TY - JOUR
AU1 - Sues, Anna
AU2 - Millati, Ria
AU3 - Edebo, Lars
AU4 - Taherzadeh, Mohammad J.
AB - Abstract Consumption of hexoses and pentoses and production of ethanol by Mucor indicus were investigated in both synthetic media and dilute-acid hydrolyzates. The fungus was able to grow in a poor medium containing only carbon, nitrogen, phosphate, potassium, and magnesium sources. However, the cultivation took more than a week and the ethanol yield was only 0.2 g g−1. Enrichment of the medium by addition of trace metals, particularly zinc and yeast extract, improved the growth rate and yield, such that the cultivation was completed in less than 24 h and the ethanol and biomass yields were increased to 0.40 and 0.20 g g−1, respectively. The fungus was able to assimilate glucose, galactose, mannose, and xylose, and produced ethanol with yields of 0.40, 0.34, 0.39, and 0.18 g g−1, respectively. However, arabinose was poorly consumed and no formation of ethanol was detected. Glycerol was the major by-product in the cultivation on the hexoses, while formation of glycerol and xylitol were detected in the cultivation of the fungus on xylose. The fungus was able to take up the sugars present in dilute-acid hydrolyzate as well as the inhibitors, acetic acid, furfural, and hydroxymethyl furfural. M. indicus was able to grow under anaerobic conditions when glucose was the sole carbon source, but not on xylose or the hydrolyzate. The yield of ethanol in anaerobic cultivation on glucose was 0.46 g g−1. Mucor indicus (rouxii), Ethanol, Mycelium biomass, Hexoses, Pentoses, Wood hydrolyzate 1. Introduction Mucor indicus (formerly M. rouxii) is well-known from a series of fundamental studies on chitin biosynthesis in fungi [1], but has not found the same use for industrial production. As a zygomycete, it is primarily saprophytic, capable of assimilating several sugars. In screening of three zygomycetous genera Rhizopus, Mucor, and Rhizomucor, we found the Mucor genus, specifically M. indicus, a good candidate for ethanol production [2]. Furthermore, some other strains of Mucor show the ability for ethanol production [[3–[5]. The dominant market for ethanol is the fuel market. Ethanol is nowadays produced industrially by Saccharomyces cerevisiae which is limited to some hexoses [6]. On the other hand, lignocellulose is an abundant potential raw material for ethanol production. It is hydrolyzed to a variety of hexoses and pentoses, including glucose, mannose, galactose, xylose, and arabinose. Several economic evaluations have illustrated that efficient utilization of pentoses is important to enhance the overall efficiency of the wood-to-ethanol conversion process [7]. Therefore, the microorganism of choice should be able to ferment all the sugars within the hydrolyzate to optimize the process. Research activities on M. indicus have mainly been conducted in connection with the physiology of its dimorphism, with the growth in the yeast-like form or as filamentous mycelium [[8–[13]. The fungus has also been studied with respect to the production from its mycelia of chitin and its deacetylated derivative chitosan. The cell walls of this species contain significant amounts of chitosan, chitin, and acidic polysaccharides [[10,[14]. Furthermore, environmental concerns are promoting its utilization for the removal and recovery of different waste materials such as toxic heavy metals [15], volatile organic compounds [16] or for polyethylene degradation [17]. The current work aimed to exploit M. indicus in achieving high yields of ethanol from different sugars including hexoses and pentoses. The influence of the medium composition was studied by designing synthetic media allowing good growth and ethanol production on hexoses and pentoses. The carbon source was then switched to a lignocellulosic hydrolyzate as a non-expensive industrial substrate for growth and ethanol production. 