BIODIESEL PRODUCTION

A DISSERTATION REPORT SUBMITTED FOR THE FULFILMENT THE DEGREE OF

BACHELOR OF SCIENCE IN BOTANY

HARI PRASAD PATEL

(Enrollment Num. GGV/20/03620, Roll no. 20203015)

Under the supervision of

VIVEK KUMAR PATEL

ASSISTANT PROFESSOR

Dept. of Botany, GGV Bilaspur, Chhattisgarh, India

DEPARTMENT OF BOTANTY
GURU GHASIDAS VISHWAVIDYALAYA
(A CENTRAL UNIVERSITY)
 CERTIFICATE

This is to certify that dissertation report entitled “BIODIESEL PRODUCTION” is based on original work
done by HARI PRASAD PATEL, student of B.Sc. Botany (Hons.) session 2020-2023, Roll No: 20203015,
Enrollment No: GGV/20/03620. It represents entirely an independent work of the candidate. This is an
original work and the report has not been submitted to any other institution for the award of any degree or
diploma.

Forwarded to the controller of the examination, Guru Ghasidas Vishwavidyalaya, Bilaspur (C.G) in partial
fulfillment of the degree of “Bachelor of Science” in Botany.

Place: Bilaspur Signature of supervisor

Date;

Forwarded to the controller of examination, Guru Ghasidas Vishwavidyalaya, Bilaspur in partial fulfillment
of the Bachelor of Science in Botany

Place: Bilaspur Prof. D. K. Patel

Date: Head of the department

Botany Department

GGV, Bilaspur
 DECLARATION
I HARI PRASAD PATEL, hereby declare that the work presented in this project dissertation entitled
“BIODIESEL PRODUCTION” submitted for partial fulfillment of the degree of Bachelor of Science in
Botany has been carried out at Department of Botany, Guru Ghasidas Vishwavidyalaya, Koni, Bilaspur
(C.G.).

The work was carried out by me under the supervision of VIVEK KUMAR PATEL, Department of Botany,
Guru Ghasidas Vishwavidyalaya, Koni-Bilaspur (C.G.). The work done in the present dissertation is original
and has not been submitted to any other institution for the award of any degree or diploma.

HARI PRASAD PATEL

B.Sc. Botany (Hons.)

Department of Botany

GGV, BILASPUR (C.G.)

Place: Bilaspur

Date:
 ACKNOWLEDGEMENT
It is my great pleasure to express my sincere gratitude to my supervisor Vivek Kumar Patel
Assistant professor, Department of Botany, GGV, Bilaspur (C.G.), under whose guidance the
present project work has been completed. His valuable time, valuable suggestions, and selfless
effort helped me a lot in completing the work.

I wish to acknowledge Dr. D. K. Patel, HOD, Dept. of Botany, GGV, Bilaspur (C.G.), who gave me
the opportunity to undergo project work.

I would like to thank Dr. A. K. Dixit, Dr. S. K. Shahi, Dr. S. K. Prajapati, Dr. S. K. Pandey, Dr S.
Shweta, Dr. Deepanker Yadav, Dr. R. K. Ahirwar, Dr. Vineeta Dixit, Dr. Neelima Maravi, Dr. Amar
Abhishek, Dr. Preeti Verma and Mr. Deepak kumar Singh for their kind encouragement.

I would like to thank Mr. Pankaj Kumar Sahu, Ph.D. scholars for his help and support.

I would like to thank all the Ph.D. scholars of the botany department, my classmates, and those who
have directly or indirectly helped me to complete my work.

