Polyunsaturated fatty acids production with a solid-state column reactor

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Polyunsaturated fatty acids production

with a solid-state column reactor

Hung-Der Jang

a

, Shang-Shyng Yang

b,c,*

a_{Department of Food Science, Yuanpei University, Hsinchu 300, Taiwan}

b_{Department of Biochemical Science and Technology, National Taiwan University, Taipei 10617, Taiwan} c

Institute of Microbiology and Biochemistry, National Taiwan University, Taipei 10617, Taiwan Received 11 September 2007; received in revised form 3 December 2007; accepted 6 December 2007

Available online 29 January 2008

Abstract

To investigate the potential production of polyunsaturated fatty acids (PUFAs), a solid-state column reactor of rice bran with Mor-tierella alpina was used. The optimal conditions for PUFAs production were rice bran supplementation with 3.75% (w w 1) nitrogen source at initial moisture content 57%, initial pH 6–7, aeration, and incubation at 20°C for 5 days and then at 12 °C for 7 days. Each gram of substrate carbon yielded 127 mg of total PUFAs, 12 mg of eicosapentaenoic acid (EPA), 6 mg of arachidonic acid (AA), 5 mg of a-linolenic acid (ALA), and 117 mg of linoleic acid (LA) after 12 days incubation. Aeration enhanced the productions of AA, EPA, and total PUFAs. Supplementation of the nitrogen source on the fourth day and then a shift to lower temperature on the ﬁfth day increased EPA production.

Keywords: Polyunsaturated fatty acid; Rice bran; Solid-state fermentation; Column reactor; Mortierella alpina

1. Introduction

The x 3 and x 6 series of polyunsaturated fatty

acids have been identiﬁed as potential food additives or as pharmaceuticals for their biological activities. The PUFAs could be used in the treatment of heart and circu-latory disorders, cancer, or participate in the inﬂammatory

reaction (Willett, 1993; Certik and Shimizu, 1999;

Demai-son and Moreau, 2002; Gil, 2002; Ratledge, 2004). The

major sources of PUFAs include microorganisms, pig liver, adrenal glands, and marine ﬁsh oils. Animal sources, how-ever, have the problems of cholesterol and diﬃculty of removing some objectionable tastes and odors. Therefore, PUFAs produced from microorganisms would be more desirable than those from animal sources for use as food

additives, supplements, or feedstocks (Cheng et al., 1999).

Extensive researches have been carried out on the produc-tion of PUFAs by the ﬁlamentous fungus Mortierella in submerged culture. Many species of the genus Mortierella have been found to yield exceptionally high amount of PUFAs depending upon the fermentation media and

cul-ture conditions (Sajbidor et al., 1990; Bajpai et al., 1991;

Jareokitmongkol et al., 1993; Yu et al., 2003; Jang et al.,

2005). However, submerged culture required high energy

input and produced much wastewater (Yang, 1988).

To be economically competitive, the production of poly-unsaturated fatty acids with rice bran should be able to be performed at the rural level. Solid-state fermentation (SSF) can achieve this purpose by reducing the cost of growing microorganisms, high product yields and low wastewater

output (Cannel and Moo-Young, 1980; Yang, 1988; Yang

and Ling, 1989; Yang and Swei, 1996; Pandey et al., 1999,

2000; Rahardjo et al., 2005). Much published information

is available on the production of enzymes of industrial importance, such as amylase, glucoamylase, protease,

*

Corresponding author. Address: Department of Biochemical Science and Technology, National Taiwan University, Taipei 10617, Taiwan. Tel.: +886 2 23621519; fax: +886 2 23679827.

E-mail address:[email protected](S.-S. Yang).

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cellulase, ligninase, pectinase and xylanase. and various

by-products have been used successfully in SSF (Pandey et al.,

1999, 2000; Haq et al., 2003; Kang et al., 2004; Rahardjo

et al., 2005). SSF might provide an alternative for PUFAs

production since the association of fungal oil with solid substrate might be used as inexpensive food and feed

sup-plement and ﬁll the marketing claims (Certik and Shimizu,

1999; Jang et al., 2000).

