4_Spirogyra Biomass for Bioethanol

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Fuad Salem Eshaq et. al. / International Journal of Engineering Science and TechnologyVol. 2(12), 2010, 7045-7054

Spirogyra biomass a renewable source for

biofuel (bioethanol) Production

*Fuad Salem EshaqDepartment of Chemistry, Centre for Environment,

Jawaharlal Nehru Technological University, Kukatpally, Hyderabad-500085,

Andhra Pradesh (India).

Mir Naiman Ali

Department of Mirobiology, Mumtaz Degree & P.G College,

Malakpet, Hyderabad-500036, Andhra Pradesh (India)

Mazharuddin Khan Mohd.

Department of Mirobiology, Mumtaz Degree & P.G College,

Malakpet, Hyderabad-500036, Andhra Pradesh (India)

ABSTRACT

Biofuels refer to renewable fuels from biological sources that can be used for heat, electricity and fuel. The fuelsobtained from algae are termed as third generation fuels. The production of fuel from algae provides manyadvantages when compared to the fuel produced from other sources like agrobased raw materials. Other thanenvironmental pollution control the algal biofuel will help in reduction of the fuel cost when compared to theagrobased and fossil fuels. In the present study algae specifically Spirogyra was used for the production of bioethanol by the fermentative process. A comparative study was carried out by using chemically pre-treated anduntreated Spirogyra biomass. The Spirogyra has a very simple cell wall made up of cellulose and starch that can beconverted to ethanol by the fermentation process. The Spirogyra biomass was subjected to saccharification process by the fungal organism Aspergillus niger MTCCC 2196 for the hydrolysis, this process was followed by the

fermentation using yeast Saccharomyces cerevisiae MTCC170 for the production of alcohol. A high yield of ethanolwas recorded for untreated Spirogyra biomass when compared to chemically pre-treated biomass. The yield ofalcohol using algal biomass is more when compared to alcohol produced from other sources like agrobased rawmaterials.

Key words: Spirogyra, bioethanol, biofuel, Saccharomyes cerevisiae, Aspergillus niger.

Introduction

In the year 2008, fossil fuel accounted for 88% of the global primary energy consumption (1). The currenttechnological progress, potential reserves and increased exploitation leads to energy insecurity and climate change by increasing greenhouse gas (GHGs) emission due to consumption of energy at higher rate. The use of fossil fuels

is now widely accepted as unsustainable due to depleting resources and the accumulation of GHGs in theenvironment that have already exceeded the “dangerously high” threshold of 450 ppm CO 2 (2). With the increase inanthropogenic GHG emission and depleting fossil reserves, mainly due to large scale use of fossil fuel for transport,electricity and thermal energy generation, it has become increasingly important to develop abatement techniques andadopt policies to promote those renewable energy sources which are capable in sequestering the atmospheric CO 2 tominimize the dependency on fossil reserves and maintain environmental and economic sustainability (1, 3, 4, 5, 6).

The biofuel that is expected to be most widely used around the globe is ethanol, which can be produced fromabundant supplies of starch/cellulose biomass. The most important bioethanol production countries in the world areBrazil, US and Canada (7). Since biomass assimilation by algal growth utilize atmospheric carbon dioxide, their

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biomass for bioethanol production can reduce green house gas levels. In addition, ethanol is less toxic, is readily biodegradable and its use produces fewer air-borne pollutants than petroleum fuel. Under the Kyoto Protocol, theGovernment of Canada has committed to reduce the greenhouse gas emissions by 6% from 1990 levels between2008 and 2012 (8). Ethanol blended gasoline has the potential to contribute significantly to reduce these emissions.It can also be used as a fuel for electric power generation, in fuel cells (thermo-chemical action) and in power co-generation systems, and as a raw material in chemical industry (9). Bioethanol can be employed to replace octaneenhancers such as methylcyclopentadienyl manganese tricarbonyl (MMT) and aromatic hydrocarbons such as benzene or oxygenates such as methyl tertiary butyl ether (MTBE) (8).

