Development of Bioconversion Techniques for Irish Grown Willow, Miscanthus and Hemp Emma Smullen1, John Finnan2, David Dowling1 and Patricia Mulcahy1 Department of Science and Health, Institute of Technology, Kilkenny Road, Carlow, Ireland. 2 Teagasc, Crop Research Centre, Oakpark, Carlow, Ireland. Corresponding Author Email Address:
[email protected]
1 EnviroCORE,
Introduction The increase in population and industrialisation in Ireland has brought with it a sharp increase in the use of fossil fuels such as coal and oil [1]. Ireland is almost completely dependent on energy imports which have grown significantly, both in quantity and volume, over the last ten years. In 2006, 92% of our energy needs were imported, while €6.6 billion was spent on energy imports during 2008 [2]. Indigenous bio-energy production can help redress our over dependence on energy imports while at the same time reducing our emissions and greenhouse gases [3]. Bio-ethanol produced from plant biomass is a viable method for manufacturing fuel and fuel additives in the petrochemical industry [4]. A substantial part of the €1.5 billion spent in 2008 on imported heating fuels with native solid fuel production can be replaced both from forestry trimmings and from energy crops [3]. Current production of bio-ethanol relies on ethanol from starch and sugars but there has been considerable debate about its sustainability [5]. In this context bio-ethanol produced from lignocellulosic biomass is an interesting alternative since the raw materials do not compete with food crops and they are also less expensive than conventional agricultural feed-stocks [5]. Biomass is naturally recalcitrant and so requires a pre-treatment step during its bioconversion to physico-chemically disrupt the hemicellulose-cellulose matrix making it more accessible to enzyme hydrolysis. Bio-energy crops willow, miscanthus and hemp were selected for bioconversion using various pre-treatment techniques and enzymatic hydrolysis. Results obtained from this analysis can be extrapolated to an industrial scale to demonstrate the potential commercial sugar yield of these crops. Research at the Institute of Technology Carlow, in collaboration with the Crop Research Centre Oakpark Carlow, is focussing on the development of biomass pre-treatment technologies which can be applied to energy crops grown in Ireland. A particular focus is being placed on willow, miscanthus and hemp in order to identify and further develop pretreatment and enzymatic techniques. Results of cellulose bioconversion are presented here which will inform future research studies. Methods Potential pre-treatment techniques for the bioconversion of willow, miscanthus and hemp were identified from a literature review of current process technologies. Samples were prepared for pre-treatment by grinding them using a 0.1mm electronic grinder. The moisture and ash content was then determined. Samples were pre-treated with 4M concentrations of NaOH, NH3, H5NO and H2SO4. Pre-treated samples of willow, miscanthus and hemp were treated for 24 hrs at 37oC . Samples were filtered, washed, dried and treated with Cellulase and enzyme complex (Novozymes) in citrate buffer for 24 hrs at 60oC. Hydrolysed samples were then neutralised, filtered and analysed using the ICS 5000 Ion Chromatograph (Dionex).
Results (A) Willow
Discussion
(D) Willow
(B) Hemp
Bioconversion of biomass is a complex process which requires a series of physicochemical, biochemical and/or microbiological processes. Pre-treatment technologies can be divided into three distant categories: physical (mechanical comminution), chemical (acid, alkaline, ammonia fibre explosion and organosolvant) and biological pre-treatments (brown, white and soft rot fungi and bacteria) [6]. The current study focussed on chemical pre-treatments including NaOH, NH3, H5NO and H2SO4
(E) Hemp
(C) Miscanthus
Willow, miscanthus and hemp, potential energy crops for bio-ethanol production, contain 40%, 43% and 70% cellulose, respectively [7]. Glucose yields for cellulose from these crops are presented in Table 1. Willow, miscanthus and hemp showed promising high yields for pre-treatment using NH3, H5NO and H2SO4. NaOH pretreatment liberated lower yields which suggests that this pre-treatment is less effective than the acid and ammonia pre-treatment. NH3 or H5NO pre-treatment showed the most promise with glucose yields of 66-67%.
(F) Miscanthus
Figure 1. Bioconversion of willow, hemp and miscanthus using various pre-treatment techniques and enzymatic hydrolysis. (A) willow (Salix), (B) hemp (Cannabis sativa) and miscanthus (x giganteus) crops provided by Teagasc Crop Research Centre, Carlow, Ireland. Representative IC chromatographs of the monomeric sugars released from (D) willow, (E) hemp and (F) miscanthus. IC experimental conditions: 21 minutes, 1 mM KOH, 2050psi, 45oC. Table 1. Glucose yields from biomass samples following various pre-treatments and enzymatic hydrolysis..
Biomass Samples
NH3
NaOH
H2SO4
H5NO
Willow
76.5%
30%
66.8%
70.8%
Miscanthus
63.9%
40.5%
62.1%
72.6%
Hemp
62.2%
61%
67.3%
56.1%
These studies will now be extended to include bioconversion conditions which will further improve sugar yields. This will include (i) identification of any limitations put on bioconversions by the enzyme preparations employed, (ii) application of simultaneous saccharification and fermentation to minimise enzyme feedback inhibition effects, (iii) other pre-treatment technologies based on those listed above and, ultimately, (iv) life cycle analysis of promising processes. The latter will be important in establishing the environmental impact of each process, from the planting of the biomass crop to its bioconversion using various approaches.
References [1] Sun, Y. (2002). Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology, 83, 1, pp. 1-11. [2] Howley, M; O’ Leary, F, O Gallachoir, B (2007). Energy in Ireland 1990-2006. 2007 Report, Sustainable Energy Ireland, energy policy statistical support unit. [3] Finnan, J. (2010). Energy crop manual 2010: The case for energy crops. Teagasc Oakpark Carlow, Crop Research Centre, Ireland. [4] Wyman, C.E and Goodman, B.J. (1993). Biotechnology for production of fuels, chemicals and materials from biomass. Appl. Biochem. Biotechnol., 39/40, pp. 4159. [5] Department of Agriculture, Fisheries and Food. Non-Food Crops in Ireland, DAFF report, Ireland 2009. [6] Zheng, Y; Pan, Z. and Zhang R. (2001). Overview of biomass pre-treatment for cellulosic ethanol production. Int. J Agri. Biol. Eng., 2:3. [7] Hon, D.N.S. (1996). A new dimensional creativity in lignocellulosic chemistry. Chemical modification of lignocellulosic materials. Marcel Dekker. INC. New York. Acknowledgement: This work was funded by the Teagasc Walsh Fellowship.