Agricultural wastes such as rice bran, sweet potato res-idue, and corncob are rich in starchy or cellulosic materials, and thus could be utilized as renewable resources for the growth of microorganisms and the production of

metabo-lites (Yang and Yuan, 1990; Yang and Swei, 1996). In

2005, the cultivation area of paddy rice was 237,000 ha. and annual production was 2,600,000 tons in Taiwan

(Council of Agriculture/Taiwan, 2006). Annual production

of rice bran was estimated at 43,160 tons. Rice bran and wheat bran are good substrates for enzyme and oil

produc-tions (Deschamps et al., 1985; Yang and Chiu, 1986), and

studies on the production of PUFAs using many

agricul-tural wastes have been reported (Jang et al., 2000).

How-ever, fewer studies have addressed the factors inﬂuencing the fungal growth and lipid formation in large-scale SSF than those with SmF. In the present study, the rice bran was used to produce PUFAs with Mortierella alpina by col-umn reactor, and the optimal conditions for PUFAs pro-duction were investigated.

2. Methods 2.1. Solid substrate

Rice bran was purchased from local market in Taiwan. It contained moisture content 12.4 ± 1.4%; organic carbon 43.9 ± 4.5%; total nitrogen 2.1 ± 0.3%; and

carbon/nitro-gen ratio 16.4–26.9 (Jang et al., 2000). Rice bran also

con-tained oleic acid (OA) 14.1 ± 0.6 mg g 1substrate carbon

and LA 12.9 ± 0.2 mg g 1 substrate carbon, which was

equivalent to 40% and 38% of the total fatty acids, respectively.

2.2. Test microbe and culture conditions

M. alpina ATCC 32222 was purchased from American Type Culture Collection and used for PUFAs production.

Mortierella was grown at 20°C in a slant containing

(g L 1): glucose, 10; yeast extract, 5; and agar, 20 at pH

6.5 ± 0.1. Mycelia were harvested and then blended with three times the volume of sterilized water in a micro-War-ing blender for the preparation of the mycelial suspension.

Submerged basal medium comprised of (g L 1) soluble

starch, 20; yeast extract, 5; KNO3, 10; KH2PO4, 1; and

MgSO4 7H2O, 0.5 at pH 6.5 ± 0.1. The broth was

inocu-lated with 10% (v v 1) mycelial suspension and shaken at

200 rpm and 25°C for 2–9 days.

The solid substrate comprised (g): rice bran, 1000; yeast

extract, 25; KNO3, 50; KH2PO4, 5; and MgSO4 7H2O,

2.5. The sterile medium was mixed thoroughly with 10%

(v v 1) mycelial suspension, the moisture content was

adjusted to 57%, and incubated statically at 20°C for 5

days and mixed once daily by rotating each ﬂask. The depth of medium in each ﬂask was about 3 cm. The fer-mentative solid substrates were used for column reactors. 2.3. Solid substrate fermentation with column reactor

The column reactors were presterilized by thorough

spraying with 70% alcohol. Ten percent (w w 1)

precul-tured solid substrates were used as inoculum, mixed with 4.5 kg of sterile rice bran solid substrate medium, and thereafter put into cylindrical column reactors (60 cm

height 15 cm i.d., total volume 8.2 L) for PUFAs

pro-duction. The diagram of column reactor and its accessories

are shown inFig. 1. During the fermentative process, the

temperature inside internal column reactor was measured with a set of k-type thermocouples at diﬀerent positions and connected to a temperature detector. The temperatures at 10, 20, and 30 cm (bottom, middle, and upper layer) far from the bottom, as well as 4.0 and 7.5 cm (surrounding and central point) far from the outer walls of column

reac-tor were measured and recorded. The ﬁltered air

(1.2 L min 1) was provided with an air pump and

trans-ferred through a perforated acrylic plate beneath the bot-tom of column reactor. The humidity of the air used was about 76%. Oxygen concentration was measured using a gas sensor connected to an oxygen analyzer. The fermenta-tive substrate of each layer was separately sampled and the moisture content, pH, and PUFAs content were deter-mined periodically. After sampling, the fermentative sub-strate was mixed thoroughly before being put into the column reactor.

