INTRODUCTION:

Bioethanol:

Bioethanol is the most common biofuel, accounting for more than 90% of total biofuel usage. Conventional production is a well known process based on enzymatic conversion of starchy biomass into sugars, and/or fermentation of 6-carbon sugars with final distillation of ethanol to fuel grade. Ethanol can be produced from many feed stocks, including cereal crops, corn (maize), sugar cane, sugar beets, potatoes, sorghum, cassava, micro and macroalgae.

The production of bioethanol involves the fermentation of sugars by microorganisms to produce ethanol. As many sugars are not freely available but form part of structural and storage carbohydrates there is a requirement for treatments such as altered temperature, pH and addition of enzymes to hydrolyse the sugars prior to fermentation. Distilled bioethanol can be blended with petrol (gasoline) and used in vehicles without alterations if mixed at 5% (v/v) (The European Parliament and the Council of the European Union 2003) or up to 85% (v/v) in flex-fuel cars currently being produced by several different vehicle manufacturers (NEVC 2008).

As the CO2 released from combusted bioethanol was previously removed from the atmosphere by the plant into organic carbon, the return of this gas is considered ‘carbon neutral’. Use of bioethanol additionally reduces the amount of petrol combusted per kilometre, lowering demand and so increasing the security of supply.

Bio-ethanol is regarded as a promising alternative energy source, which is both renewable and environmentally friendly. During bio-ethanol production, the composition of media affects the physiological state and, consequently, the fermentation performance of the microorganism employed (Hahn-Hägerdal et al., 2005).

Macroalgae:

Macroalgae are multicellular plants growing in salt or fresh water. Macroalgae is one of the economically and ecologically living resources in the oceans. Macroalgae are present low-cost cultivation and harvesting possibilities, but most species are low in lipids as well as carbohydrates With processes such as cellulosic fermentation (for deriving ethanol), gasification (for deriving biodiesel, ethanol and a wide range of hydrocarbons), or anaerobic digestion (for methane or electricity generation), it is possible today to usemacroalgae as the feedstock for biofuels. Macroalgae culture has been recognized as an eco-friendly treatment and natural biodiversity mechanism in reducing pollution load from water, as well as can be used for controlling CO₂ emissions from the atmosphere.

Seaweeds have been used since ancient times as food, fodder, fertilizer and as source of medicine today seaweeds are the raw material for many industrial productions like agar, algin and carrageenan but they continue to be widely consumed as food in Asian countries. They are nutritionally valuable as fresh or dried vegetables, or as ingredients in a wide variety of prepared foods [2]. In particular, certain edibl seaweeds contain significant quantities of protein, lipids, minerals and vitamins, although nutrient contents vary with species, geographical location, season and temperature

The nutritional properties of seaweeds are not yet noted and they are usually estimated from their chemical composition alone [8, 9]. Compared to land plants, the chemical composition of seaweeds has been poorly investigated and most of the available information deals only with traditionally Japanese seaweeds. The chemical composition of seaweeds varies with species, habitats, maturity and environmental conditions.

The protein content in the marine algae was estimated by. Chidambaram and Unny et al., analyzed proteins in the species of Sargassum, Turbinaria and Gracilaria Neela et al., estimated the protein, fat, calcium phosphorous, iron, iodine and vitamin-C contents in Gracilaria sp. Gracilaria lichenoides, Hypnea sp. And Ulva lactuca. In CMFRI, studies were carried out on the chemical composition of the marine algae growing in the vicinity of Mandapam.

Brown algae as a seaweed is evolutionarily diverse and abundant in the world’s oceans and coastal waters. The seaweed industry has an estimated total annual value of 5.5 to 6 billion US$, with 7.5 to 8 million tons of naturally grown and cultivated seaweed harvested worldwide. Seaweed is mainly used in food products for human consumption, which generates approximately 5 billion US$ per year, with the remainder used for production of extracted hydrocolloids, fertilizers, and animal feed additives (Adams et al., 2009; McHugh et al., 2003). Brown seaweed has a high content of easily degradable carbohydrates, making it a potential substrate for the production of liquid fuels. The carbohydrates of brown seaweed are mainly composed of alginate, laminaran, mannitol, fucoidan and cellulose in small amounts (Horn et al., 2000).

Applications of bioethanol:

Bioethanol essentially has different types of application:

· It can be used as a motor fuel, in practically pure state or blended in different proportions with conventional 95-octane fuel (depending on whether the percentage of biofuel is 5%, 10% or 85%, these blends are E5, E10, E85 respectively). In this form it can be used to power automobiles. In some countries it is also used in buses and industrial vehicles.

