MURDOCH RESEARCH REPOSITORY

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1 MURDOCH RESEARCH REPOSITORY This is the author s final version of the work, as accepted for publication following peer review but without the publisher s layout or pagination. The definitive version is available at Lever, M., Ho, G. and Cord-Ruwisch, R. (2013) Simplifying cellulase production by using environmental selection pressures and recycling substrate. Environmental Technology, 34 (4), pp Copyright: 2012 Taylor & Francis. It is posted here for your personal use. No further distribution is permitted.

2 Title: Simplifying ethanol from cellulose by using environmental selection pressures and recycling substrate Journal: Environmental Technology Date: 5 October 2011 Authors: Corresponding author Mitchell Lever School of Environmental Science, Murdoch University, South St Murdoch, 6150, Western Australia Tel ; fax: address: mlever@westnet.com.au Co-authors Goen Ho School of Environmental Science Murdoch University, South St Murdoch, 6150, Western Australia address: g.ho@murdoch.edu.au Ralf Cord-Ruwisch School of Biological Sciences and Biotechnology Murdoch University, South St Murdoch, 6150, Western Australia address: r.cord-ruwisch@murdoch.edu.au 1

3 Simplifying ethanol from cellulose by using environmental selection pressures and recycling substrate Mitchell Lever a,, Goen Ho a, Ralf Cord-Ruwisch b a School of Environmental Science, Murdoch University, South St, Murdoch, 6150, Western Australia b School of Biological Sciences and Biotechnology, Murdoch University, South St, Murdoch, 6150, Western Australia Abstract The production of crude cellulase in solid-state fermentation was simplified by using environmental selection pressures as an alternative to specialised strains, and by re-using cellulose to ethanol fermentation substrate for cellulase production. The performance of wild strains of Trichoderma viride isolated from wheat and bark were found not to differ significantly from a cellulase enhanced strain of Trichoderma reesei. The filter paper activity of a strain of Aspergillus niger isolated in the laboratory was more than two times higher than the specialised T. reesei at ten days. Activities of enrichment cultures obtained from compost and horse manure were more than double T. reesei after 10 days. Using the solid residue of previous simultaneous saccharification and fermentations of wheat straw to ethanol as substrate for cellulase production brought forward the onset of cellulase production and increased cellulase levels by 50%. The findings may help to simplify the on-site manufacture of cellulase for cellulose to ethanol conversion and reduce the amount of substrate required for the overall process. Keywords: cellulase, fermentation, solid-state, Trichoderma reesei, Trichoderma viride Introduction Ethanol from cellulose is of interest for a number of reasons. Firstly, cellulose is the most abundant organic compound on earth. Secondly, there is an abundance and diversity of common wastes containing cellulose which, if used as ethanol feedstock, could eliminate the need for the land, energy and resources required to grow a specialised crop. Such waste streams include paper, cardboard, straw, wood, corn stover, sugarcane bagasse and municipal solid waste. Finally, cellulosic crops can be grown with limited inputs on land which is not suitable for food production, which brings the additional benefits of remediating degraded land and sequestering carbon [1]. The cost of commercial cellulase preparations is often cited as an economic barrier to cellulose ethanol production [2, 3, 4]. In addition to economic considerations, commercial cellulase production is energy intensive. According to one report, cellulase production requires a significantly higher energy input than Corresponding author. Tel ; fax: address: m.lever@murdoch.edu.au 2

4 the cellulose to ethanol conversion process itself [3]. It is therefore desirable to consider alternative methods of cellulase production which are less complex and hence use less energy. In a previous study the feasibility of eliminating some of the steps involved in commercial cellulase production (purification, concentration, addition of buffers, stabilisers and preservatives, freeze drying and packaging) by making and using a crude unprocessed cellulase via solid-state fermentation at the site of ethanol production was demonstrated [5]. In this study it is attempted to further simplify the cellulose to ethanol process by the use of environments selective for the growth of high cellulase producers, as an alternative to specialised strains maintained in culture collections. Most cellulase production for research or commercial purposes involves the use of hypercellulolytic fungi which have been developed by traditional or molecular methods. This practice can be associated with a number of complexities, costs, energy expenditures and technical difficulties. Firstly there are those which relate to the infrastructure required to carry out strain development and maintain culture collections. Secondly there are on-site issues related to the maintenance of the chosen micro-organism in pure culture over long periods. Modified strains have been shown to be less robust than their wild ancestors, requiring great care with inoculum preparation [6], and cellulase activity can reduce over time. Further, there are labour, energy and technical problems associated with sterilising substrates and trying to maintain sterile conditions during solid-state fermentation at scale-up [7]. The literature on mixed culture solid-state fermentation shows varying results, with some studies showing synergistic gains in cellulase production compared to monocultures [8, 9], and others showing the reverse [10]. While it is known that in nature cellulolytic microbes act in naturally selected communities, the mixed-culture studies generally involve researchers selecting the species to be used and growing them in pure culture under sterile conditions. Measurement of cellulase activities of undefined, naturally occurring communities has not been reported. It was hypothesised that, by deliberately incorporating the use of such environments selective for the growth of high cellulase producing microorganisms, high enough cellulase activities for the production of ethanol from cellulose might be obtained. 3

