5.0 with H2SO4, 62.9% of DOC, 38.4% of DTN, and 44.5% of DTP were stably removed from non-diluted BSW, and about 16.7 kg of freeze-dry yeast biomass was obtained. Furthermore, mixed with relatively thin SSW 1:1, removal ratios of DOC, DTN, and DTP were increased, and 14.4 kg of freeze-dry yeast biomass was obtained. Yeast sludge biomass generated in BSW and MSW contains about 60% of crude protein. Thus, they are useful for yeast extract source and animal feed protein. Furthermore, using H2SO4 to control pH supplied the wastewater with sulfur (S) appeared to raise the methionine content of yeast sludge biomass.
Shohu is a Japanese traditional liquor and its method of production is similar to the methods of whisky and brandy. Whisky distillery wastewater can be treated with yeast (Yamamoto et al. 1986). Thus, yeast should have the same advantages for alcohol distillery wastewater as it has for SW.
We thank Komasa Shuzou Co. Ltd. for kindly providing shochu wastewater. This work was supported by a Research Fellowship of the Japan Society for the Promotion of Science (JSPS) for Young Scientists, and by JSPS KAKENHI Grant no. 23 · 10107.
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