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International Journal of Applied Engineering Research, ISSN 0973-4562 Vol. 10 No.67 (2015) © Research India Publications; httpwww.ripublication.comijaer.htm

A study on the potential of oleaginous yeast in producing lipid from garbage S. Murugan, M Bharathipriyadharsini

Dr S. Jayanthi

Department of Environmental Engineering Government College of Technology Coimbatore, India [email protected]

Department of Environmental Engineering Government College of Technology Coimbatore, India [email protected]

become one of the major obstacles for its development and wide applications. On the other hand, consumption of a large amount of vegetable oils as raw material for biodiesel production would result in a shortage in edible oils and leads to the soar of food price. Adoption of animal fat, used frying oil, and waste cooking oil as feedstock is a good strategy to reduce the cost. However, these limited resources cannot meet the increasing needs for clean renewable fuels. Recently, there has been an increasing interest in looking for new oil sources for biodiesel production. Among them, microbial oils, namely single cell oils (SCOs), have attracted great attention worldwide. Oils from oleaginous microorganisms including bacteria, yeasts, moulds, and microalgae are now considered as promising candidates due to their specific characteristics such as being unaffected either by seasons or by climates, having high lipid contents, ability to be produced from a wide variety of sources with short period of time, and their similar fatty acid compositions to that of vegetable oils. However, the high production cost of SCO makes microbial oils less economically competitive. As a result, the production of microbial oils from wastes or renewable materials is significantly important. The purpose of this study was to investigate the possibility of using cheap and easily available sources as feedstock. Garbage is a biodegradable waste available plenty India as well in all over world. Garbage ( vegetable market waste ) hydrolysate as a nutrient source for L.Starkeyi for microbial oil production. The preparation of Garbage hydrolysate (GH), comparison of lipid production by L.Starkeyi in synthetic medium and Garbage Hydrolysate and trans–esterification of obtained lipids into FAME (biodiesel) were experimented in this study.

Abstract— Biodiesel is a fuel comprised of mono alkyl esters traditionally derived from vegetable oils and animal fats. Biodiesel production has many important technical advantages over petro diesel, such as inherent lubricity, low toxicity, superior flash point, negligible sulphur content, and lower exhaust emissions. The production of conventional biodiesel from vegetable oil questions the ethics of using food crops for fuel generation. Hence, the lignocellulosic wastes can be used as an alternative source for producing biodiesel. Garbage (lignocellulosic material) rich in carbon content can be used as a substrate for growing micro organisms, which accumulates lipids during their metabolism under limited nitrogen content. Studies showed that oleaginous yeast accumulates more lipids as high as 60% of their biomass dry weight when compared to other micro organisms. The lipid can then be converted to fatty alkyl methyl ester (FAME) by trans esterification. The main objective of this study is to stabilize the garbage by using eco-friendly technique along with the production of biodiesel. Based on the previous study, Lipomyces starkeyi has been chosen because of its easy handling and high production of lipids. Keywords—Biodiesel, Garbage; lipid,

I. INTRODUCTION In the present communication world even an atom may not be moved without Energy. Energy is the basis of human life. The conventional Non renewable energy sources are becomes costlier and the long term availability of which is questioned and also the pollution problems due to these energy sources are more serious than earilier. The search for alternative energy sources is strongly rooted in the realisation due this matter. The possibility of deriving biofuels from locally grown sources and using them as alternatives to petrol products is attractive in many countries. Investment in biofuels could lead to a significant boostin economic development, including the creation of new jobs and new sources of income for farmers. Biodiesel is a clean, biodegradable, renewable, and Non-toxic fuel produced from vegetable oil, which contributes no net carbon dioxide or sulphur to the atmosphere and emits fewer pollutants than conventional diesel. However, the high cost of raw material (70–75%) for biodiesel production has

II. LITERATURE REVIEW The alternative of traditional raw materials, vegetable oils is the crying need to solve the huge demand of vehicle fuel. Because of the similar fatty acid composition of vegetable oil, microbial oil (also known as single cell oil) is considered as most potential alternative ( Angerbauer et al., (2008) Li et al., (2007); Zhu et al., (2008) and others). In the economic point of view, the production of microbial oils from waste or renewable materials is of significant importance (Huang et al., (2009)). To overcome the economic problems

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in the production of microbial oils, some inexpensive substrates such as cane molasses (Zhu et al., 2008), wheat straw and wheat bran (Peng and Chen, 2008), sewage sludge (Angerbauer et al., 2008), rice straw (Huang et al., 2009), sweet Sorghum (Economou et al., 2010) have been used.