2. Material and methods 2.1. The fungus M. indicus CCUG 22424, obtained from Culture Collection, University of Göteborg (Göteborg, Sweden) was used in all experiments. The strain was maintained on potato dextrose agar slants (pH 5.6), prepared with 10 g l−1 neopeptone (Difco, Sparks, MD, USA), 15 g l−1 agar, and 40 g l−1d-glucose as the additional carbon source by incubation for four days at 30 °C. The slants could be stored for one year at 4 °C. 2.2. Dilute-acid hydrolyzate The dilute-acid hydrolyzate (DAH) used in the experiments was produced from forest residues originating mainly from spruce. Hydrolysis of the raw material was carried out in a 350-l rebuilt masonite gun batch reactor located in Rundvik, Sweden. In the hydrolysis process, an amount of material corresponding to 10 kg of dry wood splinter was impregnated with sulfuric acid and water to obtain an initial acid concentration of 5 g l−1, and a solid concentration of 33% (w/w). The impregnated wood was charged into the reactor and the reaction was started by direct steam injection, with a heat-up time of approximately 20 s. After the heat-up period, the reactor was held at a pressure of 15 bar for 10 min, followed by rapid decompression and discharge of the material into a collecting vessel. The solid residue was separated from the liquid hydrolyzate by filtration. The DAH contained altogether 52.5 g l−1 sugar, viz. in g l−1 : galactose 4.5, glucose 9.8, mannose 25.3, xylose 12.9; furthermore acetic acid 6.3, furfural 0.6, and hydroxymethyl furfural (HMF) 1.2. The hydrolyzate was kept at 4 °C until use. 2.3. Cultivation in shake flasks The effect of medium composition was initially tested by cultivation in Erlenmeyer flasks. In addition to the carbon sources, the effects of (NH4)2SO4, KH2PO4, MgSO4, ZnSO4, CaCl2, EDTA, and FeSO4 as well as some trace metals and vitamins (Table 1) were investigated. For the investigation on the medium composition, a base composition (c.f. Section 3) was selected and the effect of variation of each component was tested. Volumes of 150 ml were inoculated with 1.0 ml of a suspension containing 5–6 × 106 spores. The incubations were carried out in 300-ml cotton-plugged conical flasks on a shaker with a shaking diameter of 12 mm and shaker speed of 114 rpm, at 30 °C for 5–8 d. Glucose was then replaced by other hexoses (galactose, mannose) or pentoses (xylose, arabinose) at concentrations of 33 g l−1. Finally, the dilute-acid hydrolyzate was used as carbon source. All experiments were run in duplicate; the average deviation of the duplicate experiments was 5.4%. The liquid samples for metabolite analyses were stored at −20 °C. Table 1 Composition of the applied trace metal and vitamin solutions Compound  Concentration (g l−1)  Trace metal solution  EDTA  3.0  CaCl2· 2H2O  0.9  ZnSO4· 7H2O  0.9  FeSO4· 7H2O  0.6  H3BO3  0.2  MnCl2· 2H2O  0.16  Na2MoO4· 2H2O  0.08  CoCl2· 2H2O  0.06  CuSO4· 5H2O  0.06  KI  0.02  Vitamin solution  p-Aminobenzoic acid  0.2  Nicotinic acid  1.0  Calcium pantohtenate  1.0  Pyridoxine, HCl  1.0  Thiamine, HCl  1.0  d-Biotin  0.05  m-Inositol  25.0  Compound  Concentration (g l−1)  Trace metal solution  EDTA  3.0  CaCl2· 2H2O  0.9  ZnSO4· 7H2O  0.9  FeSO4· 7H2O  0.6  H3BO3  0.2  MnCl2· 2H2O  0.16  Na2MoO4· 2H2O  0.08  CoCl2· 2H2O  0.06  CuSO4· 5H2O  0.06  KI  0.02  Vitamin solution  p-Aminobenzoic acid  0.2  Nicotinic acid  1.0  Calcium pantohtenate  1.0  Pyridoxine, HCl  1.0  Thiamine, HCl  1.0  d-Biotin  0.05  m-Inositol  25.0  View Large Table 1 Composition of the applied trace metal and vitamin solutions Compound  Concentration (g l−1)  Trace metal solution  EDTA  3.0  CaCl2· 2H2O  0.9  ZnSO4· 7H2O  0.9  FeSO4· 7H2O  0.6  H3BO3  0.2  MnCl2· 2H2O  0.16  Na2MoO4· 2H2O  0.08  CoCl2· 2H2O  0.06  CuSO4· 5H2O  0.06  KI  0.02  Vitamin solution  p-Aminobenzoic acid  0.2  Nicotinic acid  1.0  Calcium pantohtenate  1.0  Pyridoxine, HCl  1.0  Thiamine, HCl  1.0  d-Biotin  0.05  m-Inositol  25.0  Compound  Concentration (g l−1)  Trace metal solution  EDTA  3.0  CaCl2· 2H2O  0.9  ZnSO4· 7H2O  0.9  FeSO4· 7H2O  0.6  H3BO3  0.2  MnCl2· 2H2O  0.16  Na2MoO4· 2H2O  0.08  CoCl2· 2H2O  0.06  CuSO4· 5H2O  0.06  KI  0.02  Vitamin solution  p-Aminobenzoic acid  0.2  Nicotinic acid  1.0  Calcium pantohtenate  1.0  Pyridoxine, HCl  1.0  Thiamine, HCl  1.0  d-Biotin  0.05  m-Inositol  25.0  View Large All the sugars were purchased from Sigma–Aldrich Co. 2.4. Cultivation in a bioreactor Batch cultivations were carried out in a Biostat – A bioreactor (B. Braun Biotech International, Melsungen, Germany) with 1.5 l liquid volume. The temperature was set at 