BIOETHANOL PRODUCTION FROM URBAN CELLULOSIC WASTE EMPLOYING Alcaligenes faecalis HI-1 ISOLATED FROM GUT OF TERMITE Heterotermes indicola

This study assessed the potential of termite gut inhabiting bacteria towards bioconversion of cellulosic waste into biofuel. Total seven bacterial isolates from the gut of Heterotermes indicola were isolated. Among all the isolates, HI-1 produced the largest zone upon primary screening. Untreated paper had more cellulose content (73.03%) than acid (0.5%) treated paper that was used as a lignocellulosic substrate for saccharification. Among all the isolates tested, glucose yield (1.08mg/mL) was high for HI-1 isolate. Several factors were considered for optimizing augmented glucose yield (8.57mg/mL) and growth (8.07×10cfu/mL), such as temperature 37°C, pH 4.5, 5% (w/v) substrate concentration, 6 % bacterial inoculum size, agitation 150 rpm with PEG 0.25 % and Ca ions 0.002 g/L. Overall 8-fold increase in glucose yield was achieved. Enzyme activity of HI-1 showed higher endoglucanase 0.29 ± 0.01 (U/mL/min) and exoglucanase 0.15±0.01 (U/mL/min) activity under optimum conditions, mentioned above. temperature 37°C, pH 4.5, substrate concentration 5%, inoculum size 6%, surfactants PEG 0.01%, ions Ca(0.002g/L) and agitation (120 rpm). Simultaneous saccharification and fermentation (SSF) of hydrolyzed office paper yielded 5.43mg/mL bioethanol. According to 16S rRNA sequence homology, the bacterial isolate H1 was identified as Alcaligenes faecalis. Bioethanol production from office paper untreated waste proved an effective strategy. Bacteria having natural tendency towards cellulosic waste consumption are promising for bioconversion of cellulosic waste to valuable products.


Introduction
Fossil fuel depletion causes increase in oil prices and petroleum products that led to a sharp interest in finding alternative energy sources. Biofuels can substitute fossil fuels and confer several advantages, ie., produced from renewable sources, lessens greenhouse gas emissions, and cost effective (Wyman 1994). In previous years, cellulases from cellulolytic bacteria have been applied in biofuel production due to their unique saccharolytic properties (Chang and Yao 2011). Cellulose, being an abundant biopolymer on earth, is the best resource for renewable energy (Rezaei et al. 2008). Bioethanol generated from cellulose in the form of agro-industrial waste has an improved eco-balance, as it does not compete with the food production, while on the other hand, it is readily accessible at low cost (Zhou et al. 2007). Paper materials are the largest

BIOETHANOL PRODUCTION FROM URBAN CELLULOSIC WASTE EMPLOYING Alcaligenes faecalis HI-1 ISOLATED FROM GUT OF TERMITE Heterotermes indicola
municipal solid waste product (Muttamara et al. 1994). Therefore, it is necessary to find ways for the conversion of wastepaper and its products into consumable value (Van Wyk 2003). Cellulose is the main synthesizing unit of paper which can enzymatically be transformed into sugar molecules that can later be fermented into ethanol. The guts of some insects like termites, beetles, leaf cutting ants and wood feeding roaches are highly adapted to diverse microflora and are considered highly competent natural bioreactors. Microtermes obesi, Microtermes mycophagous, Odontotermes obesus, Eremotermes paradoxalis and Microtermes unicolor are the most documented termite species from ecosystems of Pakistan (Ahmed et al. 2004). The microflora of the termite gut has a considerable impact on the degradation of cellulose from complex biopolymers like wood and other materials containing cellulose and hemicelluloses (Varma et al. 1994). Several facultative anaerobes have been isolated from the gut of different species of termites including Clostridium nzayombii and Homoacetogens termitida (Kane et al. 1991). Cellulolytic species of bacteria from certain phyla such as Thermotogea, Spirochaetes, Proteobacteria, Firmicutes, Actinobacteria, Bacteroids and Fibrobacteres have been reported from termite gut (Bergquist et al. 1999).
To digest cellulose, cellulolytic microorganisms produce cellulases, an extracellular complex of multienzymes or as single polypeptides having multiple cellulosic domains. Cellulases have gained much attention because of their diversity and practical applications in pharmaceutical, fiber, textile industries, laundry detergents and in fermentation of biomass for biofuel production. To overcome the global energy crisis, the present study aimed to develop a new approach to investigate the potential of cellulolytic bacteria isolated from termite gut and employed to eco-friendly conversion of highly cellulosic urban waste (office paper) into reducible sugars that can later be fermented into bioethanol.