 Detoxification of hydrolysates

In the present study the experimental activity has been aimed to the optimization of the conversion of Garbage (lignocellulosic biomass) into fermentable sugar, as well as the achievement of satisfactory yields in terms of triglycerides (Zhu et al., 2008). The overall objective of this study is to enhance the economic attractiveness of waste materials especially garbage for getting biodiesel through the production of lipids by oleaginous yeasts.

 Overliming

The Garbage hydrolysates (GH) were subjected to detoxification process in order to remove the inhibitor compounds and to break the complex sugar substances into simple sugar molecules (Chao Huang et al., 2012).

Calcium hydroxide, Ca(OH)2, was added to the samples containing 2%, 4%, 6% (w/v) solids until the pH reached 10.5. Precipitate formed was removed by filtration.  Activated charcoal detoxification The Garbage hydrolysates detoxified by lime were subjected to activated charcoal treatment where 3% w/v of activated charcoal was added to the samples. The samples were heated at 80˚C for 1 hour. It was then kept in rotary shaker for about 30 minutes and filtered.

III. MATERIALS AND METHODS A. Culture of microrganisms Lipomyces starkeyi was obtained from Microbial type culture collection and Gene bank (MTCC), Chandigarh in the form of freeze dried culture. It was revived in distilled water. Further culturing of L.starkeyi in broth was done in the nitrogen limiting medium containing, Malt extract Yeast extract Peptone Glucose

-

C. Optimisation  Optimization of solid loading based on C/N Ratio 3 samples (each containing Garbage powder 2%, 4%, 6% w/v) taken from dilute acid hydrolysis pretreament, overliming process and activated charcoal pretreatment were subjected to C/N analysis. 1 mL of sample was manually diluted with 99 mL distilled water and an auto-dilution of about 50 times was set in the Shimadzu TOC analyser. The sample was injected in TOC analyser and the results were obtained.

3.0 g/L 5.0 g/L 5.0 g/L 10.0 g/L

The microorganism was grown under aerobic conditions at 25°C in a rotary shaker at 150 rpm and was stored as mother culture. Slant tube was prepared with the same medium along with 20.0 g/L nutrient agar. Growth curve test was performed initially during the growth of micro organism in synthetic medium in order to obtain the growth profile of L.starkeyi.

 Optimization of temperature and pH The sample containing comparatively high C/N ratio was chosen to adjust the pH and temperature. L.starkeyi was inoculated from the mother culture into six sets of sample with varying pH ranging from 4 to 10. Each set is placed in varying temperature such as 20⁰c, 25⁰c, 30⁰c, 35⁰c, 40⁰c, 45⁰c. This optimization process results in the determination of the pH and temperature at which the micro organism growth is maximum.

B. Pretreatment of garbage Use In most instances, pretreatment is a prerequisite condition to use agro-based residues in bioconversion to fuels and chemicals. The main purpose of pretreatment is to separate the components of lignocellulosic biomass (Oh et al., 2002), as well as to reduce lignocellulosic biomass crystallinity, render cellulose accessibility, and remove lignin (Sun and Cheng, 2002). Since lignocellulosic materials are very complicated, their pretreatment is not simple either. The best method and conditions of pretreatment depend greatly on the type of lignocelluloses (Taherzadeh and Karimi, 2008). Different types of pretreatment methods were applied depending on the properties of substrate. Garbage (collected from vegetable market)is initially dried and ground into powder. The varying solid loading ratio of Garbage such as 2%, 4%, 6%, (w/v) were mixed with 3% (v/v) sulphuric acid. The mixture was autoclaved at 121ᴼC for 20 minutes and filtered through Whatman no.1 paper. This filtrate was termed as (Garbage Hydrolysate). The pH of the samples was found to be 1.30±0.20.

D. Cell dry weight determination The sample containing comparatively high C/N ratio was chosen as a medium for culturing L.starkeyi. The pH of the medium was maintained at 8.0. The growth of yeast in this medium was compared with the growth of yeast that was cultured in a standard medium containing, Glucose

-

10.00 g/L

Yeast extract

-

3.00

g/L

Peptone

-

5.00

g/L

Malt extract 3.00 g/L 7 conical flasks containing yeast were incubated for 7 days (1 flask for 1 day) at 30˚C in a rotary shaker at 150 rpm. The readings were taken for every 24 hours. After every 24 hours,

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about 25 mL of sample was taken in falcon tubes and centrifuged in laboratory centrifuge at 5000 rpm for 10 minutes. The supernatant was discarded, bottom pellet was dried and the cell dry weights were noted

(w/v)

E. Lipid production The dried biomass was mixed with 5 mL of 4M HCl. It was then boiled in water bath for 10 minutes and placed in ice for 20 minutes. About 10 mL of n-hexane was added to the mixture. This mixture was centrifuged at 5000 rpm for 10 minutes in laboratory centrifuge. The layer separations that was observed after adding n-hexane to the samples. The bottom layer was discarded. The top layer containing lipids was mixed with 2.5 mL isopropanol. The cell suspension, nhexane and isopropanol were in the ratio of 1:2:0.5 (Chi z et al., 2011). The sample was centrifuged and evaporated to obtain lipid yield. Initial weight of the crucible and weight of the crucible after evaporating the solvent was noted. From that lipid yields were calculated.