30 °C (±0.1 °C) and the pH at 5.5 (±0.07) by addition of 2-M NaOH. Fermentation using wood hydrolyzate started with 500 ml of synthetic medium with a glucose concentration of 5 g l−1 to prepare enough biomass. The medium was inoculated with 10 ml of the same spore suspension as used for the shake flasks. The stirrer at this stage was set at a low rate of 110 rpm to prevent damage of the fungal growth. After total consumption of the initial glucose, one liter of hydrolyzate solution was added to give a total volume of 1.5 l. The stirring rate for hydrolyzate cultivation was switched to 200 rpm. Aerobic conditions were maintained by continuous air sparging at a flow rate of 0.5 l min−1, controlled by a mass flow controller (Hi-Tech, Ruurlo, The Netherlands). 2.5. Analysis of metabolites Metabolites were analyzed by high-performance liquid chromatography (HPLC). An Aminex HPX-87P column (Bio-Rad, Hercules, CA, USA) was used at 85 °C for the analysis of samples containing a mixture of glucose, xylose, galactose, and mannose. Ultra-pure water was used as eluent at a flow rate of 0.6 ml min−1. Ethanol, acetic acid, xylitol, furfural, and HMF concentrations were analyzed by an Aminex HPX-87H column (Bio-Rad, USA) at 60 °C using 5-mM H2SO4 at a flow rate of 0.6 ml min−1. A refractive index (RI) detector (Waters 410, Millipore, Milford, CA, USA) and UV absorbance detector at 210 nm (Waters 486) were used in series. Concentrations of all metabolites except furfural and HMF were determined from the RI chromatograms. 2.6. Biomass determination Due to the often inhomogeneous distribution of the fungal mycelium, only the final biomass dry weight was determined by filtering the entire culture medium over a tea screen, washing with distilled water and drying at 105 °C during 24 h. The biomass yield was calculated based on the total formation of biomass and the consumption of sugars. 3. Results 3.1. Medium composition The effect of medium composition on ethanol production and growth was investigated by cultivating M. indicus in shake flasks, and the results are summarized in Table 2. The fungus was able to grow in a basal medium composition (BMC) including 7.5 g l−1 (NH4)2SO4, 3.0 g l−1 KH2PO4, 0.5 g l−1 MgSO4· 7H2O, as well as 33 g l−1 glucose as carbon and energy source. However, the cells needed more than 8 days for complete consumption of glucose, and yielded ethanol as 0.20 g g−1 glucose. Addition of zinc sulfate could reduce the incubation period from 192 to 87 h. However, zinc was not the only trace metal to improve the performance of the fungus, since the addition of all trace metals (Table 1) further decreased the time for achieving the maximum ethanol yield. The best separate performance was obtained by the addition of yeast extract to BMC, while addition of only the vitamins did not show any improvement (Table 2). However, the best results were obtained when zinc sulfate, trace metals, yeast extract, and the vitamins were added together to the BMC (Table 2). This rich medium composition containing 7.5 g l−1 (NH4)2SO4, 3.0 g l−1 KH2PO4, 0.5 g l−1 MgSO4· 7H2O, 0.2 g l−1 ZnSO4· 7H2O, 6.7 ml l−1 trace metals solution, 2.5 g l−1 yeast extract, and 0.7 ml l−1 vitamins solution (Table 2: A + B + C + D) will further be called optimum medium composition (OMC). The fungus needed only 23 h to take up 33 g l−1 glucose in OMC, and yielded ethanol, glycerol, and biomass as 0.40, 0.05, and 0.20 g g−1 glucose. Table 2 The effect of addition of (A) 0.2 g l−1 ZnSO4· 7H2O, (B) 6.7 ml l−1 trace metal solution, (C) 2.5 g l−1 yeast extract, and (D) 0.7 ml l−1 vitamin solution to the BMC on the cultivation time and metabolites and biomass yield Added componentd  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  BMCc  0.20  192  0.02  0.11  192  Pellet  A  0.29  87  0.02  0.10  87  Cotton-like  B  0.32  46  0.03  0.19  87  Pellet  C  0.40  37  0.04  0.14  37  Cotton-like  D  0.19  118  0.05  0.09  Consumed only 50% of glucose  Pellet  A + B  0.35  40  0.03  0.19  40  Cotton-like  A + C  0.38  37  0.03  0.14  37  Cotton-like  B + C  0.39  40  0.04  0.15  40  Cotton-like  B + D  0.36  40  0.03  0.19  40  Cotton-like  C + D  0.37  24  0.04  0.09  24  Cotton-like  A + B + C  0.33  24  0.03  0.18  24  Cotton-like  A + B + D  