Isolation of cellulolytic bacteria from termite gut
Termites collected from different localities of Lahore were identified and their guts were removed according to Akhtar (1974). For isolation of cellulolytic bacteria, Carboxymethyl cellulose (CMC) media having 7 pH was used (Kasan et al. 2008).

Primary screening of cellulase producing bacteria
Congo red assay was performed to determine the cellulolytic activity of the isolates (Upadhaya et al. 2012). Streaked plates of CMC media were incubated at 37°C for 48 hours. Colonies were flooded with Congo red stain (0.1%) for up to 20 min, later the stain was removed by flushing the plates with 1M NaCl. Diameters of clear zones around the colonies were measured.

Pretreatment and proximate analysis of substrate
Waste office paper was used as a cellulosic substrate, they were shred into small pieces (5 to 10mm) and were exposed to different pretreatments to analyze the % cellulose, moisture, dust, and lignin content as reported previously (Wayman et al. 1992). Pre-treatment was done by two methods, where in one method paper was exposed to steam in the presence of dilute sulphuric acid (0.5%) to remove lignin and hemicelluloses while the other was only steam treatment (Anwar et al. 2012).

Enzyme activity: CMCase assay
For CMCase assay 100µL of crude enzyme was mixed with 900µL of 1% CMC (1g of CMC dissolved in 20mM of phosphate buffer having pH 7.0) kept at 50°C for 30 minutes. After incubation, the reaction was stopped by adding 1mL 3, 5-Dinitrosalicylic acid solution and kept in boiling water for 5 minutes and then cooled at room temperature. Reducing sugar was measured as reported previously (Miller 1959). Endo-β-1, 4-glucanase activity was defined in one unit as; "amount of enzyme that can hydrolyze CMC and liberate 1µL glucose per minute at 50°C".

Enzyme activity: FPase assay
For FPase assay, 1mL of culture aliquot was centrifuged at 10,000rpm for 10 minutes and then mixed with 50mg strip of Whatman filter paper (1×6cm), immersed in 1mL of 0.05M sodium phosphate buffer at pH 5.0 and incubated at 50°C for 60 minutes (Mandels et al., 1976). After incubation, 1mL of DNS solution was added and boiled for about 5 minutes and later cooled to room temperature. The reducing sugar was measured by DNS method (Miller 1959). The activity of exoglucanase in one unit was described as "enzyme amount that can release 1μmole reducing sugar in one min from filter paper".

Simultaneous saccharification and fermentation (SSF)
The yeast (Saccharomyces cerevisae) inoculum of 24 hours (10 6 ) was prepared in YPD media containing 1% yeast extract, 2% peptone and 5% glucose (Anwar et al. 2012). Simultaneous saccharification and fermentation was carried out in 250mL flasks that contained 100mL S-I medium. Media was aseptically inoculated with 5% yeast suspension (Saccharomyces cerevisae). Anaerobic conditions were established by fitting a rubber cork over each flask and one end of capillary was inserted into it whereas the outer tip was dipped in Ca(OH)2. SSF experiments were performed under optimum conditions temperature 37°C, pH 4.5, substrate concentration 5%, inoculum size 6%, surfactants PEG 0.01%, ions Ca 2+ (0.002g/L) and agitation (120 rpm) for a period of 3 days. The ethanol produced during SSF was estimated by HPLC.

Ethanol estimation by HPLC
The aliquots of fermented products were analyzed by HPLC (Shimadzu Corporation Koyoto, Japan) for estimation of ethanol yield. The samples were quantified by using the column CLC-NH2. The mobile phase used was acetonitrile and water (75:25, v/v) at 1mL/min. The injection volume was 10µL and column temperature was maintained at room temperature.

Statistical analysis
All experiments were performed in triplicates. Data was analyzed using software SPSS 16.0. Duncan's Multiple Range test was performed on the data to calculate significance difference (p≤0.05) between values obtained at different conditions. Intergroup differences in proximate analysis and secondary screening were calculated by applying t-test with significance at p≤0.05.

