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Production of hydrocarbon compounds by endophytic fungi Gliocladium species grown on cellulose
Bioresource Technology 102 (2011) 9718–9722
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of hydrocarbon compounds by endophytic fungi Gliocladium species grown on cellulose Aftab Ahamed, Birgitte K. Ahring ⇑ Center for Bioproducts and Bioenergy, Bioproducts Sciences and Engineering Laboratory, Washington State University, Richland, WA 99354-1671, USA
a r t i c l e
i n f o
Article history: Received 31 March 2011 Received in revised form 5 July 2011 Accepted 13 July 2011 Available online 28 July 2011 Keywords: Biofuel Hydrocarbons Green energy Bio-products Endophyte
a b s t r a c t Endophytic fungi belonging to the genus Gliocladium are able to degrade plant cellulose and synthesize complex hydrocarbons under microaerophilic conditions. These fungi could thus be used to produce biofuels from cellulosics without the need for hydrolytic pretreatments. Gas chromatography–mass spectrometry–solid-phase micro-extraction (GC–MS–SPME) of head space gases from Gliocladium cultures demonstrated the production of C6–C19 hydrocarbons including hexane, benzene, heptane, 3,4dimethyl hexane, 1-octene, m-xylene, 3-methyl nonane, dodecane, tridecane, hexadecane and nonadecane directly from the cellulosic biomass. Hydrocarbon production was 100-fold higher in co-cultures of Gliocladium and Escherichia coli than in pure Gliocladium cultures. The dry mycelia weight is stable at stationary period in co-culture condition which may lead to synthesize more hydrocarbons. These fungi could potentially be developed into cost-effective biocatalysts for production of biofuels. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
2. Methods
Microbes synthesizing extracellular long-chain hydrocarbons are of interest since they could be exploited for biofuel production. Some fungal species have been shown to synthesize such hydrocarbons depending on environmental conditions and growth media (Sunesson et al., 1995; Wackett, 2008). For example, Gliocladium roseum (NRRL 50072) (now designated as Ascocoryne sarcoides (Stadler and Schulz, 2009; Strobel et al., 2010; Griffin et al., 2010)), which lives inside the Ulmo trees in the rain forests of Patagonia (Argentina and Chile) was able to directly degrade plant cellulose and synthesize a broad array of hydrocarbon compounds which are remarkably similar to diesel fuel (Strobel et al., 2008). In this fungus, hydrocarbon production appears to be a defense mechanism since the production of gases rich in hydrocarbons and hydrocarbon was stimulated by exposure to antibiotics (Strobel et al., 2008). The current study was undertaken to determine if co-cultivation with bacterium would be a means to increase hydrocarbon production by three Gliocladium species. Escherichia coli strain Nissle 1917 (DSM 6601) was chosen as the test bacterium because it is non-pathogenic, metabolically versatile and able to grow under the microaerophilic conditions required by the fungus.
2.1. Fungal and bacterial test strains
⇑ Corresponding author. Tel.: +1 509 372 7682; fax: +1 509 372 7690. E-mail addresses: [email protected], [email protected] (B.K. Ahring). URL: http://www.tricity.wsu.edu/bsel (B.K. Ahring). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.073
Gliocladium 62724, G. roseum 1165 and G. roseum 62726 were obtained from DSMZ (German collection of microorganisms and cell cultures, Inhoffenstrabe, Germany), ATCC (American type culture collection, Manassas, VA, USA) and from a private Danish collection at Copenhagen Institute of Technology at Aalborg University respectively. E. coli strain Nissle 1917 (DSM 6601) was obtained from ATCC. The freeze dried fungal spores were suspended in sterile 15% (v/v) glycerol, plated on potato dextrose agar (PDA, Difco Laboratories) and incubate at 23 °C for 18 days until good sporulation occurred. The spores were harvested by washing the petri plates with sterile water containing 1% Triton X-100 (Fisher Scientific, Orangeburg, NJ, USA). 2.2. Culture media Cellulose pre-culture medium (Ahamed and Vermette, 2008) consisted of: 10 g L1 avicel (Sigma–Aldrich, St. Louis, MO, USA), 5 g L1 corn steep liquor (Sigma–Aldrich, St. Louis, MO, USA), 1.5 g L1 proteose peptone (Fischer Biotech, Fair Lawn, NJ, USA), 0.5 g L1 yeast extract (Fischer Biotech, Fair Lawn, NJ, USA), 1.7 g L1 (NH4)2SO4, 2 g L1 KH2PO4, 0.3 g L1 CaCl22H2O, 0.3 g L1 MgSO47H2O, 0.005 g L1 FeSO47H2O, 0.0037 g L1 CoCl26H2O, 0.0016 g L1 MnSO4H2O, 0.0014 g L1 ZnSO47H2O. The composition of production medium was the same as that
A. Ahamed, B.K. Ahring / Bioresource Technology 102 (2011) 9718–9722
of the cellulose pre-culture medium, except that it contained 35 g L1 avicel, 10 g L1 corn steep liquor, 3 g L1 proteose peptone, 2 g L1 yeast extract, 2 g L1 (NH4)2SO4, 4 g L1 KH2PO4 and 0.2 ml L1 tween 80. The production medium was sterilized at 121 °C for 45 min.