· Bioethanol is also used as an additive for traditional petrol in the form of Ethyl tertiary butyl ether (ETBE). The ethanol is mixed with isobutene (a non-renewable petroleum derivative) to form ETBE. Because of its high octane rate.

· as a fuel for power generation by thermal combustion

· as a fuel for fuel cells by thermo chemical reaction

· as a fuel in cogeneration systems

· as a feedstock in the chemicals industry

MATERIALS AND METHODS:

Sample collection:

YS1and YS2were collected from the Mandapam coast of Rameshwaram. The samples were thoroughly rinsed with fresh water to remove salt and foreign materials such as epiphytes, shells, sand, etc. All cleaned seaweeds were dried at 60°C in an air oven until they had constant weight. After being ground into fine powder that could pass through a 0.5 mm mesh sieve, the samples were stored cold condition (4°C) for further analysis.

PROXIMATE BIOCHEMICAL ANALYSIS:

Estimation of moisture content:

The moisture content of the macroalgae was estimated by drying the known quantity of wet samples in glass container and YS1 and YS2 samples are dried in hot air oven at 60⁰C until samples are dry properly.

The difference in weight between wet weight and dry weight was calculated and expressed as percentage of moisture content of the sample. Percentage was calculated by formula

Moisture% =

Wet weight of sample – X100

Estimation of total lipid:

10 mg of dried sample 10 mL of Chloroform: Methanol mixture {2:1r/r}. The homogenate was centrifuged at 200 rpm for 10 min. The supernatant then washed with 0.9% KCl solution to remove the non lipid contents and allowed to separate.

The upper phase was discarded. The lower phase was allowed to dry in an oven and the weight was taken. The lipid content is expressed as

Lipid % = X100

Estimation of protein by Biurette methods:

0.2-10 mL of working standard solution was pipette out into a series of test tube as S1, S2, S3, S4 and S5.

0.25mL of test sample was pipette out into test tube marked as T1 and T2.

The volume of standard and test solution was diluted to 1mL using distilled water.

The test tube marked as blank contains 1ml of distilled water.

Add 4ml of Biurette solution in all test tubes.

Keep at room temperature for 10 min.

Read intensity at 600nm.

Estimation of carbohydrates by Phenol –Sulphuric acid methods:

Weigh 100mg of the sample into a boiling tube.

Hydrolysate keeping it in boiling water bath for 3 hrs with 5mL of 2.5N HCl and cooled to room temperature.

Neutralize it with solid sodium carbonate until the effervescence ceases.

Make up the volume to 100 mL and centrifuge.

Pipette out 0.2 to 1mL of working standard into series of test tubes.

Pipette out 0.1 and 0.2 mL of sample solution in two separate test tubes. Make up the volume in each tube to 1mLwith water.

Set the blank with 1mL of water.

Add 1mL of phenol solution to each tube.

Add 5mL 96% sulphuric acid to each tube and shake well.

After 10 min shake the contents in the tube and place in a water bath at 25-30⁰C for 20 min.

Read the color at 490nm.

Calculate total carbohydrate content from the standard graph juice.

Estimation of ash content:

Weigh about 5g of sample into the crucible. Heat over oe Bunsen flame with lid half covered. When fumes were no longer produced. Place crucible and lid in furnace.

Heat at 550°C over night. During heating; do not cover the lid. Cool down in the desiccator.

Weigh the ash with crucible and lid when the sample turns to gray. If not the crucible and lid to the furnace for the further ashing. The ash content is expressed as

Ash (%) = X 100

ISOLATION AND IDENTIFICATION OF MICROORGANISMS:

(a) Isolation of YS1:

Rotten grape fruit sample were taken and each variety of 1g was taken and diluted serially upto10-6 about 0.1ml of serially diluted sample was taken and done the spread plate technique by using Yeast extract chloramphenicol agar plate. The inoculated plates were incubated for 48hr at 30ºC.

(b) Isolation of YS2:

Rotten orange fruit sample were taken and each variety of 1g was taken and diluted serially upto10-5 about 0.1ml of serially diluted sample was taken and done the spread plate technique by using Rose Bengal chloramphenicol agar plate. The inoculated plates were incubated for 48hr at 32ºC.