Fig. 1. Diagram of the column reactor and its accessories for solid-state fermentation.

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Column reactor 1 was without aeration and without nitrogen supplementation; column reactor 2 was with aer-ation but without nitrogen supplement. Both column

reac-tors 1 and 2 were incubated at 20°C. Column reactor 3 was

with aeration and with nitrogen supplement and incubated

at 20°C for 12 days; column reactor 4 was with aeration

and with nitrogen supplement and incubated at 20°C for

5 days and then shift to 12°C for 3–7 days. The

fermenta-tive substrates of column reactors 3 and 4 were

supple-mented with a mixture of 3.75% KNO3and yeast extract

(2:1, w w 1) on the fourth day. After inoculation, the

col-umn reactors were put into an temperature-controlled incubator for PUFAs production.

2.4. Fatty acids analysis

After fermentation, the substrate sample was mixed with

5 times the volume of chloroform/methanol (2:1, v v 1),

and lipids were extracted by ultrasonic transistor for 2 h

and concentrated by rotary evaporation at 50°C. The

res-idue of extracted lipid was dissolved in 1 mL of 0.5 M KOH–MeOH solution, and methylated by 1 mL of 14%

BF3 in MeOH (w v 1). Saturated NaCl and anhydrous

Na2SO4were added to the aqueous layer, and the

methyl-ated fatty acids were extracted with n-hexane. PUFA con-tent was determined using a GC–8A gas chromatography (Shimadzu Co., Japan) equipped with a 5% diethylene

gly-col succinate (60/80 mesh) and 1% H3PO4packed glass

col-umn (2.0 m 0.26 mm i.d.) and a ﬂame ionization

detector. Nitrogen was used as a carrier gas. Column

tem-perature was programmed from 160°C to 200 °C with an

increasing rate of 4°C min 1. Injection port and detector

temperatures were maintained at 230°C. Methyl esters of

palmitic acid, palmitoleic acid, stearic acid, oleic acid, lin-oleic acid, a-linolenic acid, c-linolenic acid (GLA), arachi-donic acid, eicosapentaenoic acid, and docosahexaenoic acid were used as standards. Methyl pentadecanoate was

added as an internal standard (Jang et al., 2000).

The degree of unsaturation of fatty acids was calculated

as the sum of concentration (%, w w 1) of the product

times the number of unsaturated double bonds of each

fatty acids as follows (Jang et al., 2000):

Degree of unsaturation = 1(wt% of monoene) + 2(wt% of diene) + 3(wt% of triene) + 4(wt% of tetraene) + 5(wt% of pentaene).

2.5. Chemical analysis

Moisture content was measured by drying a sample at

105°C for 24 h to constant mass. pH of substrate was

determined directly by immersing the electrode into sub-strate. Organic carbon content of the solid substrate was

measured byNelson and Sommers (1982)and total

nitro-gen was determined by a modiﬁed Kjeldahl method (Yang

et al., 1991). The biomass of M. alpina was estimated by

determining the protein content, which was calculated as 6.25 times the total nitrogen content.

2.6. Statistical analysis

Treatments were performed in triplicate, and experimen-tal data were subjected to the analysis of variance and Duncan’s multiple range test (p = 0.05) using the Statistical

Analysis System (SAS Institute, 2002).

3. Results and discussion

3.1. Physical properties and fatty acids production in column reactor without aeration or nitrogen supplementation

Physical properties and fatty acids production without aeration or nitrogen supplementation are presented in

Fig. 2. The temperature at the central area of bottom layer

had a maximum value of 43°C at day 2 and then gradually

decreased to a constant value after 4 days incubation

(Fig. 2A). The other parts of reactor 1 also attained the

maximal temperatures (28–33°C) at day 2, and the

temper-ature profiles had no significant difference among these parts. The oxygen concentration sharply decreased at day 1, increased at day 4 and day 8 due to the remixing of solid substrate, and gradually declined to 8% during cultivation. Substrate pH increased from an initial value of 6.3–7.4 at day 4 and then decreased to 5.3 after 8 days incubation

(Fig. 2B). The pH drop might be due to the accumulation

of organic acid during fermentation (Matsushima et al.,

1981; Yang and Huang, 1994). Moisture content at the

upper layer increased from 57% to 62% at day 4 and then gradually decreased; while the moisture content at the mid-dle and bottom layers gradually increased to the maximal

value of 61% at day 12 (Fig. 2C). The increase of moisture

content might be because of the production of metabolic water. Same phenomena were also found in protein

enrich-ment, enzyme, antibiotic, and compost productions (Yang,

1988, 1997, 2005; Yang and Ling, 1989; Yang and Huang, 1994; Yang and Yuan, 1990; Yang and Swei, 1996; Yang

and Wang, 1996).

