Identi cation And Quanti cation Of Hydrocarbons Produced From The Acid-Pretreated Kitchen Waste By Using Fungal And Bacterial Strains

Nayab Zahara (  nayab.zahra@uettaxila.edu.pk ) University of Engineering and Technology, Taxila https://orcid.org/0000-0003-0506-8592 Muhammad Irfan Jalees University of Engineering and Technology, Lahore Muhammad Umar Farooq University of Engineering and Technology, Lahore Arfa Iqbal University of Engineering and Technology, Lahore Sadaan Umais Malik University of Engineering and Technology, Taxila


Introduction
Abundant usage of fossil fuel for transportation has adverse effects on environment, not only climate changes are visible, but depletion of fossil fuel is also a threat. This leads to use of alternative energy recourses. Environmental friendly and renewable fuel always attracts attention for the protection of the environment and supplies our needs by reducing dependence on non-renewable energy sources and petroleum [1]. Many countries, including Pakistan, are moving towards bio-fuel to reduce the emission of gasses and economic burden (imported petroleum for transportation and industrial plants).
Bio-fuel, which is an attractive alternative fuel, has the potential to meet the increasing demand of energy for different industrial processes, power generation and transportation [2]. Biofuel may be produced by food crops like sugar beet and rapeseed (1st generation) and nonedible byproduct of food crops e.g. agricultural residue, grass, sawdust, wood chips, waste cooking oil and municipal solid waste (MSW) etc.
(2nd generation) [3]. In many countries, biofuel is produced from sugarcane and approximately 60% of global ethanol production based on this raw material while in United States 90% ethanol is produced from corn [4]. Many other sources of biofuel production are also known e.g. starch, palm oil [5], canola oil [6], animal fat [7], waste cooking oil [8], wheat straw [9], rice husk [10] and algae [11]. Moreover, production of biofuel from food source like sugar and corn cause increase in the crop price. Therefore, it is essential to research inexpensive and alternative biomass for ethanol production at a reduced cost.
This issue turned the concern of researchers toward Municipal Solid Waste (MSW) as a raw material to produce biofuel.
Kitchen waste (KW) is a major portion of MSW and it is disposed of from restaurants, hotels, industrial and household kitchen at increasing amount all over the world. In Pakistan 90% of the collected waste goes for open dumping and cause environmental pollution. About 18,113 tons per day KW produced in Pakistan that is approximately 30.7% of municipal solid waste if treated or used properly then we can save our mother land from pollution [12,13]. Current practices for KW treatment involves composting, incineration, anaerobic digestion, land lling, open dumping as well as drying for animal feed [14,15].
Composting of KW provides a valuable soil conditioner and it reduces the mass and volume of waste as well. As KW has high moisture content so causes unusual level of leachate in composting that affects the performance of whole process by dropping oxygen availability and weak the strength of pile. KW can be utilized as an animal feed but due to its high moisture content and variable composition which favors microbial contamination it can be environmental unfriendly [14]. Incineration of solid waste causes emission of noxious gases and dioxins that are source of air pollution [16]. Land ll that is an engineered practice for waste disposal also causes serious environmental pollution. Its gas emissions are one of the largest anthropogenic sources of methane (cause of air pollution and ultimately global warming) especially because of KW. Leachate production from land lls is another serious environmental issue that causes ground water pollution and soil contamination with dangerous chemicals [17].
The biomass, in case of KW, has a great potential to be used as sustainable energy source if converted to ethanol or another biofuel, while simultaneously treating kitchen waste. KW is primarily composed of cellulose (insoluble carbohydrate), hemicelluloses, lignin, proteins, fat, soluble sugars such as glucose, fructose and sucrose. Cellulose, glycogen and starch components of kitchen waste can be hydrolyzed to monomeric sugars. Therefore, due to abundant source of fermentable carbohydrates this sugar can be used as a substrate in microbial fermentation for the production of useful products such as biofuel [16,18]. Young et. al [19] used food residue for ethanol production and maximum yield of 25 g/L ethanol per 100 g/L food residues was achieved using Saccharomyces cerevisiae. In another study it has been reported that hydrothermal pretreatment enhanced the 13.16% of ethanol production levels and 107.58 g/kg of nal ethanol yield was achieved using household food waste [20]. However, in the literature there are very few studies on the utilization of KW for the production of ethanol.
The production of biofuel from biomass includes two main processes: hydrolysis of polysaccharide sugars into reducing sugars and fermentation of the reducing sugars to biofuel [21]. The conventional methods for hydrolysis process are acid hydrolysis and enzymatic hydrolysis. Dilute acid hydrolysis is successful for pretreatment of biomass. High reaction rates can be achieved by the dilute sulfuric acid pretreatment and it signi cantly improves hydrolysis of cellulose and glycogen [22]. Moreover, dilute acid hydrolysis is favorable at high temperature [23]. Dilute acid hydrolysis of KW converts polysaccharides into reducing sugars like sucrose and fructose [24]. These can be converted into valuable products such as hydrocarbons (HC's), Lactic Acid (LA) and Ethanol (ET) after microbial and fungal fermentation [3].
Yanuar et. al. investigated that 87-90 g/L sugar concentration from hydrolysate was achieved at optimum conditions using lignocellulose biomass [25]. Several microbial species have the ability to ferment the hydrolysate of KW into biofuel. Aspergillus Niger, Lacto-bacillus and Escherichia coli are commonly used microbial strains in fermentation of biomass as these ferment the hexose sugars in to high ethanol yield [26]. Published studies have reported ethanol production from agro-industrial biomass using Aspergillus Niger and Trichoderma reesei [27]. Bruce et al. reported ethanol production from lignocellulose biomass using Escherichia coli [28]. Moreover, there are only few studies in the literature of microorganisms grown in monoculture on KW hydrolysates with the aim of producing renewable chemicals or biofuels. Usually, conversion of biofuel from KW is performed using open fermentation. Open fermentation has reduced production of furfural compounds which is inhibitor for bacterial growth [29,30] but produces less hydrocarbons as macromolecules are di cult to ferment. To increase the quantity and quality of hydrocarbon produced as biofuel, this study was performed. The aim of this study was to investigate the effect of hydrolysis temperature, hydrolysis reaction time and acid concentration toward reducing sugar recovery from KW. The acid hydrolysate produced after acid hydrolysis of KW was fermented by Aspergillus Niger, Lacto-bacillus and Escherichia coli for biofuel production and production of biofuel from these strains was compared. Moreover, the quantity and quality of produced hydrocarbons were analyzed on GC-MS and calorimeter.