0.38  40  0.03  0.14  40  Cotton-like  A + C + D  0.38  24  0.04  0.13  24  Cotton-like  B + C + D  0.38  40  0.04  0.15  40  Cotton-like  A + B + C + D  0.40  23  0.05  0.20  23  Cotton-like  Added componentd  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  BMCc  0.20  192  0.02  0.11  192  Pellet  A  0.29  87  0.02  0.10  87  Cotton-like  B  0.32  46  0.03  0.19  87  Pellet  C  0.40  37  0.04  0.14  37  Cotton-like  D  0.19  118  0.05  0.09  Consumed only 50% of glucose  Pellet  A + B  0.35  40  0.03  0.19  40  Cotton-like  A + C  0.38  37  0.03  0.14  37  Cotton-like  B + C  0.39  40  0.04  0.15  40  Cotton-like  B + D  0.36  40  0.03  0.19  40  Cotton-like  C + D  0.37  24  0.04  0.09  24  Cotton-like  A + B + C  0.33  24  0.03  0.18  24  Cotton-like  A + B + D  0.38  40  0.03  0.14  40  Cotton-like  A + C + D  0.38  24  0.04  0.13  24  Cotton-like  B + C + D  0.38  40  0.04  0.15  40  Cotton-like  A + B + C + D  0.40  23  0.05  0.20  23  Cotton-like  a Time needed to reach maximum ethanol production. b Time needed for total glucose consumption. c Base medium (BMC) contains only 7.5 g l−1 (NH4)2SO4, 3.0 g l−1 KH2PO4, 0.5 g l−1 MgSO4· 7H2O and 33.3 g l−1 glucose. d Added components to base case as: A, ZnSO4· 7H2O, B, trace metals; C, yeast extract; D, vitamins. e Max. ethanol yield on glucose consumed. f Glycerol yield on glucose consumed. g Biomass yield on glucose consumed. View Large Table 2 The effect of addition of (A) 0.2 g l−1 ZnSO4· 7H2O, (B) 6.7 ml l−1 trace metal solution, (C) 2.5 g l−1 yeast extract, and (D) 0.7 ml l−1 vitamin solution to the BMC on the cultivation time and metabolites and biomass yield Added componentd  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  BMCc  0.20  192  0.02  0.11  192  Pellet  A  0.29  87  0.02  0.10  87  Cotton-like  B  0.32  46  0.03  0.19  87  Pellet  C  0.40  37  0.04  0.14  37  Cotton-like  D  0.19  118  0.05  0.09  Consumed only 50% of glucose  Pellet  A + B  0.35  40  0.03  0.19  40  Cotton-like  A + C  0.38  37  0.03  0.14  37  Cotton-like  B + C  0.39  40  0.04  0.15  40  Cotton-like  B + D  0.36  40  0.03  0.19  40  Cotton-like  C + D  0.37  24  0.04  0.09  24  Cotton-like  A + B + C  0.33  24  0.03  0.18  24  Cotton-like  A + B + D  0.38  40  0.03  0.14  40  Cotton-like  A + C + D  0.38  24  0.04  0.13  24  Cotton-like  B + C + D  0.38  40  0.04  0.15  40  Cotton-like  A + B + C + D  0.40  23  0.05  0.20  23  Cotton-like  Added componentd  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  BMCc  0.20  192  0.02  0.11  192  Pellet  A  0.29  87  0.02  0.10  87  Cotton-like  B  0.32  46  0.03  0.19  87  Pellet  C  0.40  37  0.04  0.14  37  Cotton-like  D  0.19  118  0.05  0.09  Consumed only 50% of glucose  Pellet  A + B  0.35  40  0.03  0.19  40  Cotton-like  A + C  0.38  37  0.03  0.14  37  Cotton-like  B + C  0.39  40  0.04  0.15  40  Cotton-like  B + D  0.36  40  0.03  0.19  40  Cotton-like  C + D  0.37  24  0.04  0.09  24  Cotton-like  A + B + C  0.33  24  0.03  0.18  24  Cotton-like  A + B + D  0.38  40  0.03  0.14  40  Cotton-like  A + C + D  0.38  24  0.04  0.13  24  Cotton-like  B + C + D  0.38  40  0.04  0.15  40  Cotton-like  A + B + C + D  0.40  23  0.05  0.20  23  Cotton-like  a Time needed to reach maximum ethanol production. b Time needed for total glucose consumption. c Base medium (BMC) contains only 7.5 g l−1 (NH4)2SO4, 3.0 g l−1 KH2PO4, 0.5 g l−1 MgSO4· 7H2O and 33.3 g l−1 glucose. d Added components to base case as: A, ZnSO4· 7H2O, B, trace metals; C, yeast extract; D, vitamins. e Max. ethanol yield on glucose consumed. f Glycerol yield on glucose consumed. g Biomass yield on glucose consumed. View Large The effect of variation in the concentration of the chemicals in OMC was investigated by applying different concentration of (NH4)2SO4 at 7.5, 9, and 12 g l−1, KH2PO4 at 2, 3, and 5 g l−1, MgSO4· 7H2O at 0.25, 0.5, and 1.0 g l−1, trace metals solution at 6.7, 13.5, 33, and 67 ml l−1, and yeast extract at 1, 2.5, and 5 g l−1. No further improvement in ethanol yield and rate of glucose consumption was obtained compared to OMC by the tested concentrations of (NH4)2SO4, KH2PO4, and MgSO4 (not shown). However, increasing the concentration of trace metals from 6.7 to 67 ml l−1 decreased the ethanol yield from 0.39 