Bioresource Technology 100 (2009) 1781–1785

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Bioresource Technology

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Treatment and phosphorus removal from high-concentration organicwastewater by the yeast Hansenula anomala J224 PAWA

Takashi Watanabe a,b, Kazuo Masaki b, Kazuhiro Iwashita b, Tsutomu Fujii a,b, Haruyuki Iefuji a,b,*

a Graduate School of Biosphere Science Hiroshima University, Kagamiyama 1-4-4, Higashihiroshima, Hiroshima 739-8527, Japanb National Research Institute of Brewing, Kagamiyama 3-7-1, Higashihiroshima, Hiroshima 739-0046, Japan

a r t i c l e i n f o

Article history:Received 6 July 2008Received in revised form 4 October 2008Accepted 6 October 2008Available online 17 November 2008

Keywords:PhosphorusFlocculent yeastWastewater treatmentAlcoholic distillery wastewaterSemi-batch continuous treatment

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.10.006

* Corresponding author. Address: National Researchyama 3-7-1, Higashihiroshima, Hiroshima 739-0046,fax: +81 82 424 0806.

E-mail address: iefuji@nrib.go.jp (H. Iefuji).

a b s t r a c t

A flocculent yeast, Hansenula anomala J224 PAWA, bred in this study, accumulated twice as much phos-phorus as the wild type. Over a 30-d period, PAWA removed 70–80% of dissolved total phosphorus fromsweet-potato and barley shochu wastewaters (alcoholic distillery wastewaters) while the wild typeremoved only 30%. Waste sludge was easily separated from effluent wastewater because PAWA cellsmade large flocks that rapidly settled. Component analysis suggested that PAWA sludge could be usedas a protein source for feedstuff and as a phosphorus source for fertilizer. Under anaerobic conditions,denitrification was rapid, resulting in the removal of large amounts of nitrogen from barley shochuwastewater. These results suggest that small shochu manufacturers could benefit from using PAWA toremove phosphorus and organic compounds and then by using a combination of the upflow anaerobicsludge blanket and the down-flow hanging sponge method (UASB-DHS method) for nitrification/denitrification.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Phosphorus needs to be removed from wastewater because itcauses eutrophication of lakes, bays and other surface watersand because it is needed as a resource since natural phosphorusdeposits are being rapidly depleted (Abelson, 1999). Conventionalbiological phosphorus removal (BPR) processes cycle betweenanaerobic and aerobic conditions (Saktaywin et al., 2005), andare useful for sewage treatment. However, BPR by itself is not suf-ficient to treat wastewater with high concentrations of organicsfrom the food and beverage industry. Broughton et al. (2008)studied about the BPR process treating a high-strength wastewa-ter (800 COD mg l�1, 80 P mg l�1). However, the COD and phos-phorus contained in the initial wastewater of each batch werenot so high (200 COD mg l�1 and 20 P mg l�1).

The National Research Institute of Brewing (NRIB) of Japan devel-oped a wastewater treatment method using a combination of yeastsand activated sludge (Yoshizawa, 1978) which could remove largeamounts of organic compounds (10,000 COD mg l�1 d�1), requireslittle space, and discharges little waste sludge. This method is usefulfor treating such as food and beverage industry wastewater(Yoshizawa, 1978), olive mill wastewater (Lanciotti et al., 2005)

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Institute of Brewing, KagamiJapan. Tel.: +81 82 424 0818;

and chemical industry (containing formaldehyde and methanol)wastewater (Kaszycki et al., 2001). However, this system was noteffective at removing phosphorus. We recently isolated yeast mu-tants that could remove about three times as much phosphorus aswild strains from wastewater (Watanabe et al., 2008). These strainswere also as effective as wild strains at removing organic com-pounds from high-concentration organic wastewater. However,these yeasts were difficult to remove from wastewater because theydidn’t flocculate.