ðCLÞ ðVLÞ CL : ðVLÞ þ ðH0 V g Þ ð1 þ H0 Þ
2.3. Inoculum and shake flask culture
ðH0 Þ ðCleÞ:
Concentrated aqueous spore inocula (106 mL1) of the Gliocladium strains preserved in 2-mL vials at 80 °C in 15% (v/v) glycerol were inoculated into modified 1000-mL graduated Erlenmeyer flasks containing 300 mL of cellulose pre-culture medium and incubated at 23 °C on a rotary shaking incubator with an agitation speed of 125 rpm. The initial pH was 5.7 and was not controlled during pre-culture. The flasks were sealed with a rubber stopper with two holes through which stainless steel tubes were inserted to allow collection of liquid and air samples. After 72 h of cultivation, the Gliocladium mycelium suspension from the pre-culture medium corresponding to 10% (v/v) of the total production medium volume was aseptically inoculated into two 500-ml flasks containing 250 mL of production medium. One of the flasks was also inoculate with 1.89 106 CFU mL1 of E. coli Nissle 1917. Microearophilic conditions were maintained in both flasks through hose cock clamp fixed on rubber tube with sterile air filter, turning these clamps on and off two times a day facilitate the microaerophilic conditions and the flasks were incubated at 23 °C for 20 days on a rotary shaker (125 rpm). Control flasks containing autoclaved cultures were also incubated. The shake flask culture experiments were carried out in duplicate. 2.4. Determination of residual cellulose and fungal dry biomass Cellulose content of Gliocladium fungal cultures were determined by a simplified version of the method of Updegraff (1969). Ten milliliter of the culture broth was centrifuged (3000g, for 20 min), the supernatant was removed, the pellet was suspended in acetic acid nitric acid reagent (3 mL: 150 mL of 80% acetic acid with 15 mL of pure nitric acid) and boiled for 30 min in a water bath. After cooling and centrifugation (3000g, for 20 min), the pellet was washed with distilled water (10 mL), and the residual cellulose was dried at 40 °C under reduced pressure until constant weight. Six replicate samples were measured and the average cellulose content was calculated. The dry fungal biomass was determined using the method developed by Ahamed and Vermette (2009). The mycelial weight was calculated as the difference between the total dry weight of the solids (comprising mycelium and residual cellulose) and that of the residual cellulose. 2.5. Dimensionless ‘‘Henry’s law constant’’ application Henry’s Law Constant (HLC) was applied to determine the concentration of volatiles in liquid and head space gases at equilibrium. The equation is as follows:
H0 ¼
H RT
H0 , Dimensionless Henry’s law constant; H, Henry’s law constant [atm – m3/mol]; R , Universal gas constant [8.20575 105 atmm3/mol K]; T, Temperature in kelvin. Equilibrium of air–water partitioning:
ðCLÞðVLÞ ¼ ðCleÞðVLÞ þ ðCgeÞðV g Þ CL, Concentration in liquid (mg/L); VL, Volume of liquid (L); Cle, Concentration in liquid at equilibrium; Cge, Concentration in head space gas at equilibrium; Vg, Volume of gas.
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Liquid phase partitioning:
Gas phase portioning:
ðH0 Þ ðCLÞ ð1 þ H0 Þ
2.6. Headspace gases analysis Ten milliliter of the culture was centrifuged at 10,000g for 20 min, and the supernatant was carefully removed with a sterilized glass pipette and used for the measurements of volatile hydrocarbons through GC/MS (7890A GC-system with 5975C inert XL E1/C1 MSD model # G3174A, Agilent Technologies, Wilmington, DE, USA) containing DB-5MS, non-polar, high temperature limit (450 °C), 30 m 0.250 lm 0.25 lm column. The CTC analysis CombiPAL robotic arm (G6500-CTC-LHS2.PAL system-CH001210757, CTC Analytics AG, Zwingen, Switzerland) was connected to GC/MS for auto sampling system. All the samples were sequenced and programmed for incubation at 50 °C in 20 mL head space screw top clear glass vials (VWR 5188-2753) with an agitation speed of 500 rpm and the total extraction time was 2 min. The volatiles from head space gases were adsorbed to sterilized Divinylbenzene/Carboxen/Polymethylsiloxane stableflex silica fiber (Gray, Supelco Cat. 57284U, Bellefonte, PA, USA) present in solid phase micro extraction (SPME) syringe (Supelco, Cat. 57315, Sigma–Aldrich) connected to CTC-PAL robotic arm for automatic insertion. SPME allows extraction and concentration to be performed in a single step and direct analyte transfer to GC by thermal desorption inside a heated injection port for 1 min and swept into the column by ultra pure helium as carrier gas and the average velocity was maintained at 42.651 cm/s, the analytes were trapped and focused at the column inlet and the stable flex silica fiber of SPME was retracted back into the fiber holder assembly and removed automatically from the GC injection port. The chromatographic separation was performed in the normal manner, where the column temperature program was 30 °C for min1 and ramped at 10–220 °C min1 and the total run time was 21 min. The injector temperature was maintained at 240 °C throughout the entire chromatographic separation. The mass spectrometer was operated in the full scan mode between 50 and 550 amu, the ion source temperature was at 230 °C and the actual electron multiplier voltage was 1047 V. Initial identification of the unknowns produced by Gliocladium was made through library comparison using the NIST database. All chemical names in this study follow the nomenclature of this database. In all cases, kill control flasks were also analyzed and the compounds found therein were subtracted from those appearing in the pure Gliocladium or the co-cultures. Tentative identification of the fungal products was based on observed mass spectral data as compared to those in the NIST database. Final confirmatory identification was made for many of the compounds by comparing GC/MS data of authentic standards with the GC/MS data of the fungal products. The retention time of an authentic compound versus the fungal volatile was also measured. The experiments were repeated at least twice with comparable results. 2.7. Identification and quantitative analysis of hydrocarbons Sixteen commercial hydrocarbon standards were prepared at 10, 20, 30 and 40 lg/mL (ppm) concentrations respectively in
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culture medium (without microorganisms). Qualitative analysis of head space gases produced from the above standards was done with GC–MS–SPME as described for the analysis of the culture medium. The standard volatile hydrocarbons peaks were subtracted with the peaks of culture medium (without microorganisms) to know the absolute peaks of hydrocarbons coming from standards alone at the respective retention time and compared with NIST data base. The amount of volatile hydrocarbons produced by Gliocladium in culture medium (after subtracted with control) was performed. All the peaks were in the dynamic range of the instrument and there is no dead time loss in the peak intensity.