Hari Prasad Patel

B. Sc. VI Semester

BOTANY (HONOURS)

Department of Botany

GGV, Bilaspur

Roll No.- 20203015

ABSTRACT

The scarceness of fossil-based fuels and the environmental impact produced by the conventional sources of
energy over the planet, has lead new research work to seek the sustainable sources of clean energy. Biofuel
is committed to becoming a worldwide leader in the development and deployment of renewable energy
resources. Biodiesel is an alternative fuel, made from renewable biological sources. Biodiesel can also be
produced from microbial sources like algae, bacteria and fungi. Here, we present an overview of biological
living things used in the biodiesel production and recent advances. The mono-alkyl esters, most commonly
the methyl esters, of vegetable oils, animal fats or other materials consisting mainly of triacylglycerols, often
referred to as biodiesel, are an alternative to conventional petro diesel for use in compression-ignition
engines. The fatty acid esters that thus comprise biodiesel largely determine many important fuel properties.
In turn, the composition of the biodiesel depends on the composition of the parent feedstock because
feedstock’s with widely varying. The use of different feedstock is also significant under aspects of increasing
biodiesel supply and socio-economic issues. In this article, biodiesel production is briefly described, followed
by the different biological sources used for biodiesel production.
 INTRODUCTION

Diesel that originates from plants or animals is referred to as "biodiesel." Biodiesel is defined as a fuel made
of monoalkyl esters of long fatty acids generated by vegetable oils or animal fats and is a renewable source
(Jungeng et al., 2010). These fuels may take the form of animal or vegetable fats that go through chemical
or natural modification in order to be used in a number of engines. Biofuels are created from environmentally
friendly energy sources like biological components from living things and can also be made from garbage
that decomposes biologically. The term "biofuel" is not the same as "fuel derived from fossil sources," as
biofuels have a higher carbon content and produce fewer emissions when consumed (Marshall et al., 1995).

Currently, diesel is the most commonly employed fuel in the world, used in applications that include
transportation, agriculture, power generation, and some industrial ones. The alternative must be observed to
provide long-term energy security. Vegetable oil has the capacity to replace diesel and serve as the diesel
engine's fuel [Venkanna K. 2013]. By 2015, biofuels will be able to supply 27% of all fuel specifications,
resulting in a nearly 2.1 Gt/year lowering of CO2 emissions [Atabani et al., 2014].

Diesel engine pollutants, which have an enormous impact on both the surroundings and human health, are
one of the most significant environmental pollution sources. Due to a variety of causes such as growing
petroleum prices, global environmental concerns, and the expected depletion of fossil diesel fuel, researchers
are investigating the clean consumption of diesel engines deploying alternative fuel sources. Diesel engine
pollutants, which have a significant influence on both the environment and human health, constitute one of
the most significant environmental pollution sources. Due to a wide range of reasons, such as escalating
prices for petroleum, worldwide environmental issues, and a projected depletion of fossil diesel fuel,
researchers are investigating the clean combustion of diesel engines making use of alternative fuel sources.
Scientists have been exploring for new alternative fuels that are technically feasible, generally readily
available lucrative for business, and healthy for the environment for decades (Valipour, 2014).

In order to satisfy the rising energy demand in the world, alternative energy sources must be present. Among
the additional advantages, it has a higher cetane index and produces fewer carbon dioxide emissions. In the
words of (Enweremadu and Mbarawa, 2009), biodiesel is a clean-burning, oxygenated mono-alkyl ester fuel
made from natural, renewable sources like in their natural state or used vegetable oils and animal fats. The
output in both Italy and the US increased significantly (in the US, production more than tripled). New rules
have helped biodiesel gain popularity and market share in Europe (Lieberz, 2021).
The production of biodiesel was rapidly growing throughout Asia, including in Indonesia, Singapore,
Malaysia, and China, as well as in Latin America, including in Argentina and Brazil. While biodiesel demand
is anticipated to increase by 7% over the following ten years, Indonesia intends to increase biodiesel output
by 23% by 2030 (Kondalamahanty, 2021). The amount of production of biodiesel was increasing quickly in
Latin America and Asia (Argentina and Brazil) (Agarwal, 2007).

Throughout the past ten years, both the security and accessibility of electrical energy have become major
global concerns. Fossil fuels generate greenhouse gases and other air pollutants that have a harmful impact
on the environment. By combining into traditional diesel fuel, biodiesel is becoming increasingly accessible
for use in the transportation sector; it has been suggested (Sarin et al., 2007).

