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Appl Biochem Biotechnol DOI 10.

1007/s12010-012-9587-x

Isolation and Characterization of Zygomycetes Fungi from Tempe for Ethanol Production and Biomass Applications
Rachma Wikandari & Ria Millati & Patrik R. Lennartsson & Eni Harmayani & Mohammad J. Taherzadeh

Received: 30 November 2011 / Accepted: 25 January 2012 # Springer Science+Business Media, LLC 2012

Abstract Mixed fungal cultures used for making tempe, a fermented soy bean food, were screened for biomass conversion. Thirty-two zygomycetes strains from two tempe cultures were isolated and identified as Rhizopus, Mucor, Rhizomucor, and Absidia species based upon morphology. The dry weight biomass of these strains contained 49% to 63% protein and 1024% chitosan. The strains with the best growth performance were selected and registered at Culture Collection of Gothenburg University as Rhizomucor CCUG 61146 and Rhizomucor CCUG 61147. These strains were able to grow both aerobically and microaerobically. Their ethanol yields were 0.380.47, 0.190.22, and 0.310.38 g/g on glucose, xylose, and a mix sugars consisting of cellobiose, glucose, xylose, arabinose, galactose, and mannose, respectively. The biomass yield of the strains varied between 65 and 140 mg dry weight/g glucose. Keywords Ethanol . Zygomycetes . Tempe . Chitosan

Introduction In 2010, world ethanol production reached more than 80 billion liters [1]. Sugars- and starch-containing crops are the main feedstock for commercial ethanol production. However, there is concern in regards to shifting traditional food and feed crops for use in producing biofuels. Lignocellulosic biomass is presently the only feedstock produced globally in quantities that rival these other crops. Sources include crop and wood industry residues
R. Wikandari : P. R. Lennartsson : M. J. Taherzadeh (*) School of Engineering, University of Bors, Bors, Sweden e-mail: Mohammad.Taherzadeh@hb.se R. Wikandari Study Program of Biotechnology, Gadjah Mada University, Yogyakarta, Indonesia R. Millati : E. Harmayani Food and Agricultural Product Technology Department, Gadjah Mada University, Yogyakarta, Indonesia

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and dedicated bioenergy crops. However, the recalcitrant nature of lignocellulosic materials has been a major obstacle for the process and demands pretreatment as a mandatory step [2]. A wide range of different proposed pretreatment methods exists today, although they all have different advantages and disadvantages [3]. Saccharomyces cerevisiae is presently used for industrial ethanol production. Among its favorable traits are the ability to produce ethanol with a high yield and tolerate high sugar concentrations [4]. However, S. cerevisiae is unable to ferment pentoses, and cellobiose, which is a dimer of -glucose and the fundamental unit of cellulose. Successful pentose utilization will considerably enhance the economic potential of the overall conversion process from lignocellulosic materials into ethanol [5]. The main pentose, xylose, is well known to be fermentable by several species of fungi, following a general fungal metabolic pathway. Xylose is first reduced into xylitol by xylose reductase using nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. It is followed by oxidation of xylitol into xylulose by xylitol dehydrogenase using NAD+ [6]. However, the different cofactors for reduction and oxidation generates an imbalance. To maintain the balance and provide NAD+ for xylitol oxidation, nicotinamide adenine dinucleotide (NADH) is reoxidized using oxygen. Therefore, xylose is normally only utilized under aerobic condition [7]. There are fungal species capable of anaerobic utilization and ethanol production of ethanol, such as the yeast Pichia stipitis, due to the presence of a xylose reductase capable of using NADH as a cofactor [8]. In addition, the capability to assimilate cellobiose can also improve the process economy by decreasing the expenses associated with adding -glucosidase enzyme. Zygomycetes fungi have been traditionally termed lower fungi and considered to be evolutionary primitive, mainly due to the lack of large visible fruiting bodies and septa [9]. The relative simple structure, however, has some advantages. It allows zygomycetes to grow rapidly and quickly colonize new areas in search for substrates. Some orders belonging to the zygomycetes, however, have the ability to produce septa under certain conditions, similar to the higher fungi [9]. Zygomycetes species can grow in a wide range of environments. Some zygomycetes, such as Rhizomucor pusillus, are thermophilic and can grow in temperatures above 50 C. Other species, such as Mucor hiemalis, can grow in temperatures below 0 C [10]. Some species are able to grow in micro-aerobic conditions, while others are fully dependant on oxygen for growth. Additionally, while most zygomycetes only grow at high water activities, a few species are able to grow in the presence of 15% salt [10]. Several genera belonging to the zygomycetes class have been shown to be able to grow on both glucose and xylose, and produce ethanol. Two Mucor species, i.e., Mucor indicus and M. hiemalis have been suggested as potential candidates for ethanol production from lignocellulosic hydrolyzates [4]. Recently, biomass of zygomycetes fungi have been gaining interest in the fish meal production [11]. Zygomycetes fungi have also been considered for chitosan production [12]. Methods have been published for extracting chitosan from the fungi M. indicus (rouxii) [13], Absidia [14], and Rhizopus [12, 15]. In Indonesia, several species belonging to the zygomycetes have been used for centuries in the production of tempe (fermented soybean). Tempe has been widely consumed by Indonesian people from all economical classes and the species involved in its production could thus be considered GRAS (generally regarded as safe) [16]. In traditional tempe production, mixed fungal cultures grown on Hibiscus and Tectona gandis leaves commonly referred to as Usar and Laru. Tempe fungi, particularly belonging to the Rhizopus genus, have been observed as first colonizers on natural organic substrates [16], which indicate their ability to assimilate different carbon sources. Although using food related microorganisms is highly advantageous in industrial applications, so far to the best of our knowledge, no reports exist regarding the potential of mixed culture tempe fungi as ethanol producers.