Microorganism and Inoculum preparation
Aspergillus Niger FCBP-0198, Lacto-bacillus FCBP-0004 and Escherichia coli FCBP-0011 purchased from Punjab University fungal bank, Lahore Pakistan, were used in fermentation. Fungus was grown with 20g/L Malt extract (CM0059) and 20g/L Agar (CM0463). The culture media for Aspergillus Niger was sterilized at 121°C, 15 psi for 15 min in an autoclave. For inoculum preparation, fungus was inoculated to 20 ml of Maltextract and Agar media and been put to an incubator for 5-7 days at 25°C to 27°C. Lactobacillus was grown with 20g/L Malt extract (CM0059) and 20g/L Agar (CM0463) and media was sterilized in an autoclave at 121°C, 15 psi for 15 min. For inoculum preparation, bacteria was inoculated to 20 ml of Maltextract and Agar media and been put to an incubator for 24 h at 35°C. While for Escherichia coli, bacteria was grown with 5g/L yeast extract (LP0021), 10g/L tryptone (LP0042), 10g/L NaCl and 20g/L Agar (CM0463) and media was sterilized in an autoclave at 121°C, 15 psi for 15 min. For inoculum preparation, bacteria was inoculated to 20 ml of media and been put to an incubator for 24 h at 35°C. All growth media were purchased from Merck Private Ltd., Pakistan. All cultures were preserved at 4 o C to maintain viability.