Edible oils make up the first generation of biodiesel feedstock. Due to their widespread availability, high
biodiesel production rate, and ease of processing as as a consequence of their low free fatty acid focused
attention, edible oils have been used to produce biodiesel in the United States and Europe. However, as can
be observed in countries with high populations like China, India, and Indonesia, their use has given rise to
issues like food vs. fuel concerns, environmental concerns like the destruction of essential soil resources,
deforestation, and the use of a large portion of the available arable land (Mahapatra and Mitchell, 1999;
Nurfatriani et al., 2019; Taheripour et al., 2019). The economic viability of the production of biodiesel from
food oils was hampered by all of such problems. The cost of the feedstock, which includes soybean oil, is
frequently thought to make about 75% of the total expenditure of biodiesel (Mizik and Gyarmati, 2021). In
developing nations where edible oils are prohibitively pricey, researching the development of novel low-cost
agricultural non-edible crops and the use of byproducts in the production of biodiesel could substantially
decrease biodiesel costs (Wang and Ding, 2012; Silitonga et al., 2019; Ambat et al., 2020; Ong et al., 2021).

Since it competes with edible vegetable oils for both the production of food and the manufacture of biodiesel,
the first generation biofuel is not viable. As a result, much effort has been invested into providing biodiesels
from vegetable oils that are not edible, which includes Jatropha curcas, Calophyllum inophyllum, Sapium
sebiferum, Euphorbia lathyris, (Zapata et al., 2012) Reutealis trisperma, (Takase et al., 2015; Saravanan et
al., 2010; Takase et al., 2015; Milano et al., 2018; Milano et al., 2018; Kusmiyati et al., 2019).

METHOD

1) DIRECT USE AND BLENDING

Direct injection engines are able to operate on animal fat or vegetable oil as fuel. However, because of its
unfavorable characteristics, it has some issues and cannot be used in direct motors without having modified.
Alternative fuel sources are mixed directly with traditional fossil fuels to prevent issues like these. This
blending is going to improve fuel quality, reduce the use of fossil fuels, and other benefits, rendering it also
attractive as the most practical approach to use alternative fuels including biofuels. The ratios of the biofuel
and diesel mixes are going to differ among them 10:1, 10:2, 10:3, etc. (Atabani et al., 2014), (Gandure et al.,
2013), (Palash et al., 2015).

2) TRANSESTERIFICATION PROCESS

The production of biodiesel (mono alkali ester) and glycerol takes place when the triglycerides in vegetable
oil react with alcohol (Raghuvanshi et al., 2014).

A catalyst has been used in this procedure to quicken the reaction while also enhancing the quality of the
finished product. The amount of the free fatty acids in the feedstock oil determines the kind and quantity of
catalyst. The production and use of biodiesel is negatively impacted by the greater free fatty acid written
material, which results in soap formation and decrease biodiesel yield efficiency (Ramaraju et al., 2011;
Venkanna et al., 2009).

Base catalyst transesterification process

In this stage potassium Hydroxide, sodium hydroxide and sodium methoxide are used as a catalyst. Alcohol
and oil are reacting in the presence of a base catalyst (Prabhakar et al., 2011). Maintain the reaction
temperature between 50° and 60°C during the process. The reaction temperature required to be lower than
the boiling point of methanol. The procedure is continuously stirred (1300 rpm) for dynamic mixing to
quicken the rate of reaction. The reaction will take over an hour for it to conclude. The solution should be
placed in a separating stock for 12 to 24 hours with the goal to separate the biodiesel and glycerol that were
created by the aforementioned reaction. Glycerol will be found within the lower layer and biodiesel in the
higher layer (Dhar et al., 2012; Ragit et al., 2011; Subramaniam et al., 2013).