Energy conversion utilization and access underlie many of the great challenges of our time, including thoseassociated with sustainability, environmental quality, security and poverty (10, 11). Biofuels are an attractivealternative to current petroleum based fuels as they can be utilized as transportation fuels with little change tocurrent technologies and have significant potential to improve sustainability and reduce GHG emissions (12).Research on improving biofuel production has been accelerating for both ecological and economical reasons, primarily for its use as an alternative to petroleum based fuels (3) Microbial fuel cells (MFCs) are also gettingattention but they need huge improvement s in technologies and also not suitable for transport (13).

Biofuels could play an essential part in reaching targets to replace petroleum based transportation fuels with a viablealternative, and in reducing long term CO2 emission, if environmental and economic sustainability are consideredcarefully (14) they can be direct and immediate replacements for the liquid fuels used for transport and can be easily

integrated to the logistic systems that are operating today (15). In recent years, the use of liquid biofuels in thetransport sectors has shown rapid global growth, driven mostly by policies focused on achievement of energysecurity, and mitigation of GHG emission (16).

First and second- generation biofuels

First generation biofuels which have attained economic levels of commercial production, have been mainlyextracted from food and oil crops (viz. rapeseed oil, palm oil, sugarcane, sugar beet, wheat, barley, maize. etc) aswell as animal fats using conventional technology (17). The liquid biofuels production and consumption growth isincreasing day by day, but their impact towards meeting the overall energy demands in the transport sector willremain limited due to competition with food and fiber production for the use of arable land, high water and fertilizerrequirements, lake of well managed agricultural practices in emerging economies, biodiversity conservation andregionally constrained market structures.

Global biofuel production has been increasing rapidly over the last decade, but the expanding biofuelindustry has recently raised important concerns. In particular, the sustainability of many first generation biofuels(primarily from food crops such as grains, sugar cane and vegetable oils) has been increasingly questioned overconcern such as reported displacement of food crops, effects on the environment and climate change.

The limitation of first generation biofuels produced from food crops have caused greater emphases to be placed by second generation biofuels produced from lignocellulosic feed stocks, although significant progresscontinue to be made to overcome the technical and economic challenges, second generation biofuels production willcontinue to face major constraints to execute commercial deployment (18). The logistics of providing a competitivesupply of biomass feedstock to a commercial plant is challenging, as is improving the performance of the conversion process to reduce costs.

The most positive impact of biofuels is the reduction of the GHGs emissions in the production and

consumption. This is because biomass production utilized atmospheric CO2 and biomass is renewable. On the otherhand, mass production of biofuel can lead to the increase of GHG emissions by the utilization of fossil transportationfuels in the complicated logistic needed for biomass cultivation, collection, transportation and distribution of biofuels. The deforestation or clearing grasslands to be used for biomass cultivation that leads to the emission ofCO2 captured in biomass and soil into the atmosphere. The lower content of sulfur reduces the SO2 emissions (9).

Bioethanol from algae, the third generation biofuels.

Algae are gaining wide attention as an alternative renewable source of biomass for the production of bioethanol,which is grouped under the “third generation biofuels” (17). The major drawbacks of first and second generation

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biofuels are overcome to a greater extent by third generation biofuels. The concept of using algae as energyfeedstock dates back to the late 1950s (19) but a concerted effort began with the oil crisis in 1970s. Over the lastthree decades there has been extensive research on algal biofuels production and the use of algae for CO2 bioremediation (20). The US Department of Energy (DOE) devoted $25 million to algal fuels research in its aquaticspecies program at the National Renewable Energy Lab (NREL) in Golden, Colorado from 1978 to 1996. The program gave way to mile stone advances that set the stage for algal biofuel research today (21).

Algae represent a vast variety of photosynthetic species dwindling in diverse environments (22, 17). They may beautotrophic or heterotrophic. The autotrophic algae use photosynthesis to harness sunlight and fix the inorganiccarbon from atmospheric CO2 which is then assimilated in the form of reserve food materials such as carbohydrate.There are many algal species which are heterotrophic and they are able to take up small organic molecules in theenvironment and turn them into the building blocks of their own which are mainly fat or oil and proteins. There arecertain algal species which can use neither inorganic carbon (CO2) from atmosphere nor organic carbon from theenvironment and this process is called mixotrophy. Through any of the three processes, algae can producecarbohydrates, lipids and proteins over a short period of time, which can then be processed to generate biofuels.Some algae can even serve as self biorefinery for ethanol production during anaerobic dark condition by utilizingtheir photosynthates. There are several reports documenting the potential of algal biomass to generate biofuels (23,24, 25).While considering algal biofuels, the first point that comes to view is about the biodiesel, as many of thealgae are oleaginous in nature and are exploited for the production of biodiesel. Besides biodiesel, algae can becultivated and can be used as feedstock for the production of bioethanol. The algal starch, cellulose or other

accumulating carbohydrates can be used for the production of ethanol after hydrolysis.