Identification of microorganisms:

The isolates were characterized morphologically, culturally and physiologically byusing satandard (CMPT mycology plus 2008) and DNA sequencing analysis. DNA sequencing was based on the dideoxymediated chain termination method using a fluorescent-labelled terminator. The DNA fragments were sequenced by the progen biotech using BioEdit Sequence Alignment Editor (Version 7.1.3.0). DNA isolation was conducted by employing a DNA extraction kit of Nucleon PHYTOpure (Amersham Life Science).Primer of YS1 UL18F (5’ - TGTACACACCGCCCGTC - 3’), UL28R: (5’ ATCGCCAGTTCTGCTTAC -3’) and UL620R (5’ - TGGTCCGTGTTTCAAGA - 3’) and primer of sample YS1were used for PCR amplification. PCR products were subsequently purified based on the polyethyleneglicol (PEG) precipitation method (Hiraishi et al. 1995) and followed with a sequencing process using Sequence Alignment Editor (Version 7.1.3.0).Sequences were further used for taxa identification using the BLAST program http://www.ncbi.nlm.nih.gov/BLAST/) and compared to he GenBank database.

Inoculam preparation:

To prepare the starter culture, 50 mL of the growth medium taken in 250 mL capacity conical flask. The medium was sterilized at 121°C and 15 psi pressure for 20 min, and inoculated with a loopful of the strain. The flasks were incubated at 30°C for 24 h.

Fermentation:

The fermentation medium containing (w/v) glucose (2.0g), yeast extract (0.5g), potassium dihydrogen orthophosphate (0.2g), ammonium sulphate (0.5g) and magnesium sulphate (0.5g) was used for the production of ethanol. In fermentation medium macroalgae hydrolysate was used in place of glucose. The medium was sterilized by autoclaving, inoculated with 24-h-old 10% (v/v) starter culture and incubated at 32⁰C for one week. Microorganisms YS1 and YS2used in the fermentation process. Baker’s yeast S. cerevicea used as the model organism for ethanol production.

Purification of ethanol:

Fractional distillation:

The fermented broth was dispensed into round-bottom flasks fixed to a distillation column enclosed in running tap water. A conical flask was fixed to the other end of the distillation column to collect the distillate. A heating mantle with the temperature adjusted to 70°C was used to heat the round-bottomed flask containing the fermented broth.

Analytical procedures:

Cell dry weight was determined by centrifugation of 20 mL of the yeast culture in a pre-weighed dried tube, washing of the pellet with 20 Ml of distilled water, drying overnight at 105 ºC and weighing. The total reducing sugars and total sugar were estimated by using anthrone method and anthrone reagent method.

3. RESULTS:

3.1 SAMPLE COLLECTION:

Gracilaria species and Sargassum species was collected from Mandapam coast of Rameshwaram.

Fig 1 Gracilaria species

Fig 2 Sargassum species

3.2 PROXIMATE BIOCHEMIOCAL ANALYSIS:

The moisture content of the macroalge varied from 74.94% to 79.5%; the maximum moisture was recorded in Gracilaria species (79.5%) and the minimum was Sargassum species (74.94%).

In that protein content varied from 7.76±0.64% to 8±0.2%; maximum protein was recorded in Sargassum species (8±0.2%) followed by Gracilaria species (7.76±0.64%).

The lipid content of seaweeds varied from1.4±0.30% to 2.33±0.31%; in that the maximum lipid content was observed from Sargassum species(2.33±0.31%) followed by Gracilaria species(1.4±0.3%)

The carbohydrate content varied from 23.5±0.62% to 54.47±0.14%, in that the maximum carbohydrate concentration was recorded from Gracilaria species (54.47±0.14%) followed by Sargassum species (23.5±0.62%).

The ash content of the macroalgae Gracilaria speciesand Sargassum specieswas recorded as 25.48% and 21.8% respectively.

The chemical composition of Gracilaria spcies and Sargassum spcies under the present study is given in table.