Acid catalyst esterification process

Some feedstock oils include more than 1% free fatty acids; in those instances, the process of base catalyst
transesterification preceding the acid catalyst esterification of the feedstock oil. The ester is formed in this
circumstance by converting the free fatty acid (Canakci et al., 2015).
The water must be removed as soon as possible because it will cause soap to form during the base catalyst
transesterification process whenever the alcohol combines with the oil using an acid catalyst in order to
generate biodiesel. Here, sulfuric or phosphoric acid has been used as a catalyst. The base case catalyst
transesterification process is used for the production of biodiesel employing the product from the acid catalyst
(Ong et al., 2014; Canakci et al; Silitonga et al., 2014).

3) THERMAL CRACKING (PYROLYSIS)

With or without a catalyst, thermal cracking transforms hydrocarbons' complicated structures to their most
fundamental forms. The density and viscosity of oil is going to reduce as a result of this procedure.

In order to produce biodiesel, alumina, zeolite, and redmud typically serve as accelerators in the thermal
cracking process (Parawira, 2010).

The oil or animal fat which requires to be converted into biodiesel is deposited inside the reactor, which has
a safety mechanism, drain pipes, temperature indicator, and other features. The reactor is then heated to
approximately 250° and 350°C. Now that the petroleum or animal fat is vaporized, it moves via the pipe to
the condenser. The liquid is collected in the beaker and is known as biodiesel whenever the condenser cools
the vapor in the liquid (Parvizsedghy et al., 2015; Jahirul et al., 2012; Vivek et al., 2004).

Production Of Biodiesel From Algae

Due to the absence of toxic relationships, biodiesel is the best fossil fuel substitute (Lapinskiene et al., 2010).
Commercial biodiesel can be created from an assortment of sources, including canola, corn, palm, palm the
kernel itself and waste cooking oil. It is contentious because using petroleum from plants for biodiesel
production necessitates an important quantity of land (Lee et al., 2011). Microalgae is a suitable alternative
raw material for the manufacturing of biodiesel (Chisti et al., 2010; Rawar et al., 2013).

Collection Of Algae Species and Their Pretreatment

Algae samples have been collected from the Khyber Pukhtunkhwa, Pakistan, districts of Swabi and Mardan.
The samples have been left out onto the open to come into contact with the sun and drain any excess water
from the biomass. The dried algae had been ground and fine powder was sieved under 500 microns to obtain
the incredibly fine powder, which can conveniently mix with biochemical solvent by physical interaction,
almost 24 hours later. Big particles were employed in compost after searching (Ihsanullah et al., 2015).
Solvent Treatment Of Sieved Algae Powder

n-Hexane and Di-ethyl ether were utilized as solvents to extract oil from algal biomass. Both solvents were
employed both alone and in mixture with n-hexane and di-ethyl ether. Treatment of the 20 gm of ground up
algal biomass required 20 ml of solvent. The algal biomass and solvent were incorporated and left at room
temperature for 24 hours. A layer of oil accomplished on the surface and isolated from the waste. The solvent
was evaporated in a rotary evaporator to separate the extracted oil. Di-ethyl ether was initially allowed to
diminish for 15 minutes at 34° C in the evaporator. Then, to eliminate n-Hexane, the temperature was elevated
to 69°C. Oil devoid of solvent was left in the evaporation flask after this kind of procedure. Percentage of
yield is directly proportional to the time of contact between solvent and biomass of algae (Ihsanullah et al.,
2015).

Transesterification -Methanol with Oil

400 cc of oil were obtained that extends from the 20 kg of biomass from algae sample during the extraction
process. Through a chemical procedure known as transesterification, the crude oil that was extracted was
transformed into biodiesel in an atmosphere of methanol. Triglycerides and alcohol are combined with alkali
in this reaction to create diesel or fatty acid ester. According to stoichiometry, 1 mole of triglycerides produces
3 moles of fatty acid ester and 1 mole of triglycerides for every mole of alcohol (IHSANULLAH et al.,
2015).

Principal Bacteria for Biodiesel Production

One-celled microorganisms function as a catalyst and source of fatty acids and alcohols to facilitate the
biodiesel synthesis process. In both now biodiesel production, grease or oleaginous bacteria are widely
researched and used as a source of fatty acids for the transesterification process. A variety of agricultural and
industrial resources can be changed into cellular lipids by oleaginous microorganisms that come from a rich
source of microbes. (Peralta-Yahya et al., 2010) The fatty acid information of the lipids derived from
oleaginous microorganisms is similar to that of vegetable oils, rendering biodiesel production simple (Kosa
et al. 2011). There are grease strains in several microorganism’s yeast, mold, and algal species (Kosa et al.,
2010).