Shochu is a traditional Japanese distilled liquor. Shochu wastehas high concentrations of organics (25,000–50,000 COD mg l�1)and phosphorus (400–1000 mg l�1). In the south Kyushu region,the annual discharge of shochu waste is about 79,000 ton. Shochuwaste was previously dumped into the ocean but this will soonbe illegal. Thus, new treatment systems are needed, especially forsmall shochu manufacturers, because they can’t afford to treatwastewater with conventional expensive methods. High-phospho-rus-accumulating yeasts might thus be well suited for suchsystems.

The upflow anaerobic sludge blanket (UASB) system is widelyused for pretreatment of activated sludge (Seghezoo et al., 1998).Yamada et al. (2006) showed that treatment of shochu wastewater(supernatants of shochu waste) with a pilot-scale thermophilicmulti-staged UASB (MS-UASB) reactor could remove COD at a highrate (60 kg COD m�3 d�1). However, because the UASB system isanaerobic, it cannot remove phosphorus or nitrogen. A physico-chemical removal treatment (such as precipitation) is needed.

1782 T. Watanabe et al. / Bioresource Technology 100 (2009) 1781–1785

The UASB system also requires the addition of chemicals to main-tain a neutral pH (Yamada et al., 2006). As a post-treatment forUASB, activated sludge treatment requires a lot of energy for aera-tion. Instead of the activated sludge treatment, the down-flowhanging sponge (DHS) system was developed as a post-treatmentfor UASB (Machdar et al., 2000). The UASB-DHS combination sys-tem is less expensive to operate and thus is better suited for devel-oping countries than the activated sludge treatment (Machdaret al., 2000; Tandukar et al., 2005).

A flocculent yeast, Hansenula anomala J224 was originally iso-lated as a wastewater treatment yeast from a wastewater treat-ment tank of a Japanese sake brewing factory (Saito et al., 1990).In this study, we attempted to enhance the phosphorus-accumu-lating activity of H. anomala J224. One mutant obtained in thisstudy, H. anomala J224 PAWA, accumulated twice as much phos-phorus as the wild type. In a semi-batch continuous experimentin this study, this strain removed not only phosphorus but alsothe majority of organic matter from shochu wastewater.

2. Methods

2.1. Strains and culture conditions

The flocculent yeast H. anomala J224 (Saito et al., 1990) was ob-tained from our stock collection. A flocculent yeast accumulatinghigh-concentration phosphorus, H. anomala J224 PAWA was de-rived from strain J224. A non-flocculent yeast that accumulateshigh concentration of phosphorus, H. anomala J224-1 PAW1, whichwas derived from strain J224-1 (Watanabe et al., 2008), was usedas a construct for a sedimentation experiment.

YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptoneand 1% dextrose) was used for pre-cultivation. High P-YPD medium(1% yeast extract, 2% peptone, 2% dextrose and 0.8% KH2PO4) wasused to screen mutants that stained blue by the acid phosphatasehydrolysis of 5-bromo-4-chloro-indolylphosphate (X-phosphate;50 mg l�1 in high P-YPD medium) (Morohoshi et al., 2002).Sweet-potato shochu and barley shochu waste were centrifugedat 7000 rpm for 10 min to obtain the supernatants. Each superna-tant was used as shochu wastewaters for batch and semi-batchcontinuous wastewater experiments.

2.2. Analytical methods

Dissolved total phosphorus (DTP), dissolved total nitrogen(DTN), dissolved organic carbon (DOC), nitrate nitrogen (NO3–N)and ammonium nitrogen (NH3–N) of the supernatant (centrifugedat 3500 rpm for 10 min.) were measured using standard methods(APHA et al., 1998). DTP was digested using the sulfuric acid–nitricacid digestion and then measured as inorganic phosphate (PO4–P)using the ascorbic methods. DTN was digested using alkali perox-odisulfate digestion method and then measured as NO3–N. NH4–Nwas measured using the indophenol method. Organic nitrogen(Org-N) was calculated as DTN minus inorganic nitrogen (NO3–Nand NH4–N). pH was measured using a pH meter (F-52, HORIBA, Ja-pan). Mixed liquor suspended solids (MLSS), sludge volume index(SVI), and were assayed using standard methods (APHA et al.,1998). SVI is defined as V30 � 10,000 MLSS�1, where V30 is the per-centage of occupancy sludge volume settled after 30 min. SVI de-scribes the volume that 1 g MLSS has after 30 min of settling.