Fatty acids contents after inoculation at time zero were 32 mg OA, 39 mg LA, 0.5 mg ALA, 0.2 mg AA, 0.6 mg EPA and 40.3 mg total PUFAs per gram of substrate car-bon. During cultivation, the biomass of microbes increased

and it had 80.7 mg protein g 1substrate after 12 days

incu-bation in the column reactor 1 (Table 1). OA content

decreased during the fermentation; whereas ALA, AA and EPA were produced after 4 days incubation. It was postulated that the microorganism converted OA to longer-chained and more highly unsaturated fatty acids such as ALA, AA and EPA. However, LA content did not show significant difference. The degrees of unsaturation increased from 1.6 at the start of incubation to 2.1 at day 12. These results indicated that more longer-chained and more highly unsaturated fatty acids were produced after 12 days cultivation in the rice bran-based substrate. The upper layer had the highest yield due to the temperature profile that was good for microbial growth and PUFAs production, followed by the middle layer, and the bottom

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layer had the least. The high temperature and the low oxy-gen concentration at the bottom layer was the limiting fac-tor for microbial growth and PUFAs production. As

shown in Fig. 2D, each gram of substrate carbon yielded

6.2 mg of EPA, 1.7 mg of AA, 3.8 mg of ALA, 102.2 mg of LA, 17.0 mg of OA and 113.9 mg of total PUFAs in the upper layer after 8 days incubation. It was obvious that the contents of ALA, AA and EPA in the fermentative product were comparatively lesser under the cultural condi-tion without aeracondi-tion or nitrogen supplementacondi-tion. 3.2. Physical properties and fatty acids production in column reactor with aeration and without nitrogen supplementation

Physical properties and fatty acids production with aer-ation and without nitrogen supplementaer-ation are shown in

Fig. 3. Temperatures at the central layer and the

surround-ing area of the bottom layer reached the maximum

temper-ature of 43°C and 40 °C at day 2, respectively, and then

decreased gradually to 20°C after 4 days incubation. The

temperature decreased from the central area of the middle layer, the surrounding area of middle layer, the central area of the upper layer and the surrounding area of the upper layer. The upper layer and the middle layer had somewhat lower temperature than those of the bottom layer during

day 1–3 (Fig. 3A). In general, the temperature proﬁles

had no signiﬁcant diﬀerence between the cultures with

and without aeration. The oxygen concentration was about

20% during the cultivation with aeration of 1.2 L min 1.

Substrate pH had the maximal values at day 4 and then

decreased to pH 5.3 after 8 days incubation (Fig. 3B).

The patterns of substrate pH change were very similar to those in the non-aeration treatment. The slightly acidic to neutral pH favored the yield of EPA, AA and PUFAs pro-ductions with Mortierella. Same phenomenon was also

found in submerged fermentation of algae (Bajpai et al.,

1991; Yongmanitchai and Ward, 1991). The moisture

con-tent increased from 57% to the maximal value 64% in the bottom layer due to the formation of metabolic water by

the tested organism (Fig. 3C). Similar phenomena were

also observed in the PUFAs production without aeration or nitrogen supplement. However, aeration enhanced the microbial growth, increased the substrate temperature and metabolic water production.

During cultivation, the biomass of microbes increased

for the aeration and it had 99.4 mg protein g 1 substrate

after 12 days incubation in column reactor 2 (Table 1).