to 0.36 g g−1, but the rate of glucose consumption remained constant. On the other hand, the presence of yeast extract had a positive effect on ethanol yield, since the addition of 5 g l−1 yeast extract to the medium increased the ethanol yield from 0.38 to 0.41 g g−1. However, no improvement in the rate of glucose consumption was obtained either at higher or lower concentrations of yeast extract. Furthermore, the additional effects of FeSO4· 7H2O at 0.3 and 1.0 g l−1, EDTA at 2, 5, and 10 g l−1, and CaCl2· 2H2O at 0.3, 0.8, 1.0, 3, 5, 10 g l−1 were also investigated separately. Further addition of FeSO4· 7H2O did neither enhance metabolite nor biomass yields. Calcium chloride, at concentrations below 1 g l−1, slightly enhanced the overall ethanol yield and its production rate during the exponential growth phase (data not shown). Moreover, calcium delayed the consumption of ethanol once the medium was depleted of sugars (cf. Fig. 2 for aerobic cultivation). EDTA had serious inhibitory effects which were only partially restored by addition of ions possibly chelated by EDTA such as CaCl2· 2H2O or ZnSO4· 7H2O. Fig. 2 View largeDownload slide Aerobic and anaerobic cultivation of M. indicus in shake flasks with glucose as the carbon source. Filled symbols represent aerobic conditions and open symbols represent anaerobic conditions: (a) glucose (•/◯); (b) ethanol (▪/□) and glycerol (♦/⋄). Arrows indicate the relevant ordinates. Fig. 2 View largeDownload slide Aerobic and anaerobic cultivation of M. indicus in shake flasks with glucose as the carbon source. Filled symbols represent aerobic conditions and open symbols represent anaerobic conditions: (a) glucose (•/◯); (b) ethanol (▪/□) and glycerol (♦/⋄). Arrows indicate the relevant ordinates. 3.2. Assimilation of sugars and product formation In order to examine the capability of the fungus to consume other types of monosaccharides, glucose was replaced by galactose, mannose, xylose, or arabinose. The most important results of these experiments are presented in Table 3. Glucose and mannose were consumed almost within the same period of time and were depleted in about 24 h. The other hexose, i.e. galactose, was exhausted in slightly more than 24 h. In contrast, xylose remained longer in the medium and arabinose was even more poorly consumed. The concentration of arabinose had decreased only about 15% from the initial concentration after 93 h cultivation. The main metabolites from the other four sugars were ethanol and glycerol. Hexoses were the preferable sugars for the fungus to produce ethanol. Among the hexoses, galactose gave the lowest maximum ethanol concentration. Although the ethanol yield from xylose was lower than that from hexoses (Table 3), the ethanol production was in line with xylose consumption (data not shown). The yields of glycerol and biomass were almost similar for all the sugars (Table 3). While no distinct metabolites were detected when arabinose was the carbon source, the fungus grew with a biomass yield of 0.17 g g−1. Xylitol appeared in the medium only when xylose was used. A yield of 0.07 g g−1 which corresponds to a concentration of 2.3 g l−1 xylitol was obtained. Table 3 The ethanol, biomass, and glycerol yields and the morphology of M. indicus in cultivation on different sugars   Sugar  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  Hexoses  Glucosec  0.40  23  0.05  0.20  23  Cotton-like    Galactose  0.34  24  0.03  0.19  >24  Cotton-like    Mannose  0.39  24  0.04  0.21  24  Cotton-like  Pentoses  Xylose  0.18  88  0.03  0.19  113  Pellets and cotton-like    Arabinose  0.00  Not reached  0.00  0.17d  Not reached  Pellets and cotton-like    Sugar  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  Hexoses  Glucosec  0.40  23  0.05  0.20  23  Cotton-like    Galactose  0.34  24  0.03  0.19  >24  Cotton-like    Mannose  0.39  24  0.04  0.21  24  Cotton-like  Pentoses  Xylose  0.18  88  0.03  0.19  113  Pellets and cotton-like    Arabinose  0.00  Not reached  0.00  0.17d  Not reached  Pellets and cotton-like  a Time needed to reach maximum ethanol production. b Time needed for total sugar consumption. c Optimal Medium Composition. d Total consumption of arabinose was only 19.4% of the initial amount. e Max. ethanol