2.3. Mutation of yeast

Yeast strains were induced by the mutagenic agent ethylme-thanesulfonate (EMS) (Lindegren et al., 1965). Yeast strains were

cultivated in 5 ml of YM medium with shaking for 24 h. An aliquotof the culture medium (250 ll), 4.6 ml of 0.2 M phosphoric acidbuffer (pH 8.0) and 150 ll of EMS were mixed in test tubes. Thetest tubes were shaken gently for 1 h at 30 �C. Hydrosulfite sodiumsolution (6%) was added to stop EMS-induced mutation and thetest tubes were left at rest for 10 min. The cell suspension was di-luted by a factor of 50 with 67 mM phosphoric acid buffer andspread on high P-YPD plates containing X-phosphate. Followingovernight cultivation, blue-stained colonies that hydrolyzed X-phosphate by acid phosphatase were picked up as mutants thatconstitutively express acid phosphatase. The mutants and theparental strain were each cultivated in 5 ml of High-P YPD mediumfor 24 h at 30 �C with shaking.

2.4. Batch wastewater treatment experiment

Each shochu wastewater was diluted to adjust DOC concentra-tion to around 8000 mg l�1. Yeast strains were pre-cultivated withshaking in 5 ml of YM medium for 24 h. Cell density was measuredby the absorbance at 660 nm (A660). The cells were harvested,washed with sterilized H2O twice, inoculated into 50 ml ofsweet-potato or barley shochu wastewater (0.2A660) in 200 mlflasks, and grown at 30 �C with shaking at 120 rpm. Every 12 h till48 h, 1 ml aliquots were harvested and centrifuged (3500 rpm,10 min) and analyzed.

To measure flocculation ability, the cells were inoculated into500 ml of shochu wastewater (0.2A660) in 2 l flasks, and grown at30 �C with shaking at 120 rpm for 48 h. One liter of culture wasput in a 1-l graduated cylinder, and the time course change ofthe volume occupied by the sludge was analyzed.

2.5. Semi-batch continuous wastewater treatment experiment

A water tank (32 cm � 20 cm � 25 cm deep) was used for thisexperiment. The total volume of water tank is 16 l, with a workingvolume of 6 l. Air was supplied from an air pump and through anair-stone sitting on the bottom of the water tank. Wastewaterwas agitated by a stirrer turning at 200 rpm. The water tank wasset in an incubator box at 30 �C.

The experiment was conducted as follows: step 1, 1 l of seedsludge of PAWA was pre-cultivated using 2-l flasks, shaking at120 rpm for 24 h. Step 2, 5 l of sweet-potato shochu wastewaterand 1 l of pre-culture were mixed in the water tank. Step 3, after6 h, 1 l of treated water with sludge was removed with a siphon.Step 4, 1 l of wastewater, 2.5 ml of 10% anti-foam (Wako) and2.5 ml of 5% HClO were added to the tank. HClO was added to keepyeast as the dominant microorganism in the tank (Yoshizawa et al.,1980). Step 5, steps 3 and 4 were repeated 60 times using sweet-potato shochu wastewater, and then 2 l of seed sludge was replacedwith 2 l of treated water. Step 6, steps 3 and 4 were repeated 60times using barley shochu wastewater.

Thus, the hydraulic retention time (HRT) and the sludge reten-tion time (SRT) were 36 h. The DOC-volume loadings of sweet-po-tato shochu wastewater and barley shochu wastewater were8 kg m�3 d�1 and 13 kg m�3 d�1, respectively.

2.6. Component analysis of dehydrated shochu waste and yeast sludge

Dehydrated shochu waste was obtained by centrifuging shochuwaste at 7000 rpm for 10 min and discarding supernatant. Yeastwaste sludge was obtained from the semi-batch continuous exper-iment and from a preliminary experiment. These by-products wereanalyzed for moisture content, protein, fat, fiber, soluble non-nitro-gen and ash according to the manual of the Association of OfficialAnalytical Chemists (AOAC, 1980).