Results and Discussion
The present study was undertaken to investigate the potential of cellulolytic bacteria isolated from the gut of termites in bioconversion of highly cellulosic urban waste, and office paper into reducible sugars that was later fermented to bioethanol. Termite specie collected for this study was identified as Heterotermes indicola. Seven bacterial strains were isolated from H. indicola. Alcaligenes faecalis HI-1 from the current study was identified as facultative anaerobe. Other facultative anaerobes Bacillus cereus (Thayer 1976;Ramin et al. 2009), Cellulomonas (Wenzel et al. 2002), Cellulomonas pachnodae (Bakalidou et al. 2002), Enterobacter aerogenes and Acinetobacter (Ramin et al. 2009) had been reported from guts of termites and were capable of lignocellulosic degradation. Alcaligenes has been reported from hind-gut of other termite specie such as Reticulitermes hesperus (Thayer 1976) and Odontotermes obesus (Devi et al. 2006) and identified as facultative wood digesting bacteria, however it is not previously reported from Heterotermes indicola.

Primary screening
In the primary screening all bacterial isolates produced clear zones against Congo red reagent which confirmed their cellulolytic nature ( Figure 1A).

Proximate analysis
In the present study an important urban cellulosic waste, an office paper, has been considered a cheap substrate for bioethanol production (Coman et al. 2012). Utilization of office paper waste is beneficial in two ways as, on one side waste is managed and on the other side a useful product i.e., biofuel is produced. Different studies have been conducted on the bioconversion of wastepaper to bioethanol. Proximate analysis of pretreated and control samples revealed that cellulose and lignin content of untreated office paper were 73% higher as compared to pretreated office paper (67%). Whereas moisture and ash content were significantly (p≤0.05) higher in pretreated office paper (Table 1). There might be a possibility that sulfuric acid not only dissolved lignin but also affected cellulose content thereby producing undesirable products. In this context, it can be concluded that in case of wastepaper, pretreatment is not necessary for efficient bioconversion to bioethanol (Zhu et al. 2011).

Secondary screening
All the isolates produced significantly higher (p≤0.05) glucose by utilizing untreated office paper as compared to pretreated office paper ( Figure 1A). The highest glucose producing bacterial isolate was Alcaligenes faecalis HI-1 that produced 1.07±0.04 (mg/mL) of glucose with significantly (p≤0.05) higher cell biomass up to 2.60×10 8 ±0.004 (cells/mL) ( Figure 1B). Previous studies focused only on the enzymatic conversion of wastepaper to sugar monomers (Van Wyk 2003). Whereas in the current study cellulolytic bacteria from the termite gut was used to convert cellulose waste to generate glucose monomers. and glucose yield (mg/mL) by cellulolytic bacterial isolates obtained from termite gut during secondary screening using office paper at different conditions.

Optimization
Enhanced cellulase activity relies heavily on various physicochemical factors and the current study focused on standardizing the physical and biochemical conditions for maximum enzyme activity and enhanced glucose concentration.

Temperature
Incubation temperature has considerable effect on the growth, survival and metabolism of microorganisms and their activities. Glucose release and bacterial growth were the two parameters studied during optimization using untreated office paper (Figure 2A). It was found that Alcaligenes faecalis HI-1 had significantly (p≤ 0.05) higher growth (3.33×10 8 ±0.02 cells/mL) with enhanced glucose yield (2.36±0.02 mg/mL) at temperature 37 °C after 48 hours of incubation. As Alcaligenes faecalis HI-1 showed both increased cellulase activity and cell biomass with respect to glucose production at 37ºC, indicating that bacteria had efficiently utilized the substrate (office paper) by producing enzymes that efficiently converted the cellulose of substrate into glucose. Bacillus subtilis, Pseudomonas fluorescence, Bacillus sp., Cellulomonas and Micrococcus sp. have been reported to exhibit maximum growth and cellulase activity at temperature range from 37-40°C (Immanuel et al. 2006).

pH
In the current study Alcaligenes faecalis HI-1 showed highest cell biomass and maximum glucose yield (mg/mL) at pH 4.5 with a fold increase in glucose yield. Increase in bacterial biomass was 3.86×10 8 ±0.02 (cells/mL) and glucose yield was 3.43 ± 0.01 (mg/mL) after 48 hours of incubation ( Figure 2B). pH had a profound effect on microorganisms and their activities. The change in the pH might have affected ionic bonding of the enzymes that may affect functional structure of the enzymes which degrade cellulose (Mussatto et al. 2008). Maximum activity of cellulolytic enzymes was reported between pH 4.2 to 5.8, whereas pH 4.8 has been considered optimal for the stability of cellulase enzymes (Pardo and Forchiassin 1999).