The following list of essential traits could serve as a background: The strains should have the following
characteristics: (1) over fifty percent of lipids in the cells should be accumulated; (2) simple apparatus may
be utilized to industrialize culture; (3) anti-pollution ability and rapid growth speed; and (4) straightforward
oil extraction. Due to their large population, various metabolic capabilities, and high fatty acid content,
microalgae are receiving a lot relevant as an economically feasible oil feedstock in the recent years
(Radakovits et al., 2010). Chlorella vulgaris, Botryococcus braunii, Navicula pelliculosa, Scenedsmus
acutus, Crypthecodinium cohnii, Neochloris oleoabundans, Monallanthus salina, and Tetraselmis sueica
have only a few examples of the numerous autotrophic microalgae that may produce oil (Li et al., 2008).

Ethanol Producers to Supply Alcohol Sources

Methanol or ethanol constituting the two most often utilized acyl acceptors. According to (Yusuf et al., 2011),
ethanol is believed to be more environmentally friendly and less harmful than other fuels and can be created
from renewable resources with ease through microbial fermentation. This has been supported by multiple
assessments and newspapers. (Weber et al., 2010; Lin et al., 2006)

Saccharomyces cerevisiae is the microbe that generates ethanol most frequently and is recommended because
it can produce ethanol at a concentration of as much as 18 percent of the fermenting broth (Lin et al., 2006).
Future biorefineries will likely use S. cerevisiae as the main platform for cell manufactures (Honk et al.,
2012).

Another thoroughly investigated strain of microbe that produces ethanol is Zymomonas mobilis, whose
genome sequence has been made public (Seo et al., 2005). In comparison to yeast, Z. mobilis manufactures
ethanol at higher yields and at faster specific rates. Numerous studies on the metabolic engineering of Z.
mobilis have been conducted with the goal of expanding the substance range for ethanol generation. The
genetic manipulation mechanisms in this organism are clearly understood (Rogers et al., 2007). In the study
by Hideshi Yanase (Yanase et al., 2012), Z. mobilis was modified by insertion of genes for mannose and
xylose catabolic enzymes to convert the wood hydrolysate, which comprised components of glucose,
mannose, and xylose, to ethanol.Within a period of 72 hours, the yield of ethanol had reached 89.8% of the
theoretical output. E. coli bacteria could be genetically engineered in order to produce ethanol.(Ingram et al.,
1987)

Recombinant Microorganisms Capable Of De Novo Biosynthesis of Biodiesel

Microbes can become "cell factories" through the use of genetic engineering and their way manipulation.
(Peralta-Yahya et al., 2010) According to some claims, metabolic engineering has resulted in the de novo
generate of biodiesel. (Elbahloul et al., 2010; Elbahloul et al., 2012; Wang et al., 2012; Duan et al., 2011) A
result of an overabundance of highly developed genetic tools on its metabolism, E. coli is an ideal host for
genetic engineering to manufacture high-value chemicals (Huffer et al., 2012) and (Handke et al., 2011)