2%

8271.00

916.20

9.027

2

4%

13637.00

655.40

20.807

3

6%

16619.00

1118.00

14.865

11.344

2

4%

9340.00

578.50

16.145

3

6%

8610.00

646.50

13.318

1

2%

2

4%

3

6%

Total Carbon

Total Nitrogen

(ppm)

(ppm)

9540.00

495.10

19.269

11766.00

599.80

19.617

11392.00

608.50

18.721

C/N

From the tabulations C/N ratio was found to be higher in 4% w/v samples. When the solid loading was increased to 6% w/v, C/N was found to decrease due to the increase in total nitrogen content. it is clear that after overliming, there was a decrease in C/N ratio which was due to the precipitation of inhibitor compounds which were filtered off. After activated charcoal treatment, some of the inhibitors were adsorbed, so the sugar compounds which were previously linked to the inhibitors were exposed. This increase in carbon content has lead to a significant increase in C/N ratio

Table 1: C/N ratio for dilute acid pretreated Garbage

1

477.80

2%

(w/v)

A. Optimisation of Solid Loading The samples with different solid loadings were analysed for C/N ratio using TOC analyser and the results were recorded.

Total Carbon Total Nitrogen (ppm) (ppm)

5420.00

1

Solid S.No. Loading

IV. RESULTS AND DISCUSSION

Solid Loading (w/v)

(ppm)

Table 3: C/N ratio for Garbage hydrolysate detoxified by activated charcoal

Ethonol Emulsuion Test The Ethanol emulsion test is a test that determines the presence of lipids. About 5 mL of ethanol was added to the lipid obtained followed by addition of distilled water. A layer of cloudy suspension was seen in the tubes. This confirmed the presence of lipids.

S.No.

(ppm)

C/N

B. Optimised temperature and pH From the process of optimization of Temperature and pH, it was found that L. starkeyi shows its maximum growth at the temperature of 30⁰C and at the pH of 8. Fig. 1: GROWTH CURVE TEST

Table 2: C/N ratio for Garbage hydrolysate detoxified by overliming Solid S.No. Loading

Total Carbon

Total Nitrogen

C/N

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V. CONCLUSION Biodiesel production has gained importance because of the increasing energy demand and depletion of fossil fuels. Different lignocellulosic materials can be used as starting materials for the growth of oleaginous microorganisms and for the production of microbial lipids. The fermentation of hydrolysates of lignocellulosic materials is of great strategically importance, due to the abundance of the agricultural and forestal residues, offering a renewable feedstock for the production of biofuels. Instead of exploiting food crops for oil production, oleaginous yeasts can be used for producing biodiesel after optimizing the conditions for obtaining higher yield of lipids. These microbial lipids can be used as feedstock for the synthesis of biodiesel by transesterification.

From the growth curve test, it was found that L. starkeyi reaches its stationery phase at fifth day, where the lipid accumulation was found to be maximum.

References

C. Lipid Yield Table 4: Comparison of Lipid yield in Standard medium and 4% w/v GH medium detoxified by activated charcoal S.No. Incubation

Lipid yield (g/25mL)

period (hours)

Standard medium

4% GH medium

1

24

0.055

0.033

2

48

0.056

0.035

3

72

0.061

0.043

4

96

0.066

0.051

5

120

0.072

0.063

6

144

0.065

0.056

7

168

0.059

0.049

From the results given in Table, the maximum lipid yield was observed at 120 hours in both standard medium as well as 4% w/v Garbage hydrolysate medium. After 120 hours the lipid yield was found to decrease in both the medium. So, the optimum incubation period for obtaining maximum lipid from L. starkeyi was 120 hours. In order to maintain growth after 120 hours, the cells had started utilizing the accumulated lipids. So a decrease in lipid yield was observed. Theoretically, maximum lipid yield in standard medium and 4% w/v GH medium detoxified by activated charcoal were 2.880 g/L and 2.520 g/L respectively. .

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