5 Materials & Methods Materials The substrate for solid-state fermentation was wheat straw ground and sieved through a 1.8 mm mesh. In two fermentations the solid residue from previous simultaneous saccharification and fermentations of (similarly treated) wheat straw to ethanol (detailed in [5]) was used. The residue was dried prior to reuse. Substrates were moistened with Toyama s mineral salt solution [11] of composition (g/l): KH 2 PO 4, 3; (NH 4 ) 2 SO 4, 10; MgSO 4.7H 2 O, 0.5; CaCl 2.2H 2 O, 0.5, added in the ratio 2 ml:1 g dry substrate. In addition, for all fermentations wheat bran and kelp powder were added at 10% and 2% dry substrate weight respectively and thoroughly mixed. Trichoderma reesei CBS (QM 9123) supplied from the Centraalbureau voor Schimmelcultures culture collection (Netherlands), described as a cellulase enhanced mutant derived from QM6 was used as the hypercellulolytic strain. Three strains of Trichoderma viride were obtained from the West Australian Culture Collection at the WA Department of Agriculture. The strains were WAC 7797 (isolated from wheat), WAC (isolated from plasterboard) and WAC (isolated from the bark of Eucalyptus viminatus). A wild strain of Aspergillus niger, isolated in the laboratory, was also used as an inoculant and measured for cellulase activity. Analytical Methods Fermented substrate was aseptically removed from the top of the fermenting bed at regular intervals for cellulase assay (which was verified as a valid method in a previous study [5]). Crude unprocessed cellulase solution was extracted by adding deionised water to the removed fermented substrate in 250 ml Schott bottles and shaking at 200 rpm for one hour at 20 C in an orbital shaker. The ratio of deionised water added for extraction to moist fermented substrate was kept constant at 100 ml : 24 g, (equivalent to a ratio of 12.5 ml : 1 g initial dry substrate, neglecting fermentation losses). The mixture was decanted into 40 ml lots, centrifuged at 2500 rpm for 10 min and the supernatant was removed and frozen for subsequent cellulase assay. 4

6 Cellulase activity was measured by the IUPAC filter paper assay [12] with the variations that 1 ml of cellulase extract was added to 2 ml citrate buffer ph 4.8, instead of adding 0.5 ml extract to 1mL buffer, and actual glucose was measured instead of reducing sugars by the DNS method. Glucose measurement was with a YSI 2700 Select Biochemistry Analyser (John Morris Scientific, Perth). An average of two readings per sample was taken. One FPU is defined as the amount of enzyme activity which releases 1 µmol/min of glucose. Units are reported in FPU/gDS (filter paper units per gram of initial dry substrate). The laboratory isolate was identified by DNA sequencing carried out by Murdoch University Plant Pathology Laboratory using the methods detailed in [13]. Procedure Inoculation of all pure cultures was carried out by growing the strains on potato-dextrose agar in Petri dishes at 30 C for one week, cutting half a plate into ca. 5 mm squares and thoroughly mixing it into the substrate/nutrient mix. Enrichment cultures were obtained by adding 20 g of wet compost or horse manure to 200 ml of mineral salt solution, placing the mixture in an orbital shaker at 200 rpm for 30 minutes, and inoculating solid-state fermentations with 25 ml of supernatant. Solid-state fermentations were carried out in 500 ml Schott bottles with cotton bungs covered with aluminium foil. 50 g ground wheat straw was mixed with 100 ml nutrient solution (plus bran & kelp). For later fermentations (Figures 4 and 5) the quantities were reduced to 20 g and 40 ml because this was measured to produce an increase in cellulase production (possibly due to increased air availability). In the case of the enrichment cultures, to keep the moisture content constant, 75 ml of nutrient solution was used and 25 ml of compost or horse manure inoculum was added. All fermentations were incubated at 30 ºC for 12 to 18 days. Solid-state fermentations were carried out using T.reesei, three native strains of T.viride, a laboratory isolate of A. niger and enrichment cultures from compost and horse manure. Non-autoclaved, noninoculated controls were carried out to check for the effects of the natural contamination of wheat straw alone. Fermentations were also carried out using the dried solid residue from previous wheat straw to ethanol fermentations under non-sterile and sterile conditions. For sterile conditions substrates and 5