The biomass of the microbes grown in the column reactor with aeration was signiﬁcantly higher that those of without aeration. OA and LA contents decreased 10.5–11.6% for the conversion to longer-chained and more highly unsatu-rated fatty acids such as ALA, AA and EPA. As a result, the degree of unsaturation also increased from 1.6 at time zero to 2.2 at day 12. Each gram of substrate carbon

Temperature (ºC) 10 20 30 40 50

Upper layer, central area Upper layer, sorrounding area Middle layer, central area Middle layer, surrounding area Bottom layer, central area Bottom layer, surrounding area

pH 4 5 6 7 Upper layer Middle layer Bottom layer

Incubation time (day)

0 10 12 Moisture content (%) 54 57 60 63 66 Upper layer Middle layer Bottom layer A C 0 10 20 30 60 90 120 OA LA ALA AA EPA Total PUFAs

Yield of OA & PUFAs (m

g g -1 substrate carbon) 0 10 20 30 60 90 120

0 10 12 0 10 20 30 60 90 120 D Upper layer Middle layer Bottom layer B 2 4 6 8 2 4 6 8

Fig. 2. Physical properties and fatty acids production of column reactors incubated at 20°C without aeration and without nitrogen source supplement: (A) temperature, (B) pH, (C) moisture content, and (D) yields of OA and PUFAs. Each value is the mean of triplicate. The standard deviation of the data fell within 5% of the mean.

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Table 1

Biomass and fatty acid proﬁles in the solid-state column reactors Number of column reactora Sampling time (day) Biomass (mg protein g 1 substrate)

Fatty acidsb(%) Degree of

unsaturation

OA LA ALA AA EPA Others

1 0 36.5 ± 1.5G 40.1 ± 1.5A 38.2 ± 1.5A nd nd nd 21.7 ± 0.9AB 1.6 ± 0.1D 4 52.4 ± 2.5F _{38.6 ± 1.3}A _{37.4 ± 1.7}A _{0.3 ± 0.1}F _{3.8 ± 0.3}E _{1.5 ± 0.1}F _{18.4 ± 0.7}B _{2.0 ± 0.1}C 8 71.8 ± 3.0E _{35.0 ± 1.7}B _{36.8 ± 1.3}A _{0.4 ± 0.1}F _{4.5 ± 0.3}D _{1.4 ± 0.1}F _{21.9 ± 0.9}AB _{2.1 ± 0.1}BC 12 80.7 ± 2.7D _{32.5 ± 1.0}C _{37.6 ± 1.5}A _{0.3 ± 0.1}F _{4.4 ± 0.3}D _{1.5 ± 0.1}F _{23.7 ± 0.9}A _{2.1 ± 0.1}BC 2 4 66.5 ± 3.0E _{35.8 ± 1.3}B _{30.5 ± 1.7}B _{1.0 ± 0.1}E _{10.9 ± 0.5}B _{5.0 ± 0.3}E _{16.8 ± 0.5}C _{2.0 ± 0.1}C 8 91.2 ± 3.5C 30.2 ± 1.1D 26.8 ± 1.0C 1.3 ± 0.1D 13.2 ± 0.7A 6.2 ± 0.3D 21.9 ± 0.7AB 2.1 ± 0.1BC 12 99.4 ± 3.8BC 28.5 ± 1.0D 27.7 ± 1.3BC 1.3 ± 0.1D 13.0 ± 0.7A 5.7 ± 0.3D 23.8 ± 0.9A 2.2 ± 0.1B 3 4 76.8 ± 3.5DE 32.1 ± 1.3C 30.8 ± 1.5B 1.9 ± 0.2BC 9.9 ± 0.5C 10.0 ± 0.7C 15.3 ± 0.5C 2.1 ± 0.1BC 8 107.3 ± 4.2A _{28.3 ± 1.0}D _{21.1 ± 1.0}D _{1.5 ± 0.1}C _{12.9 ± 0.9}A _{14.2 ± 0.7}B _{22.0 ± 0.9}A _{2.2 ± 0.1}B 12 115.2 ± 5.2A _{26.6 ± 0.7}E _{22.0 ± 0.5}D _{1.7 ± 0.1}C _{13.2 ± 0.9}A _{14.4 ± 0.9}B _{22.1 ± 0.7}A _{2.3 ± 0.0}A 4 4 74.3 ± 3.0E _{33.5 ± 1.0}C _{30.4 ± 0.7}B _{2.0 ± 0.1}B _{8.5 ± 0.1}C _{10.2 ± 0.3}C _{16.4 ± 0.7}C _{2.1 ± 0.0}B 8 93.5 ± 3.8C _{27.5 ± 0.9}D _{21.4 ± 0.7}D _{2.8 ± 0.1}A _{8.7 ± 0.3}C _{16.6 ± 0.7}A _{23.0 ± 0.5}A _{2.2 ± 0.1}B 12 105.6 ± 4.2B _{25.3 ± 0.7}E _{22.1 ± 0.5}D _{2.7 ± 0.3}A _{8.7 ± 0.5}C _{16.9 ± 0.7}A _{24.3 ± 1.0}A _{2.3 ± 0.0}A a