80.0
A
3. Results and discussion 3.1. Cellulose consumption and fungal biomass accumulation Pure cultures of Gliocladium and co-cultures with E. coli degraded 72% and 52% of the cellulose, respectively. Fungal biomass was higher in stationary phase of Gliocladium co-cultures than in pure cultures (Fig. 1A and B), which suggest that the stable mycelial mass is also a key component for the production of secondary metabolites (Benhamou and Chet, 1993). It has also suggested that shortage of easily accessible nutrients for microorganisms, especially for those living in soil and on plant surfaces, could result in explicit nutrient competition among microorganisms (Lewis and Papavizas, 1991).
Cellulose utilized (%)
70.0
3.2. Qualitative and quantitative production of fungal hydrocarbons
60.0 Control Mono-culture (62724) Mono-culture (1165) Mono-culture (62726) Co-culture (62724) Co-culture (1165) Co-culture (62726)
50.0 40.0 30.0 20.0 10.0 0.0 1
3
5
7
9 11 13 15 17 19 Days
14.0
B Dry mycelial wt. (g/L)
12.0 10.0
Mono-culture (62724) Mono-culture (1165) Mono-culture (62726) Co-culture (62724) Co-culture (1165) Co-culture (62726) Control
8.0 6.0 4.0 2.0 0.0 1
3
5
7
9
11 13 15 17 19
Days Fig. 1. Cellulose utilized (A) and biomass accumulation (B) in pure and co-cultures of Gliocladium species 62724; 1165 and 62726 grown on cellulose rich medium in shake flasks under microaerophilic conditions at 23 °C and pH 5.7.
In co-culture conditions, Gliocladium strains efficiently produced C6–C19 extracellular hydrocarbons while consuming cellulose a main source of food in culture medium. Hydrocarbon production varied with culture conditions and Gliocladium strain. Headspace gas analysis showed twice the number of hydrocarbons in Gliocladium strains co-cultured with E. coli than in pure Gliocladium culture (Table 1). These hydrocarbons from the co-culture were identified as benzene, heptane, 1-octene, octane, m-xylene, 3-methylnonane, dodecane, tridecane, hexadecane and nonadecane. Pure cultures of Gliocladium produced hexane, benzene, 3,4-dimethylhexane, 1-octene and m-xylene (Table 1). Head space analysis using GC–MS–SPME gave good quantitative performance of volatile hydrocarbons produced by Gliocladium fungi and has the potential to overcome many of the difficulties associated with conventional extraction methods (Langenfeld et al., 1996; Bland et al., 2001). It eliminates the use of extraction solvents, reduces sample preparation time, has excellent detection limits and identifies large numbers of volatile compounds in complex samples. In order to further quantify the concentration of hydrocarbons present in Gliocladium culture broth, Henry’s law constant (HLC) or air–water partition coefficient (Fig. 2A–L) was adopted. HLC influences the rate of evaporation of organic solutes from the culture supernatant, determines the head space gases, purge and trap analysis, and controls the effectiveness of gas stripping operations which are used to remove volatile organic solutes from culture broth (Sedlbauer et al., 2002). The head space gas analysis revealed that 0.001 and 0.002 mg L1 of hexane produced only in pure cultures of G. catenulatum (62724) and G. roseum (1165), respectively whereas, hexane was not produced in co-culture condition
Table 1 GC–MS–SPME analysis of Gliocladium species 62724; 1165 and 62726 co-cultured with E. coli and Gliocladium pure cultures grown on cellulose in shake flasks under microaerophilic conditions at 23 °C and pH 5.7. Compounds
Hexane Benzene Heptane 3,4-Dimethyl hexane 1-Octene Octane m-Xylene 3-Methyl nonane Dodecane Tridecane Hexadecane Nonadecane
Molar mass
Retention time (min)
62724
1165
62726
Mono-culture (mg/L)
Co-culture (mg/L)
Mono-culture (mg/L)
Co-culture (mg/L)
Mono-culture (mg/L)
Co-culture (mg/L)
86.2 78.0 100.0 114.2
2.00 2.57 2.97 4.01
0.0010 0.0248 0 0
0 0 0 0
0.0020 0.0227 0 0.0017
0 0.0231 0 0
0 0.0154 0 0
0 0.0601 0.0000 0
112.0 114.0 106.0 142.2 170.0 184.0 226.0 268.0
4.37 4.50 5.58 7.27 10.87 12.29 16.08 19.31
0.0034 0 0 0 0 0 0 0
0.0005 0 0 0 0.0519 0 0 0.0093
0 0 0.1161 0 0 0 0 0
0 0.0011 0.0262 0 0.0790 1.4394 0.2130 0.0034
0 0 0 0 0 0 0 0
0.0007 0 0 0.1046 0.0230 4.3598 0.1759 0
A. Ahamed, B.K. Ahring / Bioresource Technology 102 (2011) 9718–9722
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Fig. 2. Hydrocarbons (A–L) calculated using Henry’s law constant in total liquid culture ( ), in liquid at equilibrium ( ) and head space gases at equilibrium ( ) from the mono and co-cultures of Gliocladium species 62724; 1165 and 62726 grown on cellulose.