7 nutrient mixes were autoclaved (121 C, 25 min), sampling and inoculation were carried out using sterile technique (Figures 1,2,3). When non-sterile conditions were under test autoclaving and the use of sterile technique was omitted (Figure 4, part of Figure 5). Fermentations were carried out in duplicate with the following exceptions. Fermentations using compost and horse manure were carried out in triplicate. The fermentation utilising the laboratory isolate of A. niger was not duplicated. Results & Discussion T.viride strains vs T. reesei Solid-state fermentations were run with T. reesei and each of the three strains of T.viride (Figure 1). After 10 days of fermentation, the highest level of cellulase activity of the T. viride strains was exhibited by WAC 7797, followed by WAC and WAC The order is consistent with the fact that the strains were isolated respectively from wheat, Eucalyptus bark and plasterboard which are in order from most similar to least similar to wheat straw, the substrate employed for solid-state fermentation. Analysis of variance showed the specialised T. reesei and the strains of T. viride isolated from wheat and bark outperformed the T. viride strain isolated from plasterboard moulding at a 5% level of significance. Although one of the T. reesei fermentations demonstrated the highest cellulase production (2.1 FPU/gDS) of all the isolates, due to a wide variation between the two trials at 14 days, overall T. reesei did not demonstrate significantly better performance than the native T. viride species isolated from wheat and bark. It is known that modified strains can be less robust than wild types (Szakacs et al. 2006) and their cellulase activity can decrease over time. Although T. reesei QM 9123 was sold as a cellulase enhanced mutant, in these trials it did not show improved cellulase performance and in fact showed reduced consistency compared to the wild strains of T.viride. 6

8 Laboratory isolate of A. niger A solid-state fermentation inoculated with Aspergillus niger, isolated as a visibly distinct contaminant (white mycelium with black spores tending to dominate the fermentation over time) from a previous solid-state fermentation inoculated with T. reesei was carried out (Figure 2). At 10 and 18 days cellulase production was higher than the T. reesei strain. At 14 days, due to the high variation between the two T. reesei trials, there was no significant difference in maximum cellulase production between the wild strain of A. niger isolated in the laboratory and T. reesei. A. niger is a well-known high cellulase producing fungus, commonly found as a contaminant in the laboratory. It is likely that the environment in which this isolate was initially noticed (i.e. a visible contaminant of earlier solid-state fermentations) is related to its high level of cellulase activity, as was the case with the strains of T.viride tested. An autoclaved, nutrient enriched bed of moistened ground wheat straw on which the cellulolytic T. reesei was growing may have provided an environment selective for the growth of high cellulase producing species. Enrichment Cultures Solid-state fermentations were inoculated with enrichments from compost and horse manure. Enrichment cultures generally displayed higher cellulase activities than cultures inoculated with T. reesei (Figure 3), T. viride (Figure 1) and the controls (Figure 3). The controls indicate cellulase production by the cultures present in the wheat straw initially with no inoculant added. Analysis of variance at 10 days (Figure 3) showed differences were significant at p = The average of the two enrichment cultures was approximately 140 % higher than the T. reesei fermentations. The enrichments were chosen from fresh, moist, aerobic environments rich in cellulose, nutrients and microbial populations. Inoculating from these environments into a nutrient enriched and moistened bed of wheat straw would probably have further enhanced the selection pressures in favour of high cellulase producers. This combined with the aforementioned possible limitations to the performance of specialised mutant strains over time may explain why the naturally enriched fermentations exhibited superior 7