Raw Material and Pretreatment
The KW utilized in this present research work was collected from houses, restaurants and cafeterias (Lahore, Pakistan) in summertime. The composition of KW was uncooked vegetables and fruits (51%), cooked meat (16%), uncooked meat (15%), bread (2%), tea leaves (5%), egg shell (6%), miscellaneous (5%). Mixed KW was dried using Laboratory hot air oven (Wise cube WON-105) at 55 ± 2°C for 48 h or more, until constant weight and then was ground in a laboratory grinder to achieve ne particles size of 0.45 mm to 1 mm by using a sieve of 200-400 micron and to increase the surface area of particles. To maintain its physicochemical characteristics during the whole period it was stored at -20 o C. The characteristics of the KW used in this present study are presented in Table 1. The dry mass of KW was mainly composed of cellulose, hemicellulose, starch sugars, protein and fat, which could be considered as a suitable biomass for ethanol production. These characteristics of KW were very similar to other studies that have been reported [31] [32] [33].

Acid Hydrolysis
Acid hydrolysis was performed to produce soluble reducing sugars from KW. The dilute acid hydrolysis of KW with 1%, 3% and 5% (w/w) sulfuric acid was conducted at two different temperatures (90 and 120°C) and four reaction times (30, 60, 90 and 100 min) at a solid to liquid ratio of 1:10 (w/w) (based on total solid) in a 500 mL Erlenmeyer asks with a working volume of 100 ml. After dilute acid hydrolysis, the ask was cooled in an ice bath, and the hydrolysate was separated from the solid by ltration. The hydrolysate was then analyzed for its reducing sugar content by using by 3,5-dinitrosalicylic acid method using glucose as the standard following Miller's method [34].

Fermentation
Reducing sugars produced after acid hydrolysis of KW were subjected to fermentation using fungal and bacterial strains i.e. Aspergillus Niger FCBP-0198, Lacto-bacillus FCBP-0004 and Escherichia coli FCBP-0011without adding of any nutrient components. After the adjustment of pH to 6.5 using 5 M NaOH, a nal volume 100 mL of hydrolysates in 250 ml Erlenmeyer asks was fermented with 2% (v/v) of Aspergillus Niger FCBP-0198, Lacto-bacillus FCBP-0004 and Escherichia coli FCBP-00. The asks were sealed with a rubber stopper. These three asks were incubated in laboratory incubator (Wise Cube WIG-105) at 25 o C (Aspergillus Niger), 37 o C (Lacto-bacillus) and 37 o C (Escherichia coli) for 7 days by using separate hydrolysis and fermentation. Reducing sugar concentration was measured after every day by collecting 20ml liquid of samples in the asks, for continuously monitoring of the fermentation substrate. All fermentation experiments were performed with three replicates.

Analytical methods
The preparation of samples for GCMS and analysis procedure is explained elsewhere [35]. In summary, the fermented product was dried under nitrogen ow and passed through magnesium sulphate column to remove any moisture contents. The dried contents then dissolved in cyclohexane and send for GCMS

Statistical analysis
Statistical analysis of the results was carried out by two-way ANOVA analysis and Tukey's test using Minitab software Version 17.0. Tukey's test was used to compare the signi cance between the means of results at the 95% con dence level (p < 0.05).