T. Watanabe et al. / Bioresource Technology 100 (2009) 1781–1785 1783

2.7. Denitrification of effluent of barley shochu wastewater treated byyeast

One liter of effluent of barley shochu wastewater treated byyeast from the continuous experiment and 1 l of waste sludge fromthe sewage treatment plant of Higashihiroshima city were put intoa 2-l glass bottle. The bottle was capped. The mixture was mixedwith a magnetic stirrer at 35 �C for 20 d. Every 24 h, 500 ml ofthe mixture was replaced with 500 ml of new wastewater.

3. Results and discussion

3.1. Comparison of wastewater treatment abilities on batchexperiment

Mutagenized samples of H. anomala J224 were spread on platesand about 40,000 colonies appeared. Of these colonies, one mutant,named H. anomala J224 PAWA (PAWA), accumulated twice asmuch phosphorus as the wild-type strain (data not shown).

PAWA and the parent strain were about equally effective inremoving DOC and DTN from sweet-potato shochu wastewaterand from barley shochu wastewater (Fig. 1). On the other hand,PAWA removed about 95% of DTP from both types of wastewater,while the parent strains removed only about 70–80%.

In S. cerevisiae, the PHO regulatory pathway senses phosphorusconcentration signals and regulates the uptake of phosphorus(Oshima, 1997; Auesukaree et al., 2004). The major phosphatetransporters and acid phosphatases are regulated in parallel bythe same transcriptional activators. We previously showed thatmutants that constitutively express phosphate transporters andacid phosphatases removed twice as much phosphorus as the wildtype (Watanabe et al., 2008). Hence, we believe that some muta-tion in the regulatory pathways of PAWA causes phosphate trans-porters and acid phosphatase to be constitutively expressed,leading to an enhanced uptake and accumulation of phosphorus.

0

DTP

DTN

DOC

Removal ratio (%)

Sweet-potato wild typeSweet-potato PAWABarley wild typeBarley PAWA

20 40 60 80 100

Fig. 1. Removal ratios of DOC, DTN and DTP from sweet-potato shochu wastewaterand barley shochu wastewater treated by yeasts. The results were shown as averageof three different experiments. The error bars shows standard deviations.

3.2. Flocculating ability

Barley shochu wastewater was treated with the non-flocculentyeast PAW1 and the flocculent yeast PAWA. While PAW1 showedlittle settling after 30 min, PAWA made large flocks that settledto about 10% of volume in 5 min. The SVI value of PAWA waslow (23–24), while that of PAW1 was high (310–340).

The flocculating mechanism of H. anomala J224 was previouslyidentified as self-aggregation between cell wall 37K-proteins (Saitoet al., 1990). Unlike brewer yeasts, H. anomala J224 does not needbivalent metal ions for flocculation and is only eliminated irrevers-ibly by pronase E or proteinase K. The SVI values of PAWA indi-cated that the PAWA sludge accumulating high-concentrationphosphorus would rapidly settle and thus would be easily sepa-rated from the sediment tank.

3.3. Stability of DTP removal in a continuous process

During cycles 1–6, the DTP removal volume (Fig. 2) was low,probably because the initial mixed liquor suspended solids (MLSS)was too low. After MLSS of the yeast sludge reached a sufficient le-vel (cycles 10–40), most of the DTP was removed. After 40 cycles,the DTP removal ability gradually decreased, apparently becauseof the presence of contaminating bacteria (Bacteria colonies wereobserved on the plate that was spread 100 ll effluents). After 60cycles, 4 l of treated water was removed, and 2 l of barley shochuwastewater and 2 l of fresh seed yeast sludge cultivated in barleyshochu wastewater were added to the tank. Then, the second phasewas started continuously from 61 cycles. The DTP removal abilityrecovered when the fresh seed sludge was replaced with 1/3 vol-ume of treated water (barley, 61–100 cycles). After 100 cycles, con-tamination of bacteria was observed again and the DTP removalability gradually decreased. These results suggest that the DTP re-moval ability and dominance of PAWA could be maintained if mostof the contaminated sludge was replaced with fresh seed yeastsludge at regular intervals.