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The goal of this work was to isolate food-related zygomycetes, capable of producing ethanol from hexoses and pentoses, possibly allowing for the production of valuable coproducts in addition to ethanol. Therefore, the fungi were isolated from Usar and Laru leaves and their key characteristics, mainly for ethanol production were evaluated. The biomass yield and its protein and chitosan content were also evaluated. The two strains with the best performance were deposited in a culture collection and were used for further investigation on the effect of different carbon sources on both ethanol and biomass production.

Materials and Methods Fungal Strains Two different mixed cultures of zygomycetes fungi growing on T. gandis (Laru) and Hibiscus (Usar) leaves were used in this work. The Laru was obtained from traditional home-made production of tempe in the Gunung Kidul District, Yogyakarta, Indonesia. The Usar was obtained from the traditional market of the Bantul District, Yogyakarta, Indonesia. As a reference to the isolates obtained from the mixed cultures, a pure strain of tempe fungi, Rhizopus microsporus obtained from The Indonesian Institutes of Science, was used in this work. The fungi were maintained on potato dextrose agar (PDA) slants containing 4 g/l potato extract, 20 g/l glucose, and 15 g/l agar. Cultures were grown aerobically at 30 C for 3 days and stored at 4 C. The cultures were renewed every three months. Isolation, Purification, and Identification of the Fungi In order to extract the fungi, the Laru and the Usar were cut by a sterilized scissor and soaked in 100-ml Erlenmeyer flasks containing 50 ml of 0.5 g/l Tween 80. After 24-h incubation in a 100-rpm shaker bath at 30 C, the spore suspensions were diluted to 105, 106, and 107 of the original concentration. Subsequently, 100 l of the diluted spore suspension was spread on the surface of PDA plates. The plates were then incubated at 30 C for 3 days. Each formed colony were transferred to new PDA plates and incubated at identical conditions. The colonies were continuously transferred into new agar plates until pure colonies (isolates) were obtained. A pure culture was rated by its homogenous morphological appearance in size, shape, spore color, and mycelium color. Furthermore, the reverse of the plates were checked to ensure they were colorless, indicating no competition between two or more different colonies. The isolates were tentatively identified based on their morphology according to Samson et al. [17]. Cultivation of the Zygomycetes Strains In order to select potential strains for ethanol production, the strains were cultivated in 40-ml medium in 100-ml cotton-plugged Erlenmeyer flasks and incubated in a shaker bath at 32 C and 125 rpm for 5 days. The medium was prepared as reported by Zamani et al. [18]. Inoculation was performed with 8107 spores per flask. During the cultivation, samples were taken at 0, 24, 48, and 120 h. After desired periods of cultivation, the wet biomass mycelium was harvested on a screen, washed with water for three times, and kept at 20 C until use. The biomass was freeze-dried (FreeZone, Labconco Corporation, Kansas City, USA) for 24 h to get dry biomass. In order to confirm the growth of the selected strains on the different carbon sources and to study the corresponding ethanol production, the strains were cultivated in liquid media.