yield on glucose consumed. f Glycerol yield on glucose consumed. g Biomass yield on glucose consumed. View Large Table 3 The ethanol, biomass, and glycerol yields and the morphology of M. indicus in cultivation on different sugars   Sugar  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  Hexoses  Glucosec  0.40  23  0.05  0.20  23  Cotton-like    Galactose  0.34  24  0.03  0.19  >24  Cotton-like    Mannose  0.39  24  0.04  0.21  24  Cotton-like  Pentoses  Xylose  0.18  88  0.03  0.19  113  Pellets and cotton-like    Arabinose  0.00  Not reached  0.00  0.17d  Not reached  Pellets and cotton-like    Sugar  YE/S maxe  ta (h)  YGly/Sf  YX/Sg  tb (h)  Morphology  Hexoses  Glucosec  0.40  23  0.05  0.20  23  Cotton-like    Galactose  0.34  24  0.03  0.19  >24  Cotton-like    Mannose  0.39  24  0.04  0.21  24  Cotton-like  Pentoses  Xylose  0.18  88  0.03  0.19  113  Pellets and cotton-like    Arabinose  0.00  Not reached  0.00  0.17d  Not reached  Pellets and cotton-like  a Time needed to reach maximum ethanol production. b Time needed for total sugar consumption. c Optimal Medium Composition. d Total consumption of arabinose was only 19.4% of the initial amount. e Max. ethanol yield on glucose consumed. f Glycerol yield on glucose consumed. g Biomass yield on glucose consumed. View Large When the sugars existed in mixtures, an interaction among the sugars was apparent (Fig. 1). With all hexoses present in the medium at 33 g l−1 each, the complete consumption of sugars was delayed to more than 24 h (Fig. 1(a)), which could be the result of higher osmotic pressure due to the high sugar content of the medium. Furthermore, the assimilation of xylose and arabinose was favoured by the existence of growing cells after glucose consumption (Fig. 1(b)). Total arabinose consumption was 31% of the initial amount, which is higher than when arabinose was used alone (Table 3). Xylose was depleted faster in the presence of glucose, compared to the experiment in which it was the sole carbon source (data not shown). Fig. 1 View largeDownload slide Consumption of different mixtures of sugars by M. indicus in shake flask: (a) glucose (•), galactose (▪), and mannose (▾); (b) glucose (•), xylose (◯), and arabinose (□); (c) galactose (▪), mannose (▾), and xylose (◯). Fig. 1 View largeDownload slide Consumption of different mixtures of sugars by M. indicus in shake flask: (a) glucose (•), galactose (▪), and mannose (▾); (b) glucose (•), xylose (◯), and arabinose (□); (c) galactose (▪), mannose (▾), and xylose (◯). 3.3. Effect of oxygen The influence of oxygen was examined in shake flasks with synthetic medium using glucose or xylose as carbon source. The optimal medium composition supplemented with 1 g l−1 calcium chloride (OMM) was used for all the experiments, because addition of calcium chloride slightly increased the ethanol yield and production rate (Section 3.1). The fungus was able to grow aerobically as well as anaerobically on glucose as carbon and energy source (Fig. 2). In contrast, the fungus failed to grow under anaerobic conditions on xylose as the carbon source (data not shown). Glucose was completely depleted almost twice as fast (in less than 24 h) under aerobic conditions as under anaerobic conditions (in less than 48 h). However, anaerobic cultivation led to a maximum ethanol yield of 0.46 g g−1 compared to aerobic cultivation yielding 0.40 g g−1. The corresponding yields of glycerol were 0.06 and 0.05 g g−1, respectively. Accordingly, aerobic conditions were preferred for biomass production. A biomass yield of 0.16 g g−1 was obtained in the aerobic experiment, whereas only 0.06 g g−1 biomass was harvested after anaerobic cultivation. Experiments in a bioreactor under more controlled environmental conditions were also performed. The better aeration in the bioreactor supported the biomass growth. A yield of 0.36 g g−1 for biomass was obtained when the fungus was cultivated aerobically. However, fungal mycelia grew on most surfaces of the bioreactor, making the operation cumbersome to be handled, and the ethanol yield dropped to 0.18 g g−1. 