(Fig. 2A). However, benzene, 1-octene and m-xylene were produced in both pure Gliocladium and in co-cultures (Fig. 2B, E and G)
whereas, G. roseum (62726) co-cultured with E. coli produced 0.0601 mg L1 of benzene as compared to the pure culture
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(0.0154 mg L1). Considerably higher amounts of dodecane (0.079 mg L1) and hexadecane (0.213 mg L1) were produced in G. roseum (1165) co-cultured with E. coli (Fig. 2I). Whereas under co-culture conditions, 4.4 mg L1 of tridecane (Fig. 2J) was produced in G. roseum (62726) and 0.0093 mg L1 of nonadecane (Fig. 2L) was produced by G. catenulatum (62724). Overall, G. roseum 1165 cocultured with E. coli was highly capable of producing hydrocarbons on cellulose culture medium. However, for the first time Strobel et al. (2008), has reported 4 ppm of hydrocarbons inG. roseum (NRRL 50072). This fungal strain has been reassigned as A. sarcoides based on the DNA sequence analysis and comparative morphological studies (Stadler and Schulz, 2009; Strobel et al., 2010; Griffin et al., 2010). The difference in hydrocarbon production capabilities between these two strains are species specific. Stadler and Schulz (2009), have reported the production of saturated volatile hydrocarbons from Gliocladium fungi and suggested that these endophytes are deserve to be studied more intensively not only for volatiles but also other products of higher market value, such as innovative products for flavors, food and cosmetic industries. No single organism has been used so far in a commercial-scale biofuel plant with the aim of simultaneous degradation of cellulose and synthesis of hydrocarbon compounds. The discovery of such endophytic fungi, which can counter attack with its competing microorganism by releasing volatile toxic substances, might help us to understand the evolutionary aspect of chemical diversity. However, producing infrastructure ready biofuels directly from biomass materials yield a much lower process cost compared to processes involving both pretreatment and enzymatic hydrolysis before the actual biofuel production. Commercial production of these hydrocarbons needs much more attention towards fermentation technology, culture conditions, induction by chemical stress and genetic manipulations. The present finding further demonstrated the potential of using Gliocladium fungal species for producing biofuels in the future. 4. Conclusion The hydrocarbons produced by Gliocladium species especially in co-culture with E. coli are very similar to diesel fuel, and one can envision production of such hydrocarbons from cellulose; however, improvements in fermentation technology, culture
conditions, fortifying aromatic compounds in culture media and genetic manipulations will be necessary to achieve commercially viable production. Acknowledgements Funding for this study came from a Washington State STAR researcher Grant to Prof. Ahring (2008–2011). References Ahamed, A., Vermette, P., 2008. Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. Biochem. Eng. J. 40, 399–407. Ahamed, A., Vermette, P., 2009. Effect of culture medium composition on Trichoderma reesei’s morphology and cellulase production. Bioresour. Technol. 100, 5979–5987. Benhamou, N., Chet, I., 1993. Hyphal interactions between Trichoderma harzianum and Rhizoctonia solani: ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology 83, 1062–1071. Bland, J.M., Osbrink, W.L.A., Cornelius, M.L., Lax, A.R., Vigo, C.B., 2001. Solid-phase micro extraction for the detection of termite cuticular hydrocarbons. J. Cromatogr. A 932, 119–127. Griffin, M.A., Spakowicz, D.J., Gianoulis, T.A., Strobel, S.A., 2010. Volatile organic compound production by organisms in the genus Ascocoryne and a reevaluation of myco-diesel production by NRRL 50072. Microbiology 156, 3814–3829. Langenfeld, J.J., Hawthorne, S.B., Miller, D.J., 1996. Quantitative analysis of fuelrelated hydrocarbons in surface water and wastewater samples by solid-phase microextraction. Anal. Chem. 68, 144–155. Lewis, J.A., Papavizas, G.C., 1991. Biocontrol of plant diseases: the approach for tomorrow. Crop Prot. 10, 95–105. Sedlbauer, J., Bergin, G., Majer, V., 2002. Group contribution method for Henry’s law constant of aqueous hydrocarbons. AIChE J. 48, 2936–2959. Stadler, M., Schulz, B., 2009. High energy biofuel from endophytic fungi. Trends Plant Sci. 14, 353–355. Strobel, G., Knighton, B., Kluck, K., Ren, Y., Livinghouse, T., Griffin, M., Spakowicz, D., Sears, J., 2008. The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072). Microbiology 154, 3319–3328. Strobel, G., Tomsheck, A., Geary, B., Spakowicz, D., Strobel, S., Mattner, S., Mann, R., 2010. Endophyte Strain NRRL 50072 producing volatile organics is a species of Ascocoryne. Mycology 1, 187–194. Sunesson, A.L., Wouter, H.J.V., Nilsson, C.A., Goran, B., Barbro, A., Rolf, C., 1995. Identification of volatile metabolites from five fungal species cultivated on two media. Appl. Environ. Microbiol. 61, 2911–2918. Updegraff, D.M., 1969. Semi-micro determination of cellulose in biological materials. Anal. Biochem. 32, 420–424. Wackett, L.P., 2008. Microbial based motor fuels: science and technology. Microb. Biotechnol. 1, 211–225.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of hydrocarbon compounds by endophytic fungi Gliocladium species grown on cellulose Aftab Ahamed, Birgitte K. Ahring ⇑ Center for Bioproducts and Bioenergy, Bioproducts Sciences and Engineering Laboratory, Washington State University, Richland, WA 99354-1671, USA