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The cultivations were performed in 500-ml Erlenmeyer flasks containing 200-ml medium at 32 C using a water bath with 125 rpm agitation. The liquid medium was prepared as previously described [18], except the carbon sources. Three different carbon sources were tested; 50 g/l glucose, 50 g/l xylose, and 15 g/l mixed sugars. The composition of the mixed sugars was chosen in order to mimic a hydrolyzate, and contained 2 g/l cellobiose, 4 g/l glucose, 2 g/l xylose, 1 g/l arabinose, 1 g/l galactose, and 5 g/l mannose. Zygomycetes Cell Wall and Chitosan Cell wall derivatives were prepared according to Synowiecki et al. [19]. The separation of the cell wall or alkali insoluble materials (AIM), and purification of its chitosan were performed according to the methods developed by Zamani et al. [20, 21] with minor modifications. The dry AIM was powdered using a Ball Mill (Retsch MM400, Retsch, Haan, Germany) and mixed with 72 mM H2SO4 (100 ml/g of AIM) in 50 ml tubes. The tubes were tightly closed and placed on a shaker at 100 rpm and room temperature. After 10 min, the mixture was centrifuged at 7,100g for 5 min, the pellet collected and washed twice with distilled water. Subsequently, the pellet was mixed with 72 mM H2SO4 (100 ml/g of initial AIM) in glass bottles, sealed, and placed in an oil bath at 120 C for 45 min. The hot mixture (ca 100 C) was vacuum filtered and the filtrate was collected. The filtrate was cooled on ice and 150 mM NaOH was added to adjust the pH to 810. The precipitate formed was recovered by centrifugation at 7,100g for 5 min, washed with distilled water to neutral pH, freeze-dried, and kept at room temperature until use. Analytical Methods The sugar consumption and metabolite production during cultivation were quantified using HPLC (Waters 2695, Waters Corporation, Milford, USA). A sugar column (Aminex HPX-87P, Bio-Rad, Hercules, USA) at 85 C with ultrapure water as eluent was used to measure the individual sugars in the mixed sugars solutions, while an organic acid column (Aminex HPX87H, Bio-Rad) at 60 C with 5 mM sulfuric acid as eluent was used to determine pure sugars and all the metabolites. A refractive index detector operating at 40 C (Waters 410) and an UV absorbance detector operating at 210 nm (Waters2487) were used in series. Glucosamine (GlcN; Sigma-Aldrich) and N-acetylglucosamine (GlcNAc; SigmaAldrich) contents were analyzed using a two-step acid hydrolysis method [18] with minor modification at the end of depolymerization and deamination process. In order to stop the depolymerization and deamination, 0.5-ml 12% of ammonium sulfamate was added to the tubes and mixed for 4 min. Subsequently, 1 ml of this solution was placed in a 100-ml volumetric flask filled with ca 80-ml ultrapure water. Then, 0.5-ml 0.5% 3-methyl-2benzothiozolinone hydrozone hydrochloride hydrate (Sigma-Aldrich) was added, the solution mixed, and left at room temperature. After 1 h, 0.5 ml FeCl3 was added, and the solution was mixed and incubated at room temperature for 1 h. The volume was adjusted to 100 ml with ultrapure water, and the absorbance was measured using a spectrophotometer at 650 nm. For determination of GlcNAc content, the concentration of the acetic acid released after the hydrolysis was measured by HPLC. The amount of substance of acetic acid in each sample is equal to that of GlcNAc. The protein content of the cells was determined using Biuret method [22]. Samples of 10 mg dry biomass were mixed with 3 ml 1 M NaOH, boiled for 10 min, and cooled to room temperature. One ml of 2.5% CuSO45H2O was added, and the solution whirly mixed for 5 min. In order to separate the protein dissolved in the liquid, the tube was centrifuged. The

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absorbance of the liquid was measured at 555 nm. Different concentrations of bovine serum albumin (Sigma-Aldrich) were used as standard solutions. In order to evaluate the extraction process of chitosan from the fungi, the spectrum of fungal chitosan was compared with commercial chitosan produced from crab shells (Sigma-Aldrich) using FTIR (SensIR Technologies, DanBurg, CT, USA). All experiments were performed in duplicate, and all reported intervals and error bars represent standard deviation.