Microalgae are thought to be one of the earliest life forms on earth (26) and they are the fastest growing plants in theworld, since they can inhabit diverse ecological habitats ranging from fresh water, brackish water, or sea water, theyare able to thrive in various extreme temperatures and P H conditions. These peculiarities make microalgae the mostabundant organism on earth. There has been remarkable surge in research to investigate the utilization of microalgaeas an advanced energy feedstock for bioethanol production (27, 28,29). Microalgae like Chlorella, Dunaliella,Chlamydomonas, Scenedesmus, Spirulina are known to contain large amount (> 50% of the dry weight) of starchand glycogen, useful as raw materials for ethanol production (30). Microalgae can assimilate cellulose which canalso be fermented to bioethanol (19).

Spirogyra is one of the commonest of green algae abundant in spring. It is found in bright green freefloating masses in the still water fresh water ponds, pools, lakes and ditches and also in flowing streams. Accordingto Randhawa1959 genus includes about 289 species and of these 94 have been reported from India. The plant body

is thallus which consists of a long green cylindrical thread about 1/10mm across and several centimeters long. It issilky, hair like unbranched and often called a filament. Each cell consists of cell wall enclosing the protoplast. Thecell wall consists of two concentric layers. The inner is cellulose in nature. The outer is a pectose layer covered withmucilage sheath. In the present study bioethanol was produced by a fermentation process using alga Spirogyra

biomass, the powdered biomass was processed and treated chemically. Fermentation was carried out in two steps-saccharification and fermentation using Aspergillus niger MTCC 2196 and Saccharomyces cerevisiae MTCC 170.

Materials & Methods:

Microorganisms and culture:

1. Spirogyra: The alga Spirogyra was collected from Mir Alam Lake-situated in Hyderabad city (India).

The algal mat was collected in sterile containers and transferred to the laboratory.

2.

Fungal cultures- Two fungal cultures Aspergillus niger MTCC2196 and Saccharomyces cerevisiae MTCC 170 were procured from MTCC (Microbial Type Culture Collection Centre and Gene Bank)Chandigarh. The fungi Aspergillus niger was cultured and maintained on potato dextrose agar mediumat 300C. The yeast Saccharomyces cerevisiae was cultured and maintained on YPD (Yeast extract, peptone and dextrose) agar media at 300C.

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Methods:

Identification of the algal sample: The algal sample was identified by the methods of Smith (31) andupon microscopic examination it was identified as Spirogyra species.

Processing of biomass: The sample was subjected to sun dryness in which about 40% moisture content

was observed. The dried Spirogyra biomass was grinded and filtered through 1mm sieve. Fine powder ofSpirogyra biomass thus obtained was used for all fermentation experiments by taking two variations: halfof the biomass was chemically pre-treated and remaining biomass was left untreated.

Chemical pre-treatment of Spirogyra biomass: The Spirogyra biomass was chemically pretreated with1%NaOH for a period of 2 hrs (32, 33).

Chemical analysis of Spirogyra biomass: Spirogyra biomass was subjected to the estimation of totalsugars (34), reducing sugars (35) and cellulose content (36).

Fermentation Studies:

For comparative studies Spirogyra biomass was used for fermentative production of bioethanol in twovariations- chemically pre-treated form and untreated form. Fermentation studies were performed in 250

ml Erlenmeyer flasks with three different variations:i. 5g of the biomass in 100ml of distilled water,

ii.