a r t i c l e
i n f o
Article history: Received 31 March 2011 Received in revised form 5 July 2011 Accepted 13 July 2011 Available online 28 July 2011 Keywords: Biofuel Hydrocarbons Green energy Bio-products Endophyte
a b s t r a c t Endophytic fungi belonging to the genus Gliocladium are able to degrade plant cellulose and synthesize complex hydrocarbons under microaerophilic conditions. These fungi could thus be used to produce biofuels from cellulosics without the need for hydrolytic pretreatments. Gas chromatography–mass spectrometry–solid-phase micro-extraction (GC–MS–SPME) of head space gases from Gliocladium cultures demonstrated the production of C6–C19 hydrocarbons including hexane, benzene, heptane, 3,4dimethyl hexane, 1-octene, m-xylene, 3-methyl nonane, dodecane, tridecane, hexadecane and nonadecane directly from the cellulosic biomass. Hydrocarbon production was 100-fold higher in co-cultures of Gliocladium and Escherichia coli than in pure Gliocladium cultures. The dry mycelia weight is stable at stationary period in co-culture condition which may lead to synthesize more hydrocarbons. These fungi could potentially be developed into cost-effective biocatalysts for production of biofuels. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
2. Methods
Microbes synthesizing extracellular long-chain hydrocarbons are of interest since they could be exploited for biofuel production. Some fungal species have been shown to synthesize such hydrocarbons depending on environmental conditions and growth media (Sunesson et al., 1995; Wackett, 2008). For example, Gliocladium roseum (NRRL 50072) (now designated as Ascocoryne sarcoides (Stadler and Schulz, 2009; Strobel et al., 2010; Griffin et al., 2010)), which lives inside the Ulmo trees in the rain forests of Patagonia (Argentina and Chile) was able to directly degrade plant cellulose and synthesize a broad array of hydrocarbon compounds which are remarkably similar to diesel fuel (Strobel et al., 2008). In this fungus, hydrocarbon production appears to be a defense mechanism since the production of gases rich in hydrocarbons and hydrocarbon was stimulated by exposure to antibiotics (Strobel et al., 2008). The current study was undertaken to determine if co-cultivation with bacterium would be a means to increase hydrocarbon production by three Gliocladium species. Escherichia coli strain Nissle 1917 (DSM 6601) was chosen as the test bacterium because it is non-pathogenic, metabolically versatile and able to grow under the microaerophilic conditions required by the fungus.
2.1. Fungal and bacterial test strains
⇑ Corresponding author. Tel.: +1 509 372 7682; fax: +1 509 372 7690. E-mail addresses: [email protected], [email protected] (B.K. Ahring). URL: http://www.tricity.wsu.edu/bsel (B.K. Ahring). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.073
Gliocladium 62724, G. roseum 1165 and G. roseum 62726 were obtained from DSMZ (German collection of microorganisms and cell cultures, Inhoffenstrabe, Germany), ATCC (American type culture collection, Manassas, VA, USA) and from a private Danish collection at Copenhagen Institute of Technology at Aalborg University respectively. E. coli strain Nissle 1917 (DSM 6601) was obtained from ATCC. The freeze dried fungal spores were suspended in sterile 15% (v/v) glycerol, plated on potato dextrose agar (PDA, Difco Laboratories) and incubate at 23 °C for 18 days until good sporulation occurred. The spores were harvested by washing the petri plates with sterile water containing 1% Triton X-100 (Fisher Scientific, Orangeburg, NJ, USA). 2.2. Culture media Cellulose pre-culture medium (Ahamed and Vermette, 2008) consisted of: 10 g L1 avicel (Sigma–Aldrich, St. Louis, MO, USA), 5 g L1 corn steep liquor (Sigma–Aldrich, St. Louis, MO, USA), 1.5 g L1 proteose peptone (Fischer Biotech, Fair Lawn, NJ, USA), 0.5 g L1 yeast extract (Fischer Biotech, Fair Lawn, NJ, USA), 1.7 g L1 (NH4)2SO4, 2 g L1 KH2PO4, 0.3 g L1 CaCl22H2O, 0.3 g L1 MgSO47H2O, 0.005 g L1 FeSO47H2O, 0.0037 g L1 CoCl26H2O, 0.0016 g L1 MnSO4H2O, 0.0014 g L1 ZnSO47H2O. The composition of production medium was the same as that
A. Ahamed, B.K. Ahring / Bioresource Technology 102 (2011) 9718–9722
of the cellulose pre-culture medium, except that it contained 35 g L1 avicel, 10 g L1 corn steep liquor, 3 g L1 proteose peptone, 2 g L1 yeast extract, 2 g L1 (NH4)2SO4, 4 g L1 KH2PO4 and 0.2 ml L1 tween 80. The production medium was sterilized at 121 °C for 45 min.