Results and Discussion Isolation and Identification of Zygomycetes After spore extraction and incubation on agar plates, colonies with different morphological forms were separated and sub-cultured to obtain isolates. No growth was observed on the control, which indicated that there was no contamination during the isolation and purification process. All isolates obtained from this work were tentatively identified based on their morphology. In total, 32 zygomycetes strains were isolated from the two traditional tempe starter cultures. Laru contained 15 strains of Rhizopus and one strain of Absidia while Usar contained ten strains of Rhizopus, one strain of Mucor, and four strains of Rhizomucor. Ethanol and Biomass Production from Glucose Under Aerobic Condition The metabolites produced by all the tempe zygomycetes strains were investigated, and the results are presented in Table 1. All the strains were able to assimilate glucose as the sole carbon source in the medium. Among the 32 strains tested, 20 strains completely consumed the glucose within 24 h (bold in Table 1), whereas the remainder required 48 h. All strains produced ethanol as the major extracellular metabolic product, with maximum yields between 0.26 and 0.41 (in g/g glucose). That the strains selectively produced ethanol suggests that they are favorable candidates for ethanol production. Glycerol production was also detected for all the strains, with yields varying between 4 and 57 mg/g glucose. Some strains also produced lactic acid with yields ranging from 2 to 45 mg/g glucose. Lactic acid production is undesirable because each molecule of lactic acid represents one less molecule of ethanol [23]. Fortunately, for the strains evaluated here lactic acid was only detected at low yields. Negligible acetic acid and succinic acid formation were detected in all the cultivations (data not shown). Biomass yield of all the strains from cultivation in glucose varied between 65 and 140 mg/g glucose. In addition, the fungal biomass content of chitosan ranged from 0.10 to 0.24 g chitosan/g dry biomass (Table 2). Chitosan has several desirable properties including being non toxic, biodegradable, biocompatible, as well as having antioxidant and in possession of antimicrobial activity [24]. For these reasons, chitosan is used by pharmaceuticals, cosmetic, biomedical, food, agricultural, biotechnology, and wastewater treatment [25]. Chitosan derived from the fungal biomass in this work was similar to the commercial chitosan, which is mostly produced from shellfish wastes, as shown in Fig. 1. The fungal biomasses also contained high protein contents ranging from 0.47 to 0.63 g protein/g dry biomass (Table 2). These zygomycetes fungi could thus be utilized as a protein source for animal and fish cultures. Two of the 32 isolates were selected for further study. Their selection was mainly based upon high ethanol yield and low lactic acid production. According to those considerations,

Appl Biochem Biotechnol Table 1 Metabolite yields of the tempe zygomycetes strains during submerged growth on glucose Strain Yields and carbon balance (mg/g consumed glucose) Ethanol Ref R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 RM1 RM2 RM3 RM4 A1 M1 SD 3949 3261 3120 3155 30815 3583 28820 2610 345103 34869 4090 28810 36326 4110 35026 4050 40412 36378 40017 3385 2849 3170 3590 3403 3425 35053 3451 3963 3235 38931 37321 38321 33826 Biomass 15228 1372 1263 1365 1324 1352 1266 14924 1278 1304 1331 11018 11528 13112 11117 1227 1353 1386 1304 1371 1295 1324 1321 1256 1331 1371 1162 11929 13813 1384 1381 1405 657 Lactic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.52 0.00 0.00 0.00 0.00 0.00 5.19 5.70 37.2 0.00 0.00 3.92 0.00 4.05 4.62 0.00 3.87 4.01 0.00 41.9 5.36 5.96 0.00 0.00 5.51 36.85 5.9% Glycerol 32.7 17.4 6.2 6.5 0.0 4.0 4.4 15.7 44.6 34.5 15.1 19.8 32.7 56.9 51.0 29.4 32.0 30.6 45.9 20.9 21.8 23.9 20.0 23.8 21.0 21.4 22.4 54.7 30.5 33.1 34.0 47.4 20.3 10% C balancea 1,116 936 875 901 872 980 825 837 981 981 1,087 807 978 1,136 970 1,121 1,100 1,022 1,100 963 846 922 992 950 967 987 992 1,080 953 1,076 1,046 1,090 865