Table 1 Biochemical composition (% w/w)

BIOCHEMICAL COMPOSTION	*Gracilaria species*	*Sargassum species*
Moisture content	79.5%	74.94%
Total protein	7.76±0.64	8±0.2
Total carbohydrate	54.47±0.14	23.5±0.62
Total lipid	1.4±0.30	2.33±0.31
Ash content	25.48	21.8

ISOLATION OF MICROORGANISM:

Table 2 Characterization of microorganisms:

CHARACTERIZATION	*Gracilaria species*	*Sargassum* species
Shape	Oval	Circle
Texture	Smooth	Smooth
Color	Cream	Cream
Elevation	Raised	Flat
Margin	Undulate	Entire
Size	0.4cm	0.9cm
Colony on the plate	Single	Cluster
Surface	Shiny	Shiny

Table 3 Carbohydrate fermentation test

S.NO	CARBOHYDRATE	*Gracilaria species*	*Sargassum species*
1	Glucose	+	+
2	Fructose	+	+
3	Galactose	_	+
4	Sucrose	+	+
5	Maltose	_	_
6	Lactose	_	_

Fig Hanseniaspora opuntiae

DNA SEQUENCING ANALYSIS:

DNA sequencing analysis of YS1:

In BLAST analysis with NCBI database, the obtained ITS region showing 100% sequence coverage and 99% sequence similarity to Zygosaccharomyces bailii (ref ITS BLAST Result). LSU region BLAST analysis also showing 79% sequence coverage and 99% similarity to YS1 DAOM (Accession No. JN938914). Therefore, this isolate may be YS1.

DNA sequencing analysis of YS2:

In BLAST ANALYSIS WITH NCBI database, the obtained ITS region showing 100% sequence coverage and 99% similarly to YS2.

FERMENTATION:

The fermentation process of this study revealed that ethanol production from Gracilaria specieswas maximum (33.4 (g/L)) while Sargassum specieswas used followed by YS1was minimum (21.06 (g/L)).Amount of ethanol produced and the biomass concentration given in the table

The fermentation process of this study revealed that ethanol production from Sargassum specieswas maximum (22.4 (g/L)) while Gracilaria specieswas used followed by YS1was minimum (17.06 (g/L)).Amount of ethanol produced and the biomass concentration given in the table

PURIFICATION OF ETHANOL:

Fractional distillation:

The purification section separates the fermentation broth into water, anhydrous ethanol, and solids. Distillation was used to recover ethanol from the raw fermentation liquid and produce 99.5% ethanol

CONCLUSIONS:

Gracilaria speciesand Sargassum species was collected from Mandapam coast of Rameshwaram. The moisture content of the macroalge varied from 74.94% to 79.5%. Maximum protein was recorded in Sargassum species (8±0.2%) followed by Gracilaria species (7.76±0.64%). The maximum lipid content was observed from Sargassum species (2.33±0.31%) followed by Gracilaria species (1.4±0.3%). The maximum carbohydrate concentration was recorded from Gracilaria species (54.47±0.14%) followed by XS2 (23.5±0.62%).The ash content of the macroalgae Gracilaria species and Sargassum species was recorded as 25.48% and 21.8% respectively. In BLAST analysis with NCBI database, the obtained ITS region showing 100% sequence coverage and 99% sequence similarity to YS1 (ref ITS BLAST Result). LSU region BLAST analysis also showing 79% sequence coverage and 99% similarity to YS1strain DAOM (Accession No. JN938914). Therefore, this isolate may be YS1. The dilute acid hydrolysis process of macroalgae Gracilaria species and Sargassum species, we found that H₂SO₄ was much better than HCl, H₃PO₄ or malic acid. The dilute alkali hydrolysis process of macroalgae Gracilaria species and Sargassum species; we found that NaOH was much better than KOH. The enzyme pretreatment process on macroalgae cellulose was much better than pectinace. The fermentation process of this study revealed that ethanol production from Gracilaria species and Sargassum species was maximum while Sargassum species was used followed by YS1was minimum.99.5% ethanol was separated in the purification section.

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Received on 27.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 157-162

S.NO	MICROORGANISM	BIOMASS CONCENTRATION (g/L)		AMOUNT OF ETHANOL (g/L)
S.NO	MICROORGANISM	INITIAL	FINAL	AMOUNT OF ETHANOL (g/L)
1	Sargassum species	0.0001	6.1±0.2	47.5
2	YS2	0.0001	6.0±0.6	21.06
3	Gracil ariaspecies	0.0001	5.6±0.9	33.4

S.NO	MICROORGANISM	BIOMASS CONCENTRATION(g/L)		AMOUNT OF ETHANOL (g/L)
S.NO	MICROORGANISM	INITIAL	FINAL	AMOUNT OF ETHANOL (g/L)
1	Sargassum species	0.0001	5.9±0.2	37.5
2	YS1	0.0001	5.2±0.6	17.06
3	Sargassum species	0.0001	5.1±0.9	22.4