HClO and HCl are sometimes added to keep yeast as the domi-nant microorganism in the wastewater treatment tank (Yoshizawa,1978; Yoshizawa et al., 1980). In this study, we added HClO, butcontamination occurred after 40 cycles. Although the initial pHwas at the preferred value (around 4.0), the effluent pH rose to5–7 as a result of greater consumption of organic acids. The higherpH, in turn, led to increased bacterial growth. Therefore, we con-cluded that adding HCl (to maintain low pH) was preferable toadding HClO to keep PAWA as the dominant microorganism.

3.4. Process performance in a continuous process

The amounts of DOC, DTN and DTP removed in the continuousexperiment (Table 1) were less than those of the batch experiment

050

100150200250300350400

0

cycle

DT

P (m

g/L

)

Inf.

Eff.

BarleySweet-potato

20 40 60 80 100 120

Fig. 2. Time course of DTP concentration of influents and effluents of Semi-batchcontinuous wastewater treatment using H. anomala J224 PAWA.

Table 1Process performances of wastewater treatment system based on Hansenula anomalaJ224 PAWA.

Parameter Sweet-potato Barley

Ave. Stev. Ave. Stev.

DOC Influent (mg l�1) 11,781 523 19,474 1834Effluent (mg l�1) 5876 475 11,164 607Removal (mg l�1) 5905 8310Day removal (mg d�1) 3937 5540

DTP Influent (mg l�1) 202.8 8.9 328.5 14.4Effluent (mg l�1) 67.1 18.0 69.8 27.0Removal (mg l�1) 135.6 258.7Day removal (mg d�1) 90.4 172.5Removal ratio (P/C) 2.3 3.1

DTN Influent (mg l�1) 861.3 201.6 2872.0 231.0Effluent (mg l�1) 228.9 30.7 2004.7 202.2Removal (mg l�1) 632.4 867.3Day removal (mg d�1) 421.6 578.2Removal ratio (N/C) 10.7 10.4

pH Influent 4.25 0.15 3.73 0.14Effluent 6.93 0.36 4.76 0.63

MLSS Influent (mg l�1) 1000 200 1500 350Effluent (mg l�1) 6370 1399 11,425 1515

SVI 27.4 1.3 19.2 2.2

1784 T. Watanabe et al. / Bioresource Technology 100 (2009) 1781–1785

(Fig. 1). Because anti-foam could not be added automatically in thisexperiment, the aeration rate was repressed to 0.67 vvm to avoidblowing out the foam. Generally, the aeration rate is around 1–2 vvm in wastewater treatment systems that use yeast (Yoshizawa,1978). The lower removal volumes were due to a lack of supplemen-tal oxygen. Despite the low aeration rate, the continuous processwas able to remove 4–6 kg DOC m�3 d�1 from sweet-potato andbarley shochu wastewater. These values are very high comparedto the amounts removed by activated sludge (0.5–1.0 kgm�3 d�1). After wastewater is treated with yeast, it is usually trea-ted with activated sludge. Treatment of wastewater with a combi-nation of yeast and activated sludge can drastically decrease thepond area and operating cost (the main operating cost is for aerationenergy).

Both the efficiency of removal of organic compounds and the ra-tios of C:N:P removal are important factors for wastewater treat-ments. The ratios of C:N:P removal of wastewater treatment byactivated sludge are around 100:5:1. The ratios of C:N removal ofsweet-potato and barley shochu wastewater by PAWA were100:10.4–10.7. These ratios indicate that nitrogen was efficientlyremoved by PAWA. The amounts of phosphorus removed fromthe sweet-potato and barley shochu wastewater by the wild type(H. anomala J224) in the same experiments (60 mg l�1 and130 mg l�1, respectively) were only half those of PAWA.