The strains Ref, R1-R13, R16, R19, R22, R23, R25, RM3 and RM4 completed glucose consumption within 24 h
a

Ref Rhizopus microsporrus, R Rhizopus, RM Rhizomucor, A Absidia, M Mucor, SD standard deviation Carbon balance was calculated as Ethanol=0:51 Biomass=0:49 Lactic acid=0:75 Glycerol=1:02

Rhizomucor 3 (RM3) and Rhizomucor 4 (RM4) were selected and registered at Culture Collection University of Gothenburg (Sweden) as Rhizomucor CCUG 61146 and Rhizomucor CCUG 61147, respectively.

Appl Biochem Biotechnol Table 2 Protein, AIM, and chitosan contents of the tempe zygomycetes strains during submerged growth on glucose Strain Content (mg/g) Protein/biomass Ref R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 RM1 RM2 RM3 RM4 A1 M1 5117 54534 48612 51523 61415 52633 52024 59135 57531 5102 59744 61243 52428 4993 54022 5941 5703 50323 6320 53544 52423 59215 49215 4768 56717 49966 54612 50134 5685 5450 56912 5657 AIM/biomass 23033 3840 26540 2182 25613 18018 29323 22320 26222 2061 29524 1598 1598 23538 2887 29718 2255 24710 2686 21713 29937 3107 25031 29633 27992 1899 2636 25513 23364 20728 1793 22923 23113 Chitosan/AIM 49514 62718 63676 6270 6067 50843 60648 57742 67644 5925 67135 62629 59811 616154 6295 758105 60620 51629 52822 58777 60422 6269 43516 57848 71312 632105 66121 64524 59497 5540 67384 56452 78755 Chitosan/biomass 11419 1290 1675 1370 15510 9217 17828 12921 1763 1221 1976 9910 956 14860 1816 22645 1368 12812 1419 1279 18129 194 10917 17233 19862 11914 1742 16514 14160 1040 12117 1281 1813

The strains Ref, R1-R13, R16, R19, R22, R23, R25, RM3 and RM4 completed glucose consumption within 24 h Ref Rhizopus microsporrus, R Rhizopus, RM Rhizomucor, A Absidia, M Mucor

Ethanol and Chitosan Production of RM3 and RM4 on Different Carbon Sources In order to study the effect of different carbon sources on metabolites production, the selected strains were further tested in submerged liquid cultures containing glucose, xylose,

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100 90 80 70 60 50 a b c d 40 30 20 10 0 100 90 80 70 60 50 a b c d 3,600 3,100 2,600 2,100 1,600 Wavenumber (cm-1) 40 30 20 10 0

RM3

RM4
1,100 600

Fig. 1 FTIR spectra of a commercial chitosan and fungal chitosan from biomass grown on b glucose, c xylose, and d mixed sugars

or mixed sugars (Tables 3 and 4). Generally, both Rhizomucor sp. behaved similarly in sugar consumption and metabolite production. They consumed all of the glucose in the medium both under aerobic and micro-aerobic conditions. In the presence of oxygen, approximately 30 g/l of xylose was assimilated by both fungi, whereas only 3 g/l of xylose was consumed under micro-aerobic condition. Aerobic cultivation on the mixed sugars resulted in complete consumption of all the sugars. However, under micro-aerobic conditions, xylose, and arabinose, remained in the medium after cultivation (data not shown). Additionally, under micro-aerobic conditions 100% and 70% of the initial cellobiose was utilized by RM3 and RM4, respectively.