Column reactor 1, incubated at 20°C without aeration or nitrogen source supplement; column reactor 2, incubated at 20 °C with aeration but without nitrogen source supplement; column reactor 3, incubated at 20°C with aeration and nitrogen source supplement; column reactor 4, incubated at 20 °C for 5 days and then 12°C, with aeration and nitrogen source supplement.

b

Data are expressed as the average of three determinations ± standard deviation. Values not sharing the same superscript letters within a column are signiﬁcantly diﬀerent by Duncan’s multiple range test (p < 0.05). nd: not detected; OA: oleic acid; LA: linoleic acid, ALA: a-linolenic acid, AA: arachidonic acid; EPA: eicosapentaenoic acid; PUFAs: polyunsaturated fatty acids.

Temperature ( oC) 10 20 30 40 50

Upper layer, central area Upper layer, sorrounding area Middle layer, central area Middle layer, sorrounding area Bottom layer, central area Bottom layer, sorrounding area

0 10 12 Moisture content (%) 54 57 60 63 66 Upper layer Middle layer Bottom layer A B C 0 10 20 30 60 90 120 OA LA ALA AA EPA Total PUFAs

0 10 12 0 10 20 30 60 90 120 D Upper layer Middle layer Bottom layer 2 4 6 8 2 4 6 8

Fig. 3. Physical properties and fatty acids production of column reactors incubated at 20°C with aeration but without nitrogen source supplement: (A) temperature, (B) pH, (C) moisture content, and (D) yields of OA and PUFAs. Each value is the mean of triplicate. The standard deviation of the data fell within 5% of the mean.

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yielded 7.3 mg of EPA, 3.7 mg of AA, 4.8 mg of ALA, 104.5 mg of LA, 6.1 mg of OA and 120.4 mg of total PUFAs in the upper layer after 12 days incubation

(Fig. 3D). The aerobic microbes could grow well in the

solid substrate under aeration, which ensured an increasing yield of ALA, AA and EPA. Aeration could increase 13– 26% of PUFAs production and they had signiﬁcant diﬀer-ences (p < 0.05).

3.3. Physical properties and fatty acids production in column reactor with aeration and nitrogen supplementation

Physical properties and fatty acids production with aer-ation and nitrogen supplementaer-ation on the fourth day are

presented inFig. 4. The substrate temperature had reached

a maximal value (50°C) at day 1 in all layers, and there was

no significant difference among the different layers

(Fig. 4A). The oxygen concentration was also about 20%

during the cultivation. The substrate pH increased from 6.3 to 7.3 in the upper layer at day 4 and then decreased to 5.9 at day 12. Same patterns of pH change were noted

in the other layers, but of lesser magnitude (Fig. 4B). The

moisture content increased from 57% to 64% at day 4, and then slightly decreased to 62–63% after 12 days

incuba-tion (Fig. 4C). The pH pattern and moisture content have

no signiﬁcant diﬀerence between the cultures with and without nitrogen supplementation. The biomass of the