Acid hydrolysis
The use of low cost and abundantly available KW is presently being recognized as raw material for the production of bio-fuel because it contains signi cant amount of carbohydrates and lipid [37]. Moreover, it also has abundant nutrition, high moisture and organic component. Their key advantages are their abundance, diversity and low cost [38]. KW used in this study was composed of cellulose (insoluble carbohydrate), hemicelluloses, lignin, proteins, fat etc. Hydrolysis of KW was performed to convert polysaccharides into monosaccharide i.e. reducing sugars, lipids to fatty acids and proteins to amino acids. Optimization of various parameters for hydrolysis was done for maximum reducing sugar production.
The dilute acid hydrolysis was performed using 1%, 3% and 5% (w/w) sulfuric acid at different temperatures (90°C and 120°C) and reaction times (30, 60, 90 and 120 min). The dilute acid hydrolysis conditions, reducing sugars and energy released from KW are summarized in Table 2. The dilute acid hydrolysis conditions were selected after a series of preliminary experiments. As shown in Table 2 at constant acid concentration and temperature, reducing sugars were increased by increasing the reaction time. Through the dilute acid hydrolysis with 1% acid at 120°C for 120 minutes, 61.287 ± 0.2 g of reducing sugars were released from each kg of KW and 0.981 ± 0.004 MJ energy was produced from each kg of KW. By increasing the temperature to 120°C from 90°C and reaction time to 120 min at 1% acid concentration, release of reducing sugars and energy are increasing. At the temperature of 90°C, when the acid concentration was increased to 3%, maximum release of reducing sugars was 62.483 ± 1.5 from each kg of KW and production of energy was 1.000 ± 0.025 from each kg of KW for 120 minutes.
Reducing sugars and energy values at 3% acid concentration at 120°C for 120 minutes are higher than the previous ones it shows that increase in concentration of acid cause increase in production of reducing sugars and energy from each kg of KW. Whereas, increasing the reaction time to 120 min increased 20.747 g reducing sugars and 0.332 MJ energy from each kg of KW. The highest amount of reducing sugars and energy was detected after treatment with 5% acid concentration at 120°C for 120 min. At 5 % acid concentration by increasing temperature to 120°C and reaction time to 120 minutes release of reducing sugars and energy are increasing and maximum 97.917 ± 0.5 of reducing sugars from each kg of KW and 1.567 ± 0.008 MJ of energy from each kg of KW was achieved. Similarly, V. Gupta et al. [39]

Fermentation
In the present study, dilute sulfuric acid hydrolysate was fermented by Fungus and bacterial species i.e. Aspergillus Niger FCBP-0198, Lacto-bacillus FCBP-0004 and Escherichia coli FCBP-001 to convert reducing sugars in bio-fuel. During fermentation of 72 h change in concentration of reducing sugars was calculated after every 24 hours as shown in Fig. 1. The amount of reducing sugars of KW fermented by Aspergillus Niger, Lacto-bacillus and Escherichia coli for 72 h showed that with the passage of time the amount of reducing sugars decreased. About 64% reducing sugar was converted into hydrocarbons after fermentation of KW by Aspergillus Niger while conversion of reducing sugar into hydrocarbons after fermentation of KW by Lacto-bacillus and Escherichia coli was 45% and 50% respectively as shown in Fig. 2. It indicates that Aspergillus Niger has more potential for conversion of reducing sugars in hydrocarbons as compare to Lacto-bacillus and Escherichia coli. Overall, maximum percentage of hydrocarbons from KW was produced after fermentation by Aspergillus Niger.

GC-MS analysis
After fermentation process samples were analyzed on GC-MS to identify and quantify the compound present in samples before and after fermentation by Aspergillus Niger, Escherichia coli and Lacto bacillus. Figure 2 shows the peaks of compounds in KW after and before fermentation. Nine different compounds were identi ed in KW before fermentation and 12 hydrocarbons were identi ed after fermentation. Figure 2 shows the compounds that were present in KW. These compounds are mostly hydrocarbons and can be used as biofuel. 5-8g of HC were produced per Kg of KW.

Calori c value of samples
Calori c values of sample before and after fermentation by different strains were determined. Table 3 shows the calori c value of KW before fermentation i.e. 0.6 MJ/kg and calori c value of KW fermented by Aspergillus Niger, Escherichia coli and Lacto-bacillus are 3.56, 3.34 and 2.67 MJ/kg. Maximum calori c value i.e. 3.56 MJ/kg was achieved by KW fermented by Aspergillus Niger. This calori c value of biofuel is before distillation process. So, this value will be improved after distillation.  (Table-4). It shows the calori c values of biofuel fermented from different waste streams. Due to pre-treatment and acid hydrolysis, the production of biofuel in enhanced quite much which in indicated by the high calori c values of KW fermentation it is because the other waste was direct fermented to produce biofuel while for KW methodology was modi ed by doing pretreatment and acid hydrolysis that will increasing the chance for conversion of reducing sugars into biofuel.

Conclusions
The KW was used as source to produce biofuel. The acid hydrolysis produced maximum of 37.673g reducing sugars per each kg of KW under optimum conditions i.e. 5 % acid concentration for 2 hours at