Appl Biochem Biotechnol Table 3 Metabolite yields of the selected strains RM3 and RM4 during submerged growth Carbon source RM3a Glucose Glucose Xylose Xylose Mixed sugars RM4b Glucose Glucose Xylose Xylose Mixed sugars
d d

Aeration condition

Ethanol (g/l)

Glycerol (g/l)

Xylitol (g/l)

Ym ethanol (g/g glucose)

Yethanol (g/g glucose)

Aerobic Micro-aerobic Aerobic Micro-aerobicc Aerobic Micro-aerobic Aerobic Micro-aerobic Aerobic Micro-aerobicc Aerobic Micro-aerobic

20.320.02 23.580.44 6.370.28 0 4.140.01 4.610.02 18.420.01 22.850.17 5.620.55 0 4.020.3 4.250.52

1.970.00 1.880.05 3.310.09 0.120.02 0.150.00 0.430.00 1.830.04 1.810.21 2.640.91 0 0.210.00 0.330.02

0 0 3.280.02 0.820.14 0 0.130.02 0 0 3.640.55 0.240.02 0 0

0.390.01 0.470.01 0.130.01 0 0.280.00 0.310.00 0.370.00 0.460.00 0.110.01 0 0.270.02 0.280.03

0.380.01 0.460.00 0.220.00 0 0.310.06 0.380.00 0.380.02 0.470.02 0.190.02 0 0.310.07 0.370.03

Mixed sugarsd

Mixed sugarsd

Both aerobic and micro-aerobic cultivation and growth on different carbon sources are reported
a

Ym metabolic ethanol yield (based on consumed sugar(s)), Y ethanol yield from initial sugar(s) Rhizomucor CCUG 61146 Rhizomucor CCUG 61147 Less than 3 g/l xylose was consumed

b c

d Mixed sugars contains 2 g/l cellobiose, 4 g/l glucose, 2 g/l xylose, 1 g/l arabinose, 1 g/l galactose, and 5 g/l mannose

Ethanol (Fig. 2) was the major product for almost all of the cultivations. Replacement of glucose with xylose and mixed sugars resulted in lower ethanol yield both under aerobic and

Table 4 Yields (Y) of biomass and chitosan from AIM and its degree of deacetylation (DD) of the strains RM3 and RM4 during growth on different carbon sources Carbon source Aeration Ybiomassa condition (g/g sugars) YAIMb (g/g DB) YGlcNAcc (g/g AIM) YGlcNc (g/g AIM) Ychitosanc (g/g AIM) DD (%)

RM3 RM4 RM3 RM4 RM3 RM4 RM3 RM4 RM3 RM4 RM3 RM4 Glucose Aerobic Glucose Microaerobic Xylose Aerobic Mixed Aerobic sugars Mixed Microsugars aerobic
a b c

0.11 0.05 0.15 0.33 0.07

0.13 0.03 0.15 0.40 0.04

0.20 0.10 0.09 0.26 0.18

0.27 0.22 0.22 0.25 0.25

0.28 0.19 0.19 0.21 0.16

0.27 0.21 0.14 0.31 0.26

0.16 0.42 0.19 0.33 0.16

0.36 0.47 0.23 0.39 0.36

0.44 0.61 0.39 0.54 0.32

0.63 0.68 0.37 0.70 0.62

37 69 50 61 51

58 69 62 56 58

Yield of fungal biomass based on consumed sugars Yield of AIM based on dry fungal biomass Yields of GlcNAc, GlcN, and chitosan based on AIM

Appl Biochem Biotechnol Fig. 2 Ethanol concentration as a function of time for RM3 and RM4 cultivated on glucose (diamonds), xylose (squares), and mixed sugars (circles) under aerobic (solid lines) and micro-aerobic (dashed lines) conditions
25

RM3

Ethanol concentration (g/l)

20

15

10

0 25

RM4

Ethanol concentration (g/l)

20

15

10

0 0 24 48 72 96 120

Time (h)

micro-aerobic condition for both strains. Ethanol yields of RM4 on xylose and mixed sugars under aerobic condition were 70% and 24% lower than those obtained from glucose. Meanwhile, ethanol yields of RM3 on xylose and mixed sugars under aerobic condition were 50% and 14% lower than those obtained from glucose. Lower ethanol production on xylose might be due to the low rate of xylose transport, production of other metabolites for maintaining the redox balance, and the NADPH balance [26, 27]. Cultivation on mixed sugars also resulted in lower ethanol yields and production (Table 3), probably due to the utilization of pentoses and low initial sugar concentrations. The results show that the ethanol yield of RM3 was higher than for RM4 in all media both under aerobic and micro-aerobic condition. In general, the ethanol yield for both strains under micro-aerobic condition was higher than those under aerobic condition. However, no ethanol was detected from cultivation on xylose under micro-aerobic condition. Glycerol