ðCLÞ ðVLÞ CL : ðVLÞ þ ðH0 V g Þ ð1 þ H0 Þ
2.3. Inoculum and shake flask culture
ðH0 Þ ðCleÞ:
Concentrated aqueous spore inocula (106 mL1) of the Gliocladium strains preserved in 2-mL vials at 80 °C in 15% (v/v) glycerol were inoculated into modified 1000-mL graduated Erlenmeyer flasks containing 300 mL of cellulose pre-culture medium and incubated at 23 °C on a rotary shaking incubator with an agitation speed of 125 rpm. The initial pH was 5.7 and was not controlled during pre-culture. The flasks were sealed with a rubber stopper with two holes through which stainless steel tubes were inserted to allow collection of liquid and air samples. After 72 h of cultivation, the Gliocladium mycelium suspension from the pre-culture medium corresponding to 10% (v/v) of the total production medium volume was aseptically inoculated into two 500-ml flasks containing 250 mL of production medium. One of the flasks was also inoculate with 1.89 106 CFU mL1 of E. coli Nissle 1917. Microearophilic conditions were maintained in both flasks through hose cock clamp fixed on rubber tube with sterile air filter, turning these clamps on and off two times a day facilitate the microaerophilic conditions and the flasks were incubated at 23 °C for 20 days on a rotary shaker (125 rpm). Control flasks containing autoclaved cultures were also incubated. The shake flask culture experiments were carried out in duplicate. 2.4. Determination of residual cellulose and fungal dry biomass Cellulose content of Gliocladium fungal cultures were determined by a simplified version of the method of Updegraff (1969). Ten milliliter of the culture broth was centrifuged (3000g, for 20 min), the supernatant was removed, the pellet was suspended in acetic acid nitric acid reagent (3 mL: 150 mL of 80% acetic acid with 15 mL of pure nitric acid) and boiled for 30 min in a water bath. After cooling and centrifugation (3000g, for 20 min), the pellet was washed with distilled water (10 mL), and the residual cellulose was dried at 40 °C under reduced pressure until constant weight. Six replicate samples were measured and the average cellulose content was calculated. The dry fungal biomass was determined using the method developed by Ahamed and Vermette (2009). The mycelial weight was calculated as the difference between the total dry weight of the solids (comprising mycelium and residual cellulose) and that of the residual cellulose. 2.5. Dimensionless ‘‘Henry’s law constant’’ application Henry’s Law Constant (HLC) was applied to determine the concentration of volatiles in liquid and head space gases at equilibrium. The equation is as follows:
H0 ¼
H RT
H0 , Dimensionless Henry’s law constant; H, Henry’s law constant [atm – m3/mol]; R , Universal gas constant [8.20575 105 atmm3/mol K]; T, Temperature in kelvin. Equilibrium of air–water partitioning:
ðCLÞðVLÞ ¼ ðCleÞðVLÞ þ ðCgeÞðV g Þ CL, Concentration in liquid (mg/L); VL, Volume of liquid (L); Cle, Concentration in liquid at equilibrium; Cge, Concentration in head space gas at equilibrium; Vg, Volume of gas.
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Liquid phase partitioning:
Gas phase portioning:
ðH0 Þ ðCLÞ ð1 þ H0 Þ
2.6. Headspace gases analysis Ten milliliter of the culture was centrifuged at 10,000g for 20 min, and the supernatant was carefully removed with a sterilized glass pipette and used for the measurements of volatile hydrocarbons through GC/MS (7890A GC-system with 5975C inert XL E1/C1 MSD model # G3174A, Agilent Technologies, Wilmington, DE, USA) containing DB-5MS, non-polar, high temperature limit (450 °C), 30 m 0.250 lm 0.25 lm column. The CTC analysis CombiPAL robotic arm (G6500-CTC-LHS2.PAL system-CH001210757, CTC Analytics AG, Zwingen, Switzerland) was connected to GC/MS for auto sampling system. All the samples were sequenced and programmed for incubation at 50 °C in 20 mL head space screw top clear glass vials (VWR 5188-2753) with an agitation speed of 500 rpm and the total extraction time was 2 min. The volatiles from head space gases were adsorbed to sterilized Divinylbenzene/Carboxen/Polymethylsiloxane stableflex silica fiber (Gray, Supelco Cat. 57284U, Bellefonte, PA, USA) present in solid phase micro extraction (SPME) syringe (Supelco, Cat. 57315, Sigma–Aldrich) connected to CTC-PAL robotic arm for automatic insertion. SPME allows extraction and concentration to be performed in a single step and direct analyte transfer to GC by thermal desorption inside a heated injection port for 1 min and swept into the column by ultra pure helium as carrier gas and the average velocity was maintained at 42.651 cm/s, the analytes were trapped and focused at the column inlet and the stable flex silica fiber of SPME was retracted back into the fiber holder assembly and removed automatically from the GC injection port. The chromatographic separation was performed in the normal manner, where the column temperature program was 30 °C for min1 and ramped at 10–220 °C min1 and the total run time was 21 min. The injector temperature was maintained at 240 °C throughout the entire chromatographic separation. The mass spectrometer was operated in the full scan mode between 50 and 550 amu, the ion source temperature was at 230 °C and the actual electron multiplier voltage was 1047 V. Initial identification of the unknowns produced by Gliocladium was made through library comparison using the NIST database. All chemical names in this study follow the nomenclature of this database. In all cases, kill control flasks were also analyzed and the compounds found therein were subtracted from those appearing in the pure Gliocladium or the co-cultures. Tentative identification of the fungal products was based on observed mass spectral data as compared to those in the NIST database. Final confirmatory identification was made for many of the compounds by comparing GC/MS data of authentic standards with the GC/MS data of the fungal products. The retention time of an authentic compound versus the fungal volatile was also measured. The experiments were repeated at least twice with comparable results. 2.7. Identification and quantitative analysis of hydrocarbons Sixteen commercial hydrocarbon standards were prepared at 10, 20, 30 and 40 lg/mL (ppm) concentrations respectively in
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culture medium (without microorganisms). Qualitative analysis of head space gases produced from the above standards was done with GC–MS–SPME as described for the analysis of the culture medium. The standard volatile hydrocarbons peaks were subtracted with the peaks of culture medium (without microorganisms) to know the absolute peaks of hydrocarbons coming from standards alone at the respective retention time and compared with NIST data base. The amount of volatile hydrocarbons produced by Gliocladium in culture medium (after subtracted with control) was performed. All the peaks were in the dynamic range of the instrument and there is no dead time loss in the peak intensity.