5g of the biomass in 100ml distilled water containing 0.5% of lactose andiii. 5g of the biomass in 100ml of synthetic media containing the following components

(g/100ml): L-Glutamic acid, 0.03; NH4 No3, 0.14; KH2PO4, 0.2; CaCl2, 0.03; MgSO4, 0.03;Proteose peptone, 0.75; FeSO4, 0.5; MnSO4, 0.16; ZnSO4, 0.14; Tween 80, 2%.

The flasks were autoclaved at 15lbs for 15 minutes and inoculated with mycelial mat of Aspergillus

niger . The same process was followed for both the chemically pretreated biomass and the untreated biomass.

Saccharifiation of Spirogyra biomass by Aspergillus niger:For the saccharification of algal biomass developed mycelial mat of Aspergillus niger was used.

Aspergillus niger is cellulolytic and amylolytic in nature as it produces cellulases and amylases. These

enzymes hydrolyze the cellulose and starch present in Spirogyra and releases free sugars. Thesaccharification was carried out for a period of six days at 300C and the process was monitored every24 hrs for sugars released by the method of Miller D.L., (37).

Fermentation by Saccharomyces cerevisiae:After six days of saccharifiation mycelial mat of Aspergillus niger was removed under asepticconditions and 10% of Saccharomyces cerevisiae was added to the flasks for fermentative productionof bioethanol. The process was carried out for a period of another six days at 30 0C during which every24 hours samples were taken for the estimation of alcohol (bioethanol) by the method of Caputi et al.,(38).

Results & Discussion:

The alga – Spirogyra biomass was selected as a substrate for bioethanol (ethanol) production in the present work as it is rich in polysaccharides- starch and cellulose. Since the industrially usedSaccharomyces cerevisiae is non celluolytic and non amylolytic in nature, the fungal culture A.niger was employed to hydrolyse and produce simple sugars which can be directly utilized bySaccharomyces cerevisiae for ethanol production. The processed Spirogyra biomass was pre-treatedwith a weak alkali instead of acid, as acid pre-treatment results in production of toxic substanceswhich decreases the fermentation efficiency of Saccharomyces cerevisiae. Enzyme hydrolysis is anatural and ideal method for conversion of cellulose materials to sugars which could be used as asource of food, fuel or chemicals Martin. et al., (39). Hence in the present study Aspergillus niger

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was used as a source of cellulase enzyme for saccharification of Spirogyra biomass into simplesugars. As discussed in the material and methods for the present study, ethanol production wascarried out by stationary fermentation method.

Renewable marine resources including macro- and microalgae could be applied to convert to energyand chemical compounds. Previous work, reported that as applications of renewable marineresources, methane production using marine microalgae biomass (40) and method utilizing floatingceramic supports for the cultivation of marine microalgae have been developed to take advantage ofthis vast resource (41). Applications utilizing a large surface area of an ocean can produce vastamounts of marine biomass and useful material.

Chemical analysis of Spirogyra biomass: Spirogyra biomass was subjected to the estimation of totalsugars, reducing sugars and cellulose content. The contents of sugars and cellulose are shown inTable-1.

Table-1- Chemical composition of Spirogyra biomass

Components Composition based on dry cell

mass (% w/w)

Analytical method

Cellulose 19 % Spectrophotometric method of

Sadasivam et al., 1992

Total sugars 4.2% Spectrophotometric method of

Hedge et al., 1962

Reducing sugars 2.7% Spectrophotometric method of

Krishnaveni et al., 1984

Saccharification and ethanol production from untreated Spirogyra biomass:

Using the fungi Aspergillus niger, a source for starch and cellulose hydrolysis, a saccharification processwas performed under optimal conditions. The degree of saccharification was evaluated by the amount ofsugar released by the liquefaction of starch and cellulose by the method of Miller, G.L. (1972). Theeffective usefulness of the pre-treated Spirogyra biomass as a medium for yeast growth was furtherestimated by checking the ethanol production by the method of Caputi et al. (1968). The results of sugarreleased are shown in Table-2 and Figure-1 and the amount of ethanol produced is shown in Table-3 andFigure-2. The highest sugar was released on 6th day of saccharification in all the flasks with distilled water,lactose and synthetic media. Accordingly the amount of bioethanol produced was also more on the 6 th day.The trend of sugar released and bioethanol produced gradually increased from 1st day to 6th day with slightfluctuations except with lactose where the highest quantity of sugar was released on the 1 st day andgradually decreased up to 6th day leading to highest bioethanol production on the 1st day and lowest on the6th day indicating that lactose is acting as inducer for cellulase activity, hence more sugar was released onthe 1st day leading to highest production of bioethanol. This trend gradually decreased as the quantity of

lactose decreased from 1st day to 6th day. In both cases trend indicates that production of bioethanol wasdirectly proportional to availability of sugar for fermentation.