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production was detected in all media and aeration conditions for both the strains. Furthermore, both strains produced xylitol in the presence of xylose. The xylitol formation is evidence that xylose metabolism by RM3 and RM4 followed the general xylose pathway of fungi via xylitol. The formation of other metabolites such as lactic, acetic, succinic, and pyruvic acid from both these zygomycetes strains were negligible. Compared with other fungi evaluated for bioethanol production, both RM3 and RM4 compete well. Paecilomyces sp., a fungus isolated from compost, has been found to produce 41 g ethanol/l from 100 g glucose/l after 7 days cultivation [28]. Other fungi include several species of Aspergillus, with ethanol concentrations ranging from 5.7 to 24.4 and 0.9 to 5.4 g/l from 50 g glucose or xylose/l, respectively [29]. The zygomycetes M. indicus has been observed to reach 15.2 g ethanol/l from 33 g glucose/l, an ethanol yield of 0.46 g/g [30]. Apart from ethanol, the biomass of these zygomycetes fungi and its chitosan content is of special interest. Therefore, the effect of cultivation in different carbon source as well as aeration on biomass, AIM (e.g., isolated fungal cell wall material), and chitosan content of RM3 and RM4 were evaluated in this study (Table 4). The properties of chitosan is determined by the degree of deacetylation (fraction of GlcN units), which determines the preferred applications of the compound [31]. When GlcNAc is in majority, the polymer is instead referred to as chitin [31]. The fractions of GlcN and GlcNAc were also followed (Table 4). The result indicated that under aerobic condition, replacement of glucose with xylose or mixed sugars increased the yield of biomass for both strains, probably caused by a more aerobic metabolism. The growth on mixed sugars was probably also under a relatively stronger influence from the added yeast extract, which was not considered for calculating the yields. Similarly, the yield of AIM obtained from mixed sugars was higher than that of glucose in RM3. In addition, replacement of glucose with xylose and mixed sugar in RM3 decreased the GlcNAc content followed by increasing GlcN content. Thus, the degree of deacetylation increased, whereas the effect on chitosan yield was not clear. Effect of aeration on the yield of biomass and AIM indicated that aeration increased both the yield of biomass and the related AIM and chitosan parameters. The latter can most likely be attributed to the general thickening of the cell wall exhibited by fungi in the stationary phase. The relatively low AIM content in the zygomycetes cultivated on xylose is thus probably caused by the cells still actively growing. Since most chitosan produced in all media for both strains had a degree of deacetylation of more than 50%, it can be inferred that replacement of glucose with mixed sugars does not change the characteristic of the chitosan. The cell biomass content of other zygomycetes have been found to be similar in terms of AIM, total GlcN and GlcNAc, and in the average degree of deacetylation; however, the values fluctuate depending on the species and the growth conditions. Zamani et al. [18] evaluated Rhizopus oryzae, R. pusillus, and M. indicus, and detected (ca) 0.21, 0.25, and 0.19 g AIM/g biomass, containing 0.71, 0.80, and 0.63 g chitosan/g AIM with an estimated average degree of deacetylation of 59%, 39%, and 65%.

Conclusions Thirty-two fungal isolates from tempe inoculums were characterized for growth and ethanol production from glucose in submerged liquid cultures. Based upon these results, two isolates were chosen for further characterization. All the isolates produced ethanol as a major fermentation product during the cultivations. The selected strains, Rhizomucor CCUG 61146 and 61147, have ethanol yields comparable with S. cerevisiae, and other fungal species, from glucose. Besides, both the strains also produced ethanol from xylose, as well

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as a mixture of other sugars including cellobiose, mannose, and galactose. Apart from ethanol, the produced fungal biomass could have further value based upon their contents of protein and chitosan.
Acknowledgments This work was financially supported by the European Erasmus Mundus External Cooperation Windows (EMECW) program and the Swedish Energy Agency.

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