The simultaneous overexpression of the the manufacturing of ethanol genes from Z. mobilis and the wax
ester synthase/acyl-CoA-diacylglycerol acyltransferase (WS/ DGAT) gene obtained from Acinetobacter
baylyi strain ADP1 in E. coli revealed the world's first report of the de novo biosynthetic pathway of biodiesel
(Kalscheuer et al., 2006). Exogenous fatty acids were called for for the biosynthesis of FAEEs in Kalscheuer's
research. According to Kalscheuer's report, a factor limiting FAEEs accumulation is the absorption of
extraneous fatty acids from the medium and how they are converted to the suitable acyl-CoA
thioesters.Endogenous fatty acids from E. coli that were given as acyl moieties of coenzyme were further
modified by Yangkai Duan and his team. fatty acid thioesters for transesterification (Steen et al., 2010). Using
hemicellulose as a source of raw materials, this recently created strain had the ability to accumulate (Fatty
Acid Ethyl Ester) FAEEs. Detailed genetic modifications that were successfully utilized for E. coli to
manufacture FAEEs from lignocellulose biomass without the need of costly enzymes had been shown.S.
cerevisiae, an intriguing prospect for genetic engineering, is able to accumulate fatty acids with a chain made
up primarily of 16 or 18 carbon atoms and is an excellent generator of ethanol (Tehlivets et al., 2007). Since
ethanol is accumulated in S. cerevisiae at a high concentration, the likelihood of ethanol does not affect the
amount of biodiesel that can be produced (Shi et al., 2012; Yu et al., 2012). Since these microorganisms have
a sufficient resource of fatty acyl-CoA, some oleaginous microorganisms may express genes for microbial
biodiesel generation without a requirement for fatty acid or lipid feeding.Rhodococcus opacus (Holder et al.,
2011) and Yarrowia lipolytica (Loira et al., 2012), for example, (Elbahloul et al., 2010) emphasize their
potential applicability in microbial biodiesel generation. Cellulosic microbes consume biomass because they
contain several cellulase enzyme systems. The cost of both raw materials and the production process may be
decreased through establishing the plasmid for FAEEs production in cellulosic microorganisms employed as
an expression host, such as cellulosedegrading or hemicellulose-degrading strains. Microalgae are also
acknowledged as a useful model for biodiesel generation through manipulation, considering the simple fact
that the eukaryotic microalgae genetic engineering is still in its infancy (Courchesne et al., 2009).

Production Of Biodiesel from Fungi

Oleaginous fungus isolation and identification (Ahmed et al., 2006)

Oleaginous fungi were isolated for the synthesis of lipids by serial dilution from the various soil locales of
Tamil Nadu. (2005) Duncan F. Five oleaginous fungi, namely 1) TR1 (Thanjavur), 2) PR (Poombukar), 3),
KM1 (Kollimalai), 4) RM1 (Rameswaram), and 5) VT1 (Vetharinyam) had been chosen in Petri plates from
among a number of fungi that were isolated (Alexopolous 1996).By using the soaking - plain water - agar -
culture processes, morphologically chosen fungus were made to produce spores (Watanabe, 1983). A single
isolate (PR) disc was injected into a plate of plain water agar. The Petri's salt solution is added to the ordinary
water agar plate after one day, and the plate is then incubated for 5 to 6 days (Tucker, 1931).The approach
stated by Ahmed et al. (2006) was used to screen oleaginous fungus. The cultures undergo lipid and biomass
(gl-1) testing.

Cell Biomass Determination for Fungi (Cerik Et Al., 1996)

Mycelia were removed by suction filtered from the incubation flask and carefully cleaned with distilled water.
Isolates were then screened through Whatman No 1 filter paper. Mycelium was then dried for 15 minutes in
an oven set at a temperature of 60 degrees Celsius. The dry biomass in this balanced sample was measured
as grams per liter.

Extraction Of Fungal Lipids

Fungal lipids were isolated from the dried mycelia using the procedure outlined by Bligh and Tyer (Bligh et
al., 1959). Different nutritional factors (nitrogen and carbon supply) and growth conditions (temperature and
pH) were researched in PR. The following composition was used to analyze the lipids and biomass
synthesized by the fungal isolate PR. Yeast extract 5.0g/l, distilled water 1000 ml, glucose 30.0 (0.16 M), pH
5.4 Different carbon sources were used to replace each individual glucose molecule in the soup (Ahmed and
others, 2006). In a single milligram of fungal isolate as mycelia suspension, different carbon sources
including glucose, fructose, sucrose, and lactose are used at a concentration of 0.16 (M).