The MLSS values of sweet-potato and barley shochu wastewa-ters were 6400 mg l�1 and 11,400 mg l�1, respectively. As a roughestimation based on DOC removal values and the growth of MLSS,approximately 36% of the removed organic compounds were incor-porated into cells and the rest was converted to CO2 and released

Table 2Components of dehydrated residuum of shochu wastewater and yeast waste sludge.

Shochu type Material Moisture content Dry base com

Protein

Sweet-potato Shochu waste 85.1 22.8Wild-type sludge 80.1 44.1PAWA sludge 80.0 42.0

Barley Shochu waste 78.0 33.0Wild-type sludge 81.0 47.5PAWA sludge 80.9 44.8

to the atmosphere. In the continuous experiments with sweet-po-tato and barley shochu wastewaters, the SVI values were remainedlow (27 and 19, respectively).

3.5. By-products

The dry base components and moisture content of dehydrateresiduum and yeast waste sludge are shown Table 2. The proteincontents of yeast sludge were high (42.0–47.5%) which indicatesthat yeast sludge could be used as a protein source for feedstuffs.The phosphorus contents of yeast sludge of PAWA (3.5–3.7%) werealmost twice those of the wild type (1.9–2.1%). These results indi-cated that PAWA yeast sludge would be a richer source of phos-phorus for fertilizer than wild-type sludge.

A large amount of dehydrated shochu waste (sweet-potato:177.5 k g t�1, barley: 157.5 k g t�1) and yeast waste sludge(sweet-potato: 31.3 k g t�1, barley: 62.5 k g t�1) were dischargedfrom shochu wastewater treatment by PAWA. Treating 1 ton of sho-chu waste costs about 10,000 yen and produces 30–60 kg yeastwaste sludge. Therefore about 2000 yen was paid to produce10 kg of yeast waste sludge (contains about 35 P g k g�1). Ten kilo-grams of phosphorus fertilizer (contains about 20 P g k g�1) is soldas about 300 yen. About 15% of the cost to treat shochu waste couldbe recovered by selling yeast waste sludge. Additionally, heatingthe sludge to 70 �C causes the sludge to release the phosphorus intothe liquid phase, where it can be precipitated by adding CaCl2 (Kur-oda et al., 2002; Takiguchi et al., 2007; Watanabe et al., 2008). Therecovered phosphorus has a high degree of purity and would beuseful in various industrial applications.

3.6. Anaerobic denitrification

The semi-batch continuous anaerobic treatment (20 cycles,20 d) removed 30% of DOC and 35% of DTN (Fig. 3). The C:N re-moval ratio was 100:22, which indicates that the semi-batch pro-cess efficiently removed nitrogen.

Barley shochu wastewater also contains a high concentration ofnitrogen (mainly Org-N compounds) and PAWA removes only asmall part of the nitrogen (30% of DTN). However, PAWA removedmainly Org-N (as the source of NH4–N) and very little NO3–N frombarley shochu wastewater (Fig. 4). A good way to remove nitrogenis to denitrify NO3–N to N2 under anoxic conditions (Smith andEvans, 1982). Thus, the remaining NO3–N of barley shochu waste-water was easily denitrified under anaerobic conditions.

The NO3–N concentrations of the effluent of the PAWA treat-ment and anaerobic treatment were 630 mg l�1 and 90 mg l�1

(Fig. 4), respectively. These data show that most of the DTN re-moval was due to denitrification of NO3–N by denitrifying bacteria.We were unable to detect nitrite-nitrogen (NO2–N) in this experi-ment. Half of the Org-N was removed by yeast treatment and mostof the remaining Org-N was decomposed to inorganic NH4–N bythe anaerobic treatment. These results indicate that a combinationprocess of PAWA treatment and nitrification/denitrification cycle

ponents (%)

Fat Fiber Soluble non-N Phosphorus Non-P ash

5.3 26.2 40.5 0.1 5.15.7 9.5 33.3 2.1 5.34.7 7.4 37.0 3.7 5.2

10.4 12.6 42.1 0.3 1.68.1 9.4 30.0 1.9 3.15.7 7.4 35.6 3.5 3.0

0

2000

4000

6000

8000

10000

12000

14000

0 5 10 15 20cycle

DO

C (m

g/L)

0

300

600

900

1200

1500

1800

2100

DTN

(mg/

L)

DOC Inf.DOC Eff.DTN Inf.DTN Eff.