9 cellulase activity at 10 days. The variations after 10 days do not significantly enhance cellulase activity suggesting there may be no point continuing the fermentations beyond 10 days in practice. While the levels of cellulase activity achieved are lower than those generally reported in the literature, which can typically be a factor of 10 higher (see [5], Table 1), the results support the hypothesis that natural environmental selection pressures may be used to enhance cellulase activity. Factors which may account for the differences in activity achieved here and elsewhere include the type and degree of pretreatment of substrate, the fermenting organisms used, nutrient mixes employed and the fermentation conditions themselves. Higher attained cellulase activities generally correspond to more energy intensive and complex methods. Also, higher cellulase producing mutants have been developed since the strain employed here. Nevertheless, the range of cellulase activities obtained in this study are similar to those obtained in a previous study in which it was shown that such levels are sufficient for the production of ethanol (Lever et al. 2010). Ethanol fermentation solid residue A potential method for reducing the total amount of straw required for an on-site cellulase production process is to re-use the spent lignocellulosic material from the cellulose to ethanol fermentation as substrate for cellulase production. Since simple sugars and easily accessible cellulose are consumed in the cellulose to ethanol fermentation it was thought cellulase production may commence sooner and reach higher levels if carried out on the used substrate. New solid-state fermentations were carried out in duplicate using dried solid residue from previous simultaneous saccharification and fermentations of wheat straw to ethanol. New controls were run on fresh wheat straw. Non-sterile conditions were used by omitting autoclaving of fresh and recycled substrates. Maximum cellulase levels were approximately 50% higher and the onset of cellulase production was more rapid on the recycled substrate compared to the controls (Figure 4). Analysis of variance showed after 4 and 8 days differences were significant at 1% and 5% respectively. Differences disappeared at 12 days. 8

10 The amount of solid residue remaining at the end of cellulose to ethanol fermentations was consistently measured to be close to half the starting amount. The cellulase activities attained on the recycled substrate were two to five times higher than the levels used in the previous study to produce ethanol from wheat straw [5]. It follows that the ethanol fermentation solid residue can provide sufficient substrate for the production of all the cellulase needed for that fermentation. Therefore, by re-using the solid residue of wheat straw to ethanol fermentation as substrate for cellulase production, not only might cellulase levels be higher and attained faster, but the need for additional substrate for cellulase production might be eliminated (with the exception of start-up). Hence recycling used solids from cellulose to ethanol fermentations could be an effective strategy to reduce the amount of wheat straw required for the overall cellulose to ethanol conversion process. To test the effects of sterilising the recycled substrate, fermentations on unsterilised recycled substrate were compared with fermentations on sterilised recycled substrate (Figure 5). The fermentation with sterilised substrate had a higher initial rate of cellulase production but after 8 days the fermentation using the unsterilised substrate achieved approximately 25% higher cellulase activity. Analysis of variance showed the differences were significant at 1% and 5% for 4 and 8 days respectively. A possible explanation for these results is that initially T. reesei grew faster on the sterilised substrate due to a lack of competition. After four to eight days synergistic microbial communities may have formed on the unsterilised substrate such that growth and cellulase production were enhanced by the additional microbes present. These findings on recycled substrate support the previous findings from the growth of wild strains and impure enrichment cultures, suggesting that microbes and microbial communities selected by the environment may enhance cellulase production. 9

11 Conclusion The use of natural environments selective for the growth of high cellulase producers enabled the achievement of cellulase levels similar to or higher than those obtained with a specialised strain in pure culture, and high enough for the production of ethanol from cellulose. The findings suggests aerobic, cellulose rich environments may be used to enhance cellulase production as an alternative to using specialised strains maintained in pure culture under aseptic conditions, which may present difficulties for scale-up. The possibilities demonstrated were: inoculating with native cellulolytic organisms isolated from environments similar to the substrate employed, inoculating fermentations with undefined enrichment cultures from compost and horse manure, and the use of non-sterile fermentation conditions. It was also shown that re-using the solid-residue from the cellulose to ethanol fermentation as substrate for cellulase production provided the dual benefits of enhancing cellulase production and reducing the amount of substrate required for the overall process. More detailed studies would help determine if these possibilities can be converted to a simplified or more energy efficient practical process. 10