80.0
A
3. Results and discussion 3.1. Cellulose consumption and fungal biomass accumulation Pure cultures of Gliocladium and co-cultures with E. coli degraded 72% and 52% of the cellulose, respectively. Fungal biomass was higher in stationary phase of Gliocladium co-cultures than in pure cultures (Fig. 1A and B), which suggest that the stable mycelial mass is also a key component for the production of secondary metabolites (Benhamou and Chet, 1993). It has also suggested that shortage of easily accessible nutrients for microorganisms, especially for those living in soil and on plant surfaces, could result in explicit nutrient competition among microorganisms (Lewis and Papavizas, 1991).
Cellulose utilized (%)
70.0
3.2. Qualitative and quantitative production of fungal hydrocarbons
60.0 Control Mono-culture (62724) Mono-culture (1165) Mono-culture (62726) Co-culture (62724) Co-culture (1165) Co-culture (62726)
50.0 40.0 30.0 20.0 10.0 0.0 1
3
5
7
9 11 13 15 17 19 Days
14.0
B Dry mycelial wt. (g/L)
12.0 10.0
Mono-culture (62724) Mono-culture (1165) Mono-culture (62726) Co-culture (62724) Co-culture (1165) Co-culture (62726) Control
8.0 6.0 4.0 2.0 0.0 1
3
5
7
9
11 13 15 17 19
Days Fig. 1. Cellulose utilized (A) and biomass accumulation (B) in pure and co-cultures of Gliocladium species 62724; 1165 and 62726 grown on cellulose rich medium in shake flasks under microaerophilic conditions at 23 °C and pH 5.7.
In co-culture conditions, Gliocladium strains efficiently produced C6–C19 extracellular hydrocarbons while consuming cellulose a main source of food in culture medium. Hydrocarbon production varied with culture conditions and Gliocladium strain. Headspace gas analysis showed twice the number of hydrocarbons in Gliocladium strains co-cultured with E. coli than in pure Gliocladium culture (Table 1). These hydrocarbons from the co-culture were identified as benzene, heptane, 1-octene, octane, m-xylene, 3-methylnonane, dodecane, tridecane, hexadecane and nonadecane. Pure cultures of Gliocladium produced hexane, benzene, 3,4-dimethylhexane, 1-octene and m-xylene (Table 1). Head space analysis using GC–MS–SPME gave good quantitative performance of volatile hydrocarbons produced by Gliocladium fungi and has the potential to overcome many of the difficulties associated with conventional extraction methods (Langenfeld et al., 1996; Bland et al., 2001). It eliminates the use of extraction solvents, reduces sample preparation time, has excellent detection limits and identifies large numbers of volatile compounds in complex samples. In order to further quantify the concentration of hydrocarbons present in Gliocladium culture broth, Henry’s law constant (HLC) or air–water partition coefficient (Fig. 2A–L) was adopted. HLC influences the rate of evaporation of organic solutes from the culture supernatant, determines the head space gases, purge and trap analysis, and controls the effectiveness of gas stripping operations which are used to remove volatile organic solutes from culture broth (Sedlbauer et al., 2002). The head space gas analysis revealed that 0.001 and 0.002 mg L1 of hexane produced only in pure cultures of G. catenulatum (62724) and G. roseum (1165), respectively whereas, hexane was not produced in co-culture condition
Table 1 GC–MS–SPME analysis of Gliocladium species 62724; 1165 and 62726 co-cultured with E. coli and Gliocladium pure cultures grown on cellulose in shake flasks under microaerophilic conditions at 23 °C and pH 5.7. Compounds
Hexane Benzene Heptane 3,4-Dimethyl hexane 1-Octene Octane m-Xylene 3-Methyl nonane Dodecane Tridecane Hexadecane Nonadecane
Molar mass
Retention time (min)
62724
1165
62726
Mono-culture (mg/L)
Co-culture (mg/L)
Mono-culture (mg/L)
Co-culture (mg/L)
Mono-culture (mg/L)
Co-culture (mg/L)
86.2 78.0 100.0 114.2
2.00 2.57 2.97 4.01
0.0010 0.0248 0 0
0 0 0 0
0.0020 0.0227 0 0.0017
0 0.0231 0 0
0 0.0154 0 0
0 0.0601 0.0000 0
112.0 114.0 106.0 142.2 170.0 184.0 226.0 268.0
4.37 4.50 5.58 7.27 10.87 12.29 16.08 19.31
0.0034 0 0 0 0 0 0 0
0.0005 0 0 0 0.0519 0 0 0.0093
0 0 0.1161 0 0 0 0 0
0 0.0011 0.0262 0 0.0790 1.4394 0.2130 0.0034
0 0 0 0 0 0 0 0
0.0007 0 0 0.1046 0.0230 4.3598 0.1759 0
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Fig. 2. Hydrocarbons (A–L) calculated using Henry’s law constant in total liquid culture ( ), in liquid at equilibrium ( ) and head space gases at equilibrium ( ) from the mono and co-cultures of Gliocladium species 62724; 1165 and 62726 grown on cellulose.