Fig. 3. Process performance of an anaerobic reactor treating barley shochuwastewater. The time course of the DOC concentration of influent (Inf.) andeffluent (Eff.) and the DTN concentration of influent (Inf.) and effluent (Eff.).

0

Anaerobictreatment

Yeasttreatment

Control

DTN (mg/L)

NO3-NNH4-NOrg.-N

500 1000 1500 2000 2500 3000 3500

Fig. 4. DTN concentration and its composition of barley shochu wastewater before(control) and after treatment. Middle bar shows wastewater treated with PAWAand bottom bar shows wastewater treated with an anaerobic reactor followed bytreatment with PAWA. The results were shown as average of three differentexperiments. The error bars shows standard deviations.

T. Watanabe et al. / Bioresource Technology 100 (2009) 1781–1785 1785

treatment was effective at removing nitrogen from barley shochuwastewater.

Accumulation of NH4–N (decomposed from Org-N) is thought toinhibit denitrification. In addition, the decrease of pH as a result ofthe accumulation of organic acids is thought to inhibit methane fer-mentation. The best method for treating barley shochu wastewaterseems to be a combination of Org-N removal by yeast and nitrogenremoval by nitrification/denitrification cycles. A combination of ananaerobic UASB reactor and an aerobic DHS reactor (UASB-DHS sys-tem) makes an economical and efficient treatment system (Machdaret al., 2000). In the UASB-DHS method, nitrification and denitrifica-tion occurred in the aerobic DHS post-treatment system (Tandukaret al., 2005). This system, which removes phosphorus and organiccompounds by PAWA combined with nitrification/denitrificationby UASB-DHS, seems well suited for small shochu manufacturers.

4. Conclusions

In this study, we bred a flocculent yeast, H. anomala J224 PAWA,that accumulated twice as much phosphorus as the wild type.PAWA efficiently removed phosphorus from sweet-potato and bar-ley shochu wastewater. PAWA also removed DOC and DTN as effi-ciently as the wild type. The yeast sludge of PAWA was easilyseparated from treated water because PAWA made large flocksand settled quickly.

In the semi-batch continuous experiment, DTP removal (sweet-potato: 90 g m�3 d�1, barley: 170 g m�3 d�1) was stable and effi-

cient. Over a 30-d period, PAWA was maintained as the dominantmicroorganism by replacing fresh seed yeast sludge at regularintervals. Additionally, the efficiency of DOC removal by PAWAwas about ten times higher than the efficiency by the conventionalactivated sludge method. PAWA yeast sludge contained a high con-centration of phosphorus and would be more valuable as a sourceof phosphorus for fertilizer than wild-type sludge.

The combination process, which removes phosphorus andorganic compounds by PAWA combined with nitrification/denitri-fication by UASB-DHS seems well suited for small shochumanufacturers.

Acknowledgements

We thank Ookuchi Shuzou Co. Ltd. and Unkai Shuzou Co. Ltd.for kindly providing shochu wastewater.

References

Abelson, P.H., 1999. A potential phosphate crisis. Science 283, 2015.AOAC, 1980. Official Methods of Analysis of AOAC, 13th ed.APHA, AWWA, WEF, 1998. Standard Methods for the Examination of Water and

Wastewater, 20th ed.Auesukaree, C., Homma, T., Tochio, H., Shirakawa, M., Kaneko, Y., Harashima, S.,

2004. Intracellular phosphate serves as a signal for regulation of the PHOpathway in Saccharomyces cerevisiae. J. Biol. Chem. 279, 17289–17294.

Broughton, A., Pratt, S., Shilton, A., 2008. Enhanced biological phosphorus removalfor high-strength wastewater with a low rbCOD:P ratio. Bioresour. Technol. 99,1236–1241.