3.4. Morphology Two different types of morphogenesis were observed, viz. filamentous mycelium (cotton-like) and pellets freely dispersed throughout the broth. Pellets were mainly obtained with poor media and glucose as carbon source, whereas rich medium containing yeast extract favoured cotton-like growth using hexoses or pentoses. The effect of yeast extract was partially overcome by further addition of CaCl2· 2H2O or FeSO4· 7H2O. Therefore, the OMC medium was supplemented with 1 g l−1 of CaCl2· 2H2O and in the shake-flask experiments, pellet morphology was obtained, when OMC medium contained calcium chloride. On the other hand, high aeration in the bioreactor impaired pellet morphogenesis. 3.5. Cultivation with wood hydrolyzate as carbon source The dilute-acid wood hydrolyzate was used for cultivation under aerobic and anaerobic conditions in OMM medium, where glucose was substituted by the hydrolyzate. The experiments were carried out by inoculating the spores in 50 ml synthetic medium for germination, followed by the addition of 100 ml of the hydrolyzate. Alternatively, the spores were inoculated directly into the hydrolyzate medium, which resulted in growth of the fungus. However, this method resulted in a longer lag phase and was not applied regularly. In contrast to the results in synthetic media with hexose, no growth was observed in anaerobic cultivation with the hydrolyzate. However, good growth of the fungus was achieved in aerobic cultivation (Fig. 3). The hexoses glucose, mannose, and galactose started to be consumed 24 h after addition of the hydrolyzate and were depleted in less than 40 h. As usual, xylose was consumed more slowly. The experiment was stopped when no more carbon dioxide was detected in measurements. Ethanol and glycerol were produced up to 10 and 1.8 g l−1, corresponding to yields of 0.42 and 0.07 g g−1, respectively. No xylitol was detected during the cultivation of the fungus on the hydrolyzate, although xylose was present in the sugar mixture of the hydrolyzate. Fungal growth resulted in a final biomass yield of 0.23 g g−1. Fig. 3 View largeDownload slide Cultivation of M. indicus in bioreactor in dilute-acid hydrolyzate. The profiles of: (a) sugars, including glucose (•), galactose (▪), mannose (▾), xylose (◯); (b) ethanol (♦), and glycerol (⋄); (c) acetic acid (▵) and HMF (★) are presented. Arrows indicate the relevant ordinates. Fig. 3 View largeDownload slide Cultivation of M. indicus in bioreactor in dilute-acid hydrolyzate. The profiles of: (a) sugars, including glucose (•), galactose (▪), mannose (▾), xylose (◯); (b) ethanol (♦), and glycerol (⋄); (c) acetic acid (▵) and HMF (★) are presented. Arrows indicate the relevant ordinates. Acetic acid, furfural, and HMF which were present in the hydrolyzate (and are inhibitory to most microorganisms) were utilized or converted. Acetic acid was present at more than 4 g l−1 and was partially consumed for about 1 g l−1 within 50 h of cultivation (Fig. 3(c)). The initial concentration of 0.6 g l−1 HMF decreased to less than 0.2 g l−1 within 24 h and to zero in less than 30 h (Fig. 3(c)). Furthermore, the conversion of approximately 0.4 g l−1 furfural was probably fast enough to become eliminated in a few minutes, since we could not detect it during the cultivation. 4. Discussion The current work explores the potential of M. indicus for ethanol production. The current preferred species for industrial ethanol production, S. cerevisiae, is able to produce ethanol from glucose in a time span of 24 h with yields of about 0.42 and 0.44 g l−1 under aerobic and anaerobic conditions, respectively [[2,[18]. Furthermore, it is limited in taking up just glucose, mannose, and perhaps galactose among the sugars abundant in wood. The present results show several advantages of M. indicus for ethanol production, including: (a) M. indicus is similar to S. cerevisiae with respect to ethanol productivity and yield, with ethanol and glycerol as the main metabolite and by-product under aerobic and anaerobic conditions; (b) M. indicus has a clear advantage over S. cerevisiae in term of its capability to metabolize several types of sugars including hexoses (glucose, mannose, and galactose) and pentoses (xylose and arabinose), although the consumption of arabinose was low. Medium composition had a great impact on both the cultivation time course and the yield of M. indicus. The base medium for cultivation of