Substrate concentration
Optimum concentration of substrate for Alcaligenes faecalis HI-1 was 5% that liberated glucose up to 5.02±0.03 (mg/mL) after 48 hours of incubation ( Figure 2C). High degree of glucose yield obtained from hydrolysis of paper materials is also reported by other authors but at low substrate concentration (Wayman et al. 1992;Solange et al. 2008).

Inoculum concentration
Glucose yield increased further to 5.86±0.03 (mg/mL) by increasing the inoculum concentration to 6%. Alcaligenes faecalis HI-1 showed 17% increase in glucose content with increased cell biomass at 6% inoculum in saccharification medium of office paper ( Figure 2D). There is currently no work available in which Alcaligenes faecalis HI-1 bacterial inoculum is optimized for production of glucose during saccharification. Currently, a different approach is undertaken in which cellulose degradation potential of cellulolytic bacteria isolated from the termite gut was studied by utilizing office wastepaper.

Agitation
A significant increase in glucose yield (6.76±0.04mg/mL) was obtained with 48 hours of agitation (120rpm) ( Figure 3A). Sufficient oxygen is necessary for the cells for efficient activity.

Surfactants
Among the surfactants used, a significant (p≤0.05) decrease in cell biomass as well as glucose production was observed with SDS, EDTA and Triton X100, while PEG (0.1%) enhanced the glucose yield to 7.88±0.06 (mg/mL) after 24 hours ( Figures 3B and 3C). PEG, because of its hydrophobic nature can modify the structure of substrate and therefore enhances the availability of cellulose for the enzyme activity. PEG adsorbs on the surface of lignin and repels the enzymes for unproductive binding (Sipos et al. 2010).

Metal ionsm
Addition of metal ions to the culture showed that Ca 2+ ions increased the cell biomass to 8.07×10 8 ±0.07(cells/mL) along with increasing the glucose yield (8.57±0.08mg/mL) to 8-fold after 24 hours of incubation ( Figure 3D). Addition of Ca 2+ ions (0.002g/L) to the media enhances cellulase production was also inferred by Shankar and Isaiarasu (2011).

Simultaneous saccharification and fermentation (SSF)
Fermentation was carried out for the saccharified substrate obtained from Alcaligenes faecalis HI-1 under optimum conditions. Fermentation conditions were: 5% substrate concentration, 6% inoculums size, pH 4.5, 5% inoculum of S. cerevisae, agitation at 150rpm at 37˚C for a period of 3 days. Although temperature was not optimum for the yeast, it showed the metabolic activity. The ethanol yield obtained by saccharification of office wastepaper by cellulolytic strain Alcaligenes faecalis HI-1 was 5.43mg/mL ( Figure  5). Bioethanol reported by enzymatic saccharification of wastepaper has been reported in another study by Ballesteros et al (2002). However, in these studies direct saccharification was done by using commercial enzymes. In the present study, the application of cellulolytic bacteria for cellulase production instead of using cellulase enzymes was a novel and cost-effective approach. By further refining the process the batch culture can be upgraded to continuous culture for continuous productivity of biofuel.

16S rRNA gene sequence analysis
The amplified 16SrDNA sequence have 99% homology with specie Alcaligenes faecalis, so the isolate was designated as Alcaligenes faecalis HI-1.

Conclusions
The current study was a novel approach to produce bioethanol from office wastepaper by utilizing cellulolytic bacteria from the termite's gut. New habitats must be searched for novel cellulolytic bacteria which can ultimately lead to novel enzymes with efficient activity. The data gathered in the present study revealed that cellulolytic bacteria Alcaligenes faecalis HI-1 isolated from gut of Heterotermes indicola was competent enough to breakdown the cellulosic content of office waste paper into glucose which can be fermented into bioethanol by Saccharomyces cerevisae. The office waste paper has the potential to be converted into bioethanol without any pretreatment and is relatively cost effective. This study provided ecofriendly bioethanol from cheap urban cellulosic waste (office paper) which can replace gasoline products and overcome the global energy crisis. In addition to bioethanol production, this study is also beneficial with respect to bioremediation of urban wastes where such bacteria can be employed for generating compost from urban waste.