cul-tures was 115.2 mg protein g 1substrate after 12 days

incu-bation in column reactor 3 (Table 1). The biomass

produced in the column reactor with aeration and nitrogen supplementation was significantly higher than those of col-umn reactors 1 or 2. Therefore, the results indicated that the cultures could grow better in the solid substrate with aeration and nitrogen supplementation. With the combina-tion of aeracombina-tion and nitrogen source supplementacombina-tion, OA and LA contents decreased to 13.5% and 16.2% in the total fatty acids, respectively after 12 days incubation; while EPA, AA and ALA contents increased to 14.4%, 13.2% and 1.7%, respectively. The increase of EPA, AA and ALA contents also leads to the increase of the degrees of unsaturation to 2.3, which were significantly higher than those of column reactors 1 or 2. Solid substrate fermenta-tion had been carried out in a 500 ml flask, the optimal conditions for PUFAs production were rice bran supple-mented with 2.3–5% nitrogen at an initial of moisture

con-tent of 65–68% and a pH range of 6–7 (Jang et al., 2000).

PUFAs production per substrate carbon was the highest in the upper layer, followed by the middle layer and the

Upper layer, central area Upper layer, surrounding area Middle layer, central area Middle layer, surrounding area Bottom layer, central area Bottom layer, surrounding area

pH 5 6 7 Upper layer Middle layer Bottom layer

0 10 12 Moisture content (%) 54 57 60 63 66 Upper layer Middle layer Bottom layer A B nitrogen supplementation nitrogen supplementation nitrogen supplementation C D Upper layer Middle layer Bottom layer nitrogen supplementation 0 10 20 30 60 90 120 OA LA ALA AA EPA Total PUFAs

0 10 12 0 10 20 30 60 90 120 2 4 6 8 2 4 6 8

Fig. 4. Physical properties and fatty acids production of column reactors incubated at 20°C with aeration and with nitrogen source supplement at fourth day: (A) temperature, (B) pH, (C) moisture content, and (D) yields of OA and PUFAs. Each value is the mean of triplicate. The standard deviation of the data fell within 5% of the mean.

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bottom layer was the least, especially for EPA and total

PUFAs. As shown inFig. 4D, each gram of substrate

car-bon yielded 6.9 mg of EPA, 1.4 mg of AA, 4.6 mg of ALA, 105.0 mg of LA, 6.0 mg of OA and 117.9 mg of total PUFAs in the upper layer for 8 days incubation. In accor-dance to our previous work, the total nitrogen and carbon/ nitrogen ratio of rice bran used as the solid substrate were

2.1 ± 0.3% and 16.4–26.9, respectively (Jang et al., 2000).

Nitrogen source supplement could provide additional nutrients for microbial growth and consequently increase

PUFAs production.Ben-Amotz et al. (1985)also reported

that high concentrations of nitrogen source could stimulate EPA production. The information was evident that nitro-gen content of the solid substrate was very important for Mortierella growth, and the cultures with nitrogen source

supplement could stimulate x 3 PUFA production,

espe-cially EPA.Sajbidor et al. (1990) and Jang et al. (2005)also

indicated that the concentrations of carbon and nitrogen sources and carbon/nitrogen ratio of solid substrate were very important in lipid production. In this study, the sup-plement of nitrogen source significantly increased the yields of EPA (p < 0.05); however, the yields of total PUFAs were not significantly different.

3.4. Eﬀect of incubation temperature on physical properties and fatty acids production

Eﬀect of incubation temperature on physical properties

and fatty acids production is shown inFig. 5. The

temper-ature increased to a maximal value 47°C at day 2 and then

decreased gradually. Temperatures in central areas were higher than those of surrounding areas, and the middle and bottom layers were also higher than those in the upper layer. However, when the incubation temperature was

shifted to 12°C at day 5, the column reactor temperature

decreased rapidly to 12.1–13.5°C (Fig. 5A). Oxygen

con-centration remained at about 20% due to aeration during fermentation process. Substrate pH of the upper and mid-dle layers increased slightly to 7.1 at day 4 and then

decreased gradually to pH 5.3 (Fig. 5B). The moisture

con-tent of the bottom layer increased from 57% to 65% at day 4 and then decreased slightly during cultivation; while the moisture contents of the upper and middle layers increased

gradually and had the highest at day 8 (Fig. 5C). The

bio-mass (105.6 mg protein g 1substrate after 12 days

cultiva-tion) in column reactor 4 increased rapidly before day 4

and increased gradually after the temperature shift (Table

Upper layer, central area Upper layer, surrounding area Middle layer, central area Middle layer, surrounding area Bottom layer, central area Bottom layer, surrounding area