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Table-2 BIOETHANOL PRODUCTION BY STATIONARY METHOD FROM UN-TREATED SUBSTRATE (Sugar released g/100g)

SUBSTRATE DAY1 DAY2 DAY3 DAY4 DAY5 DAY6

Spirogyra Biomass

With Distilled Water

2.5g 5.0g 7.0g 7.5g 15.0g 19.0g

Spirogyra Biomass

With Synthetic media

9.0g 10.0g 14.0g 16.0g 15.5g 20.0g

Spirogyra Biomass

With Lactose

19.5g 17.0g 12.0g 10.5g 8.5g 5.0g

Figure-2

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Table-3

BIOETHANOL PRODUCTION BY STATIONARY METHOD FROM UN-TREATED SUBSTRATE (ETHANOL PRODUCED g/100g)


Spirogyra

Biomass With

Distilled

Water

0.15g 0.35g 0.80g 0.93g 1.27g 1.92g

Spirogyra

Biomass With

Synthetic

media

0.23g 0.78g 2.43g 2.67g 5.80g 8.0g

Spirogyra

Biomass With

Lactose

7.0g 5.9g 3.5g 1.8g 1.0g 0.3g

Figure- 3

Saccharification and ethanol production from chemically pre-treated Spirogyra biomass:

In these sets of experiments, also Aspergillus niger was used for saccharification of Spirogyra biomass andSaccharomyces cerevisiae was used for fermentative production of bioethanol as these two organisms were provedvery promising in the present study and also in the previous study. In stationary fermentation with chemicallytreated Spirogyra biomass, highest sugar released was observed on the 5th day of saccharification in all flaskscontaining distilled water, lactose and synthetic media. Accordingly the alcohol was also produced more on the 5th day of fermentation.

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Table-4

BIOETHANOL PRODUCTION BY STATIONARY METHOD FROM PRE-TREATED SUBSTRATE (Sugar released g/100g)


Spirogyra

Biomass With

Distilled

Water

3.5g 4.05g 4.25g 4.5g 5.0g 4.25g

Spirogyra

Biomass With

Synthetic

media

3.0g 3.25g 4.0g 5.5g 7.0g 5.0g

Spirogyra

Biomass With

Lactose

5.25g 6.0g 9.0g 13.25g 16.25g 7.0g

Fig – 3

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Table-5

BIOETHANOL PRODUCTION BY STATIONARY METHOD FROM PRE-TREATED SUBSTRATE (ETHANOL PRODUCED g/100g)


Spirogyra Biomass With

Distilled

Water

1.0g 1.20g 1.27g 1.71g 3.86g 1.42g

Spirogyra

Biomass With

Synthetic

media

1.59g 1.95g 2.50g 3.32g 3.64g 3.15g

Spirogyra

Biomass With

Lactose

0.85g 0.66g 0.62g 0.46g 0.35g 0.23g

Fig – 4

Conclusion:

From the present study it can be concluded that algal biomass is more beneficial raw material than agrobased rawmaterials for bioethanol production as it is available abundantly in fresh water as well as marine eco-system andmore importantly it is renewable. The studies also conclude that in general, pretreatment with chemicals are notrequired for the algal material particularly for Spirogyra. In practice chemical treatments were employed to removeor denature unwanted materials (biomass) which are present along with cellulose and starch in agriculturally basedraw materials which are extensively used in bioethanol production. As Spirogyra cell wall is made up of purecellulose and simple starch it is not demanding any type of pretreatment. In fact pretreatment will damage thecellulose leading to less yield of alcohol when compared with untreated spirogyra biomass (Table-5 and Figure 4).Further it paves the way for less expensive method by cutting off the pretreatment cost.

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4_Spirogyra Biomass for Bioethanol