Cultural Conditions

50 ml of this soup was distributed, sanitized, and incubated for 7 days at 30°C and 200 rpm in shaker
incubators in 250 ml conical flasks. Through Whatman No. 1 filter paper, the fungus mycelium was filtered.
The structure and composition of the filtered fungal mycelium's liquids and biomass were examined.
According to their earlier research, glucose was discovered to be the optimum source for making lipids. In
order to determine the optimal level for the maximal synthesis of lipids, glucose was subsequently ingested
at various concentrations.
Lipids Production And Glucose Utilization By Fungal Isolate (Pr)

The 250 ml Erlenmeyer flask was filled with the screening broth holding 0.6 M of glucose and sterilized.
Using Whatman No. 1 filter paper, 1 g of fungal isolate (PR) was inoculated as mycelial suspension,
incubated at 220 rpm in an environment shaker for 7 days, and then filtered. Mycelia were examined for lipid
and biomass content, and the filtrates were examined for glucose content. The methodology of (13) was used
to measure the glucose in the culture filter. Following centrifuging the culture filtrate at 6000 rpm and 30°C,
0.2 ml of the supernatant was pipette into a test tube.1 ml of alkaline metal tartarate reagent was added to
that, and the mixture was heated in a water bath for 10 minutes. Arseno molybdate reagent (described below)
was added after freezing for 1 ml. After 10 minutes, the absorbance of the generated orange-red color was
assessed using a colorimeter at 620 nm. The standard curve had been created using the standard's absorbance
value. To determine the amount of glucose present, the absorbance value of the samples was plotted
appropriately. The broth's yeast extract was each replaced with a different nitrogen source, that includes yeast
extract, ammonium chloride, or ammonium sulphate. Nutritional and growth factors were used for the large-
scale generation of lipid by a fungal isolate (PR) in a bio reactor. The vitamins and minerals used were
glucose (0.6 M), yeast extract (10.0 g), pH 6.5, and 30°C (Lindberg et al., 1993). A 5-L bioreactor (Lark
Innovative Technologies, India) fitted with a disc impeller, oxygen and pH electrodes were used for the
fermentation process. The apparatus additionally maintained track of the vessel's level, antifoam addition,
agitation speed, gas purging flow rate, and temperature. Throughout the duration of the experiment, the pH
level and temperature occurred constant (Dai et al., 2007). Rhodotorula glutinis was inoculated into
fermentation broth for 24 hours at a 10 percent level having (28x10 cfu/ml) in the broth. Five days of
fermentation accompanied the inoculation process. After five days, the biomass was extracted by
centrifugation, and the amount of lipids made by the various yeast strains has been determined. By using
GC-MS, fatty acid profiles for the lipids were further examined. Gas chromatography- mass spectrometry
analysis of fatty acid methyl esters (16) To examine the efficiency of fungi as a source of biodiesel, their
physical and chemical properties were applied to various fungal strains. The following qualities were
evaluated in accordance with the American standards for testing of materials (ASTM) 2003 method.

Lipids produced through the five fungal isolates during study PR sample exhibits high lipid production and
biomass (28% and 10g/l, respectively), hence PR sample was chosen for additional studies.
Effect Of Temperature On Lipids Production By Fungal Isolate

The fungus had been cultured at several different temperatures of 20, 25, 30, 35, and 40°C in order to find
the ideal temperature for the fungal isolate (PR) to produce the most lipids. At 30°C, the fungus considerably
accumulated more lipids (43.2%), whereas at 40°C, the smallest amount of lipids (14.3%) was discovered.
In accordance with the results of the current investigation, the fungal isolate (PR) produced the most biomass
and lipids at a temperature of 30 °C, as reported by (Sumner et al., 1996).

Effect Of Ph On Lipids Production By Fungal Isolate (Pr)

The impact of pH on the generation of lipids by the fungal isolated (PR) was examined at several pH ranges
between 5.5, 6.5, 7.5, and 8.5. The amount of biomass (10.7gl-1) and lipids (43.5%) at their maximum was
much higher when the fungus was produced at a pH of 6.5. At pH 7.5, there was the least number of lipids
(10.2%) and biomass (5.6%), while at pH 8, there was neither growth nor lipid accumulation. In accordance
with (Bajpai et al., 1991; Yamada et al., 1987) research, a pH of around 6.0 is ideal for producing biomass
and lipids whilst also having a substantial amount of PUFAs (poly unsaturated fatty acids) (Devi et al., 2011).