0 10 12 Moisture content (%) 54 57 60 63 66 Upper layer Middle layer Bottom layer A B nitrogen supplement nitrogen supplement temperature shift nitrogen supplement C Bottom layer Middle layer D

Upper layer nitrogen supplement

temperature shift 0 10 20 30 60 90 120 OA LA ALA AA EPA Total PUFAs

0 10 12 0 10 20 30 60 90 120 temperature shift temperature shift 2 4 6 8 2 4 6 8

Fig. 5. Physical properties and fatty acids production of column reactors incubated at 20°C for 5 days and then shift to 12 °C with aeration and with nitrogen source supplement at 4th day: (A) temperature, (B) pH, (C) moisture content, and (D) yields of OA and PUFAs. Each value is the mean of triplicate. The standard deviation of the data fell within 5% of the mean.

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1). The degrees of unsaturation of the final products were 2.3, which were not significantly different from those of col-umn reactors 3. However, the degrees of unsaturation of the column reactor with temperature shift were higher than those of column reactors 1 or 2.

PUFAs production of the fungi was signiﬁcantly inﬂu-enced by the moisture content of substrate in solid

sub-strate fermentation.Jang et al. (2000)reported that initial

moisture content of solid substrate ranging from 60% to 65% was good for PUFAs production. Moisture content

at 70–75% favored the production of x 6 series PUFAs;

while moisture content at 60–65% was good for the

pro-duction of x 3 series PUFAs. Zandrazil and Brunert

(1981)indicated that low moisture levels reduced the

solu-bility of nutrients and the swelling of substrate and increased the water tension. Correspondingly, high mois-ture contents decreased the porosity and the gas exchange, induced the loss of particle structure and the production of stickiness, reduced the gas volume, and enhanced the aerial

mycelium formation (Lekha and Lonsane, 1994).

The low temperature shift enhanced the concentration of OA and increased the yields of EPA, ALA and total PUFAs, however, AA production was reversed. The yields of EPA at the upper and middle layers were signiﬁcantly higher than those of the bottom layer (p < 0.05). Low

tem-perature cultivation increased the production of x 3

ser-ies PUFAs such as EPA. Each gram of substrate carbon yielded 12.0 mg of EPA, 1.7 mg of AA, 4.1 mg of ALA, 95.7 mg of LA, 5.0 mg of OA and 113.3 mg of total PUFAs

in the upper layer for 12 days incubation (Fig. 5D). The

yield of EPA increased 24% higher than that at 20°C after

incubation for 12 days. EPA and ALA contents were 16.9% and 2.7% of total PUFAs, respectively. When the

cultivation temperature was shifted to 12°C on ﬁfth day.

Microbial growth at low temperature increased the degree of unsaturation of fatty acids in membrane, decreased the carbon-chain length of fatty acid, and helped maintain

membrane function for optimal metabolism (Toivonen

et al., 1992; Terrados and Lopez-Jimenez, 1996).

In summary, PUFAs production of rice bran in column reactor with M. alpina is a feasible process. The OA in rice bran can be metabolized and converted to long-chained and highly unsaturated PUFAs, such as AA, EPA and ALA. Aeration, supplementation with nitrogen source,

and shift to 12°C at late growth stage increased the

PUFAs yields. Each gram of substrate carbon yielded 12 mg of EPA, 6 mg of AA, 5 mg of ALA, 117 mg of LA, 9 mg of OA and 127 mg of total PUFAs. PUFAs pro-duction with SSF oﬀers advantages over that of SmF. The PUFAs productivity of each gram of substrate carbon was higher in SSF than that in SmF. The cultures in SSF

favored the production of a higher content of x 3 series

of PUFAs in total fatty acids than those in SmF. Finally, the product of SSF could be dried and consumed directly without further extraction treatment. Agricultural waste such as rice bran could be used as solid substrates for fer-mentative production of high-valued products. Therefore,

PUFAs production with solid-state column reactor might be a feasible and economic process in agricultural waste treatment and reutilization.

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