(Fig. 2A). However, benzene, 1-octene and m-xylene were produced in both pure Gliocladium and in co-cultures (Fig. 2B, E and G)
whereas, G. roseum (62726) co-cultured with E. coli produced 0.0601 mg L1 of benzene as compared to the pure culture
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(0.0154 mg L1). Considerably higher amounts of dodecane (0.079 mg L1) and hexadecane (0.213 mg L1) were produced in G. roseum (1165) co-cultured with E. coli (Fig. 2I). Whereas under co-culture conditions, 4.4 mg L1 of tridecane (Fig. 2J) was produced in G. roseum (62726) and 0.0093 mg L1 of nonadecane (Fig. 2L) was produced by G. catenulatum (62724). Overall, G. roseum 1165 cocultured with E. coli was highly capable of producing hydrocarbons on cellulose culture medium. However, for the first time Strobel et al. (2008), has reported 4 ppm of hydrocarbons inG. roseum (NRRL 50072). This fungal strain has been reassigned as A. sarcoides based on the DNA sequence analysis and comparative morphological studies (Stadler and Schulz, 2009; Strobel et al., 2010; Griffin et al., 2010). The difference in hydrocarbon production capabilities between these two strains are species specific. Stadler and Schulz (2009), have reported the production of saturated volatile hydrocarbons from Gliocladium fungi and suggested that these endophytes are deserve to be studied more intensively not only for volatiles but also other products of higher market value, such as innovative products for flavors, food and cosmetic industries. No single organism has been used so far in a commercial-scale biofuel plant with the aim of simultaneous degradation of cellulose and synthesis of hydrocarbon compounds. The discovery of such endophytic fungi, which can counter attack with its competing microorganism by releasing volatile toxic substances, might help us to understand the evolutionary aspect of chemical diversity. However, producing infrastructure ready biofuels directly from biomass materials yield a much lower process cost compared to processes involving both pretreatment and enzymatic hydrolysis before the actual biofuel production. Commercial production of these hydrocarbons needs much more attention towards fermentation technology, culture conditions, induction by chemical stress and genetic manipulations. The present finding further demonstrated the potential of using Gliocladium fungal species for producing biofuels in the future. 4. Conclusion The hydrocarbons produced by Gliocladium species especially in co-culture with E. coli are very similar to diesel fuel, and one can envision production of such hydrocarbons from cellulose; however, improvements in fermentation technology, culture
conditions, fortifying aromatic compounds in culture media and genetic manipulations will be necessary to achieve commercially viable production. Acknowledgements Funding for this study came from a Washington State STAR researcher Grant to Prof. Ahring (2008–2011). References Ahamed, A., Vermette, P., 2008. Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. Biochem. Eng. J. 40, 399–407. Ahamed, A., Vermette, P., 2009. Effect of culture medium composition on Trichoderma reesei’s morphology and cellulase production. Bioresour. Technol. 100, 5979–5987. Benhamou, N., Chet, I., 1993. Hyphal interactions between Trichoderma harzianum and Rhizoctonia solani: ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology 83, 1062–1071. Bland, J.M., Osbrink, W.L.A., Cornelius, M.L., Lax, A.R., Vigo, C.B., 2001. Solid-phase micro extraction for the detection of termite cuticular hydrocarbons. J. Cromatogr. A 932, 119–127. Griffin, M.A., Spakowicz, D.J., Gianoulis, T.A., Strobel, S.A., 2010. Volatile organic compound production by organisms in the genus Ascocoryne and a reevaluation of myco-diesel production by NRRL 50072. Microbiology 156, 3814–3829. Langenfeld, J.J., Hawthorne, S.B., Miller, D.J., 1996. Quantitative analysis of fuelrelated hydrocarbons in surface water and wastewater samples by solid-phase microextraction. Anal. Chem. 68, 144–155. Lewis, J.A., Papavizas, G.C., 1991. Biocontrol of plant diseases: the approach for tomorrow. Crop Prot. 10, 95–105. Sedlbauer, J., Bergin, G., Majer, V., 2002. Group contribution method for Henry’s law constant of aqueous hydrocarbons. AIChE J. 48, 2936–2959. Stadler, M., Schulz, B., 2009. High energy biofuel from endophytic fungi. Trends Plant Sci. 14, 353–355. Strobel, G., Knighton, B., Kluck, K., Ren, Y., Livinghouse, T., Griffin, M., Spakowicz, D., Sears, J., 2008. The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072). Microbiology 154, 3319–3328. Strobel, G., Tomsheck, A., Geary, B., Spakowicz, D., Strobel, S., Mattner, S., Mann, R., 2010. Endophyte Strain NRRL 50072 producing volatile organics is a species of Ascocoryne. Mycology 1, 187–194. Sunesson, A.L., Wouter, H.J.V., Nilsson, C.A., Goran, B., Barbro, A., Rolf, C., 1995. Identification of volatile metabolites from five fungal species cultivated on two media. Appl. Environ. Microbiol. 61, 2911–2918. Updegraff, D.M., 1969. Semi-micro determination of cellulose in biological materials. Anal. Biochem. 32, 420–424. Wackett, L.P., 2008. Microbial based motor fuels: science and technology. Microb. Biotechnol. 1, 211–225.