Kaszycki, P., Tyszka, M., Malec, P., Kołoczek, H., 2001. Formaldehyde and methanolbiodegradation with the methylotrophic yeast Hansenula polymorpha. Anapplication to real wastewater treatment. Biodegradation 12, 169–177.

Kuroda, A., Takiguchi, N., Gotanda, T., Nomura, K., Kato, J., Ikeda, T., Ohtake, H., 2002.A simple method to release polyphosphate from activated sludge forphosphorus reuse and recycling. Biotech. Bioeng. 78, 333–338.

Lanciotti, R., Gianotti, A., Baldi, D., Angrisani, R., Suzzi, G., Mastrocola, D., Guerzoni,M.E., 2005. Use of Yarrowia lipolitica strains for the treatment of olive millwastewater. Bioresour. Technol. 96, 317–322.

Lindegren, G., Hwang, Y.L., Oshima, Y., Lindegren, C.C., 1965. Genetical mutantsinduced by ethylmethanesulfonate in Saccharomyces. Can. J. Genet. Cytol. 7,491–499.

Machdar, I., Sekiguchi, Y., Sumino, H., Ohashi, A., Harada, H., 2000. Combination of aUASB reactor and a curtain type DHS (downflow hanging sponge) reactor as acost-effective sewage treatment system for developing countries. Water Sci.Technol. 42, 83–88.

Morohoshi, T., Maruo, T., Shirai, Y., Kato, J., Ikeda, T., Takiguchi, N., Ohtake, H.,Kuroda, A., 2002. Accumulation of inorganic phosphate in phoU mutants ofEscherichia coli and Synechocystis sp. strain PCC6803. Appl. Environ. Microbiol.68, 4107–4110.

Oshima, Y., 1997. The phosphate system in Saccharomyces cerevisiae. Gene Genet.Syst. 72, 323–334.

Saito, K., Sato, S., Shimoi, H., Iefuji, H., Tadenuma, M., 1990. Flocculation mechanismof Hansenula anomala J224. Agric. Biol. Chem. 54, 1425–1432.

Saktaywin, W., Tsuno, H., Nagare, H., Soyama, T., Weerapakkaroon, J., 2005.Advanced sewage treatment process with excess sludge reduction andphosphorus recovery. Water Res. 39, 902–910.

Seghezoo, L., je Zeeman, G., Hamelers, H.V.M., Lettinga, G., 1998. A review: theanaerobic treatment of sewage in UASB and EGSB reactors. Bioresour. Technol.65, 175–190.

Smith, M.S., Evans, M.R., 1982. The effects low dissolved oxygen tension during theaerobic treatment of piggery slurry in completely mixed reactors. J. Appl.Bacteriol. 53, 117–126.

Takiguchi, N., Kishino, M., Kuroda, A., Kato, J., Ohtake, H., 2007. Effect of mineralelements on phosphorus release from heated sewage sludge. Bioresour.Technol. 98, 2533–2537.

Tandukar, M., Uemura, S., Ohashi, A., Harada, H., 2005. A low-cost municipal sewagetreatment system with a combination of UASB and the ‘‘fourth generation”downflow hanging sponge (DHS) reactors. Water Sci. Technol. 52, 323–329.

Watanabe, T., Ozaki, N., Iwashita, K., Fujii, T., Iefuji, H., 2008. Breeding of wastewatertreatment yeasts that accumulate high concentrates of phosphorus. Appl.Microbiol. Biotechnol. 80, 331–338.

Yamada, M., Yamaguchi, M., Suzuki, T., Ohashi, A., Harada, H., 2006. On-sitetreatment of high-strength alcohol distillery wastewater by a pilot-scalethermophilic multi-staged UASB (MS-UASB) reactor. Water Sci. Technol. 53,27–35.

Yoshizawa, K., 1978. Treatment of waste-water discharged from sake brewery usingyeast. J. Ferment. Technol. 56, 389–395.

Yoshizawa, K., Momose, H., Hasuno, T., Suzuki, O., Takano, K., 1980. Disinfection ofmicroorganism in a yeast tank using sterilizers during wastewater treatment.Hakkokogaku 58, 139–144 (in Japanese).