12 References [1] D. Tilman, J. Hill, and C. Lehman, Carbon negative biofuels from low input high diversity grassland biomass, Science 314 (2006), pp [2] R. Sukumaran, R. Singhania, G. Mathew, and A. Pandey, Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production, Renewable Energy 34 (2) (2009): pp [3] H. MacLean and S. Spatari, The contribution of enzymes and process chemicals to the life cycle of ethanol, Environmental Research Letters 4 (2009), pp [4] M. Galbe and G. Zacchi G, Pretreatment of lignocellulosic materials for efficient bioethanol production, Advances in Biochemical Engineering and Biotechnology 108 (2007), pp [5] M. Lever, G. Ho, and R. Cord-Ruwisch, Ethanol from lignocellulose using crude unprocessed cellulase from solid-state fermentation, Bioresource Technology 101(18) (2010), pp [6] G. Szakacs, R. Tengerdy, and V. Nagy, Cellulases, in Enzyme Technology, A. Pandey, C. Webb, C. Soccol, and C. Larroche, eds., Asiatech Publisher, Delhi, (2006) [7] B. Lonsane, G. Saucedo-Castenada, M. Raimbault, S. Roussos, G. Viniegra-Gonzalez, and N. Ghildyal, Scale-up strategies for solid-state fermentation, Process Biochemistry 27 (1992), pp [8] M. Muhannad, W. Yusoff, O. Omar, and J. Kader, Synergism of cellulase enzymes in mixed culture solid-substrate fermentation, Biotechnology Letters 23 (2001), pp [9] M. Gutierrez-Correa and R. Tengerdy, Production of cellulase on sugar-cane bagasse by fungal mixed culture solid substrate fermentation, Biotechnology Letters 19 (1997), pp [10] Y. Yang, B. Wang, Q. Wang, L. Xiang, and C. Duan, Research on solid-state fermentation on rice chaff with a microbial consortium, Colloids and surfaces B: Biointerfaces 34 (2004), pp [11] N. Toyama and K. Ogawa, in International Course on Biochemical Engineering and Bioconversion, T. Ghose, ed., New Delhi: Biochemical Engineering Research Centre, IIT; (1977) pp [12] T. Ghose, Measurement of Cellulase Activities. Pure and Applied Chemistry 59(2) (1987), pp [13] T. Burgess, P. Barber, and G. Hardy, Botryosphaeria spp. associated with eucalypts in Western Australia, including the description of Fusicoccum macroclavatum sp. nov, Australasian Plant Pathology 34 (2005), pp

13 Figure Captions Figure 1. Comparison of cellulase production in solid-state fermentation by T. reesei and three Western Australian strains of T.viride. Strain WAC 7797 was isolated from wheat, WAC from plasterboard and WAC from the bark of Eucalyptus viminalus. Fermentations were run in duplicate. Standard error bars shown. Figure 2. Comparison of cellulase production in solid-state fermentation by T. reesei and a laboratory isolate of A. niger. T. reesei fermentations run in duplicate. A. Niger profile based on a single fermentation. Figure 3. A comparison of cellulase production in solid-state fermentation by T.reesei with enrichment cultures inoculated with compost (EC) and horse manure (EH) liquor. T. reesei fermentations and controls were run in duplicate. Enrichment cultures were run in triplicate. Substrates and nutrients were autoclaved prior to inoculation, except in the case of the controls. Figure 4. Comparison of cellulase production on ground wheat straw (GWS) and ethanol fermentation solid residue (EFSR). Fermentations were carried out in duplicate, inoculated with T. reesei. Figure 5. Comparison of cellulase production on the solid residue of wheat straw to ethanol fermentation under sterile and non-sterile conditions. Fermentations were inoculated with T. reesei and run in duplicate. For sterile fermentations substrate and nutrients were autoclaved and aseptic technique was employed when inoculating and sampling. For non-sterile fermentations these steps were omitted. 12

14 Figure 1. Comparison of cellulase production in solid-state fermentation by T. reesei, three Western Australian strains of T. viride and a laboratory isolate of A. niger. Strain WAC 7797 was isolated from wheat, WAC from plasterboard and WAC from the bark of Eucalyptus viminalus. T. reesei and T. viride fermentations were run in duplicate. A. Niger profile based on a single fermentation. Standard error bars shown.

15 Figure 2. A comparison of cellulase production in solid-state fermentation by T. reesei with enrichment cultures inoculated with compost (EC) and horse manure (EH) liquor. T. reesei fermentations and controls were run in duplicate. Enrichment cultures were run in triplicate. Substrates and nutrients were autoclaved prior to inoculation, except in the case of the controls.

16 Figure 3. Comparison of cellulase production on ground wheat straw (GWS) and ethanol fermentation solid residue (EFSR). Fermentations were carried out in duplicate, inoculated with T. reesei.

17 Figure 4. Comparison of cellulase production on the solid residue of wheat straw-to-ethanol fermentation under sterile and non-sterile conditions. Fermentations were inoculated with T. reesei and run in duplicate. For sterile fermentations substrate and nutrients were autoclaved and aseptic technique was employed when inoculating and sampling. For non-sterile fermentations these steps were omitted.