the fungus (BMC) contained MgSO4· 7H2O and KH2PO4 at standard concentrations [11]. Furthermore, (NH4)2SO4 and glucose were chosen as nitrogen and carbon sources, respectively. The very long time needed for complete consumption of glucose indicated deficiencies in the medium composition which were partially supplied by zinc (Table 2: A). Zinc is an important trace metal which participates in the activity of zinc-dependent alcohol dehydrogenase (ADH) in the fermentative pathway. In S. cerevisiae ADH occurs as four isoenzymes that seem to be zinc-dependent [19]. Further stimulation of ethanol production was achieved by addition of trace metals (Table 2: B), indicating a contribution by other trace metals, and further stimulation of both glucose consumption and ethanol production when the two were combined (Table 2: A + B), implying that the standard concentration of zinc in the trace metal solution (Table 1) was suboptimal. The mixture of vitamins had a negligible effect (Table 2: D), indicating that the standard vitamins were well synthesized by M. indicus, in accordance with previous findings [20]. Yeast extract was the most efficient supplement (Table 2: C), however, glucose consumption and ethanol production were still speeded up by trace metals and vitamins. The favourable influence of yeast extract on the growth of the fungus implies that there are growth factors or stimulating nutrients contained in such a complex medium. Bartnicki-Garcia and Nickerson [20] have reported that vitamins such as thiamine or nicotinic acid are necessary to support anaerobic growth of M. indicus. However, the results of the current work show that the other tested components are more important than the vitamins to support cell growth and ethanol production. M. indicus consumed the hexoses faster than the pentoses, when present in synthetic medium as well as in wood hydrolyzate. Regardless of a long lag phase, the fungus was able to consume xylose. However, M. indicus was poor with respect to arabinose consumption. Arabinose is metabolized by engineered yeast but its fermentation needs more reaction steps than that of xylose [21]. This might explain why no metabolites were detected from the cultivation on arabinose whereas a small amount of biomass was obtained. Xylose was not assimilated under anaerobic conditions. Previous reports have shown that Mucor would not grow anaerobically in the absence of a hexose [[11,[4]. Yet, even with the presence of both hexoses and pentoses in the hydrolyzate, batch fermentation of the wood hydrolyzate under anaerobic conditions could not be performed. This failure is worth further investigation. M. indicus might need not only a hexose to support anaerobic growth in complex media like hydrolyzate, but also some growth factor as vitamins or ergosterol. Furthermore, the inhibitors in hydrolyzate may play a crucial role under anaerobic conditions. The traditional problem with cultivation of fungal mycelium in a conventional bioreactor such as CSTR is the filamentous morphology. This obstacle might be reduced by choosing a medium promoting pellet morphology. Since agitation in CSTR might even damage the pellets, other techniques such as immobilization of the fungus in porous materials can be applied. A rotating fibrous-bed bioreactor (RFB) has been successfully applied for Rhizopus oryzae to produce lactic acid [22]; fungal mycelia were immobilized on cotton cloth, resulting in clear fermentation broth. For that reason, utilization of M. indicus in industrial applications becomes more feasible. Therefore, M. indicus is a good alternative to S. cerevisiae for ethanol production from sources that contain hexoses and pentoses, not only on a laboratory scale but also on an industrial scale. Acknowledgements This work was financially supported by Swedish Energy Agency and Kristina Stenborg Foundation. 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TI - Ethanol production from hexoses, pentoses, and dilute-acid hydrolyzate by Mucor indicus
JF - FEMS Yeast Research
DO - 10.1016/j.femsyr.2004.10.013
DA - 2005-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ethanol-production-from-hexoses-pentoses-and-dilute-acid-hydrolyzate-fTWqw0edRq
SP - 669
EP - 676
VL - 5
IS - 6
DP - DeepDyve
ER -