Self sufficiency in energy requirements is critical to the success of any developing country. Investing in research and development of biomass energy for this self sufficiency is investing in its farmers, economy and environment [1]. This investment, in turn, ensures the sustainable development of the growing economy like India. India is focussing on the production of biofuel more from the cellulosic biomass that constitutes second generation biofuels considering the present debate of fuel vs food [2].

Biofuels are categorized into four types of generation based on the type of raw material used for their production viz first generation biofuels (produced from the food crops such as corn, soya, sugarcane and palm), second generation biofuels (produced from lignocellulosic biomass such as agricultural residue, forestry waste, grasses and rice straw, etc), third generation biofuels (produced from designer feed stocks such as recombinant microbes, genetically modified crop plants and trees) and fourth generation biofuels (produced from genetically optimized feed stocks designed to capture large quantities of carbon) [3-4].

Though the research is going on all the four generation biofuels, the second generation biofuel production has gained momentum in India [5]. The bioethanol production from biomass involves pretreatment, hydrolysis, fermentation and eventually distillation. The hydrolysate obtained from pretreatment and hydrolysis of the substrate, at times, needs to be detoxified as it contains several inhibitors along with pentoses and hexoses. These inhibitors are either the by– product of sugar or lignin degradation or are obtained directly from the raw material [6-8].

In the present investigation, feasibility of Saccharum spontaneum as a lignocellulosic substrate for ethanol production has been exploited because of its attributes of less economic value, drought and flood tolerant, its capacity to grow on sandy soils and light weight seeds, which allows its establishment to distant places easily [9]. Pichia stipitis is preferred for fermentation of hydrolysate because of its industrially required attributes such as short generation time, capacity to ferment even pentose sugars present in lignocellulosic substrate, negligible amount of inhibitor production and biochemical mechanism to ferment broad range of substrate containing sugars to ethanol in short period of time.

The complete production strategy of bioethanol from lignocellulosic substrate is depicted in Figure 1. The pretreatment method followed in this project is chemical (dilute acid) hydrolysis. Optimization of chemical hydrolysis of the substrate was also carried out in order to release maximum reducing sugars. Different parameters that were studied for maximum sugar recovery include temperature, acid concentration and time. The substrate released maximum sugars (17.4g/l corresponding to 85% hydrolysis) at 120⁰C at 2.5% of sulphuric acid concentration when the process is carried out at 60°C. Sugars released at different time intervals at 120⁰C are represented in figure 2.

During chemical hydrolysis several inhibitors like phenolics were released and are depicted in the form of histograms in figure 3. Direct neutralization is one such detoxification strategy that serves the purpose of removing inhibitors from the hydrolysate and enables P. stipitis to give ethanol production of 5.8 g/l from 12.0 g/l of sugars giving ethanol yield of 0.487 that is appreciably a good yield. After detoxification of the hydrolysate, it was converted to ethanol using P. stipitis in fed-batch cultivation. A fed –batch fermentation of the hydrolysate on 2% xylose was carried out. Fresh sugar solution was added into medium after every 24 hours. In the fed –batch fermentation, ethanol production was much higher as compared to batch fermentation reaching the concentration of 26.2 g/l in 72 hours and remained constant for 120 hours after which the ethanol production decreased as shown in the figure 4.

Among the lignocellulosic biomass, forests, agricultural and agro-industrial residues, which are widespread and inexpensive source of hexose and pentose sugars, Saccharum spontaneum has proven to be an ideal substrate for ethanol production.

Optimum conditions for chemical hydrolysis are 120⁰C for 1 hour with acid concentration of 2.5% that releases 17.4 g/l of sugar. At incubation time less than one hour sugar yield is less because of reduced level of harsh treatment provided to loosen the hemicellulosic backbone. At temperatures below 120⁰C, heat is not able to penetrate deep into the hemicellulosic backbone to release sugars thereby yielding less amount of sugar. At temperatures above 120⁰C, sugars produced are getting charred due to more penetration of the substrate and extensive exposure of released sugars to the intense heat. This is further corroborated with the finding of Sun et al [10].

Dilute sulfuric acid was employed for the purpose of chemical hydrolysis as it has the advantage of not only solubilizing hemicellulose but also converting solubilized hemicellulose to fermentable sugars [15]. The dilute acid pretreatment, thus, eliminates or reduces the need for use of hemicellulase enzyme mixtures. Phenolics estimation was the principle method for estimating the efficiency of detoxification strategy being used. Phenolics were used as the standard because conversely to what occur with the majority of the other toxic compounds, phenolics were not removed by evaporation [14]. Among the xylose-fermenting yeasts Pichia stipitis has shown promise for industrial application as it ferments xylose rapidly with a high ethanol yield (0.47w/w) and ethanol production of 22.3 g/l [16-17] and with insignificant amount of xylitol production (0.03g/l) [18]. Furthermore, P. stipitis has no absolute vitamin requirement for xylose fermentation [19-20] and is able to ferment a wider range of sugars including cellobiose. In all fed-batch experiments, fermentation was first carried out in batch mode until the carbon concentration had dropped to a preselected value. The substrate, an aqueous solution of xylose, was then added in discrete pulses [21].

Saccharum spontaneum is among those lignocelluloses that have little or no economic value. Also, it is robust enough to colonize even in extreme conditions. This suggests that exploitation of the waste hemicellulose from various sources for production of ethanol would lead to a cost effective process without causing global warming. Further study is required to improve the chemical and enzymatic hydrolysis of Saccharum spontaneum to provide higher yields of pentose sugar and further the pentose sugar fermentation by pentose metabolizing yeast.

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