Physico-Chemical Properties of Fungal Lipids

In order to be used as biodiesel, lipids produced in a bioreactor were studied for their physical and chemical
characteristics, and results were recorded. The calculated values have been compared against ASTM
standards. Physical characteristics were obtained, including specific gravity of 0.92 g/cc, viscosity at 40 °C
of 54.81 cSt, calorific value of 32.05 kcal/kg, flash 218 °C, fire point 230 °C, cloud 4 °C, and pour point 7
°C, as well as the chemical composition of lipids, such as free fatty acids (FFA) of 14.55%, acid value 28.2.
The fungal lipids viscosity (54.81 cSt) and acid value (28.2) were both larger than the standards (1.9-6.0 cSt
and 0.8, respectively). Other variables, such as flash point and carbon residue, were within the acceptable
range. Because fungal lipids' flash and fire points are within acceptable ranges. It can be employed in the
same way as vegetable oil. It must be decreased through transesterification since the fungal isolate's viscosity,
FFA, and acid value are higher than the levels that are needed, causing engine blockage. Based on their GC-
MS retention durations, fatty acids profiles (palmitic (33.4 min), stearic (34.34), and oleic acid (34.60)) had
been identified. (Devi et al., 2011)

Biodiesel Production from Plants

The oils of plants commonly used for the manufacture of biodiesel are Jatropha curcas, Simarouba glauca,
Sesamum indicum (Sesame), Ricinus communis (Castor), Glycine max (Soybean), Brassica napus
(Rapeseed), Gossypium sp. (Cotton) seed oil, Oryza sativa (Rice) bran oil, Pongamia pinnata (Karanja),
Madhuca longifolia (Mahua), Elaeis guineensis (Palm) kernel oil and Brassica juncea (Indian Mustard) oil.
These lipids are transesterified to produce biodiesel. The main biodiesel manufacturers are known to be
Germany and France, with sporadic assistance from the US. Argentina, Brazil, China, India, Indonesia, and
Malaysia are only a few additional nations that generate biodiesel. By 2025, the projected production is
anticipated to rise by 56 percent (OPEC, 2012). There are numerous manufacturing plants that use base
catalyzed transesterification to convert plant oils into biodiesel. MeOH is used as the lower alcohol in this
process, while KOH or NaOH is used as the homogenous catalyst. The goal of developments in biodiesel
production is typically to produce high-quality fuel that complies with international standards like ASTM or
EN standard. The production of biodiesel from bioresources can be done in four different ways: blending,
micro-emulsion, pyrolysis, and transesterification. Early methods of using plant oils for powering engines
directly or in combination with petroleum diesel include direct usage and combining of raw oils.

Since they are mainly created in many parts of the world, plant oils make up more than 95% of the feedstocks
for the manufacturing of biodiesel. Since edible oils are utilized to make food and are now very expensive to
use, they cannot be used to make biodiesel. As a result, hazardous non-edible vegetable oils have been used
in the manufacturing of biodiesel. First generation biodiesel was produced using plant oils and was
established up of residues such as rice bran, peanut, palm, soybean, and palm oil. Pongamia pinnata,
Jatropha curcas, or used vegetable oils constitute non-edible plant oils. The features of the feedstock’s affect
whether the biodiesel derived can be utilized as a diesel fuel a substitute (Singh, 2014).

Since jatropha is an unsuitable for eating plant and won't compete with human food, it is crucial in the
manufacturing of biodiesel. According to (Peterson et al., 1999), improvements in the use using biodiesel as
a substitute fuel aim to provide cheap, renewable energy that is simple to produce, clean, and more
environmentally friendly. To help everyday Nigerians utilize biodiesel as an alternative fuel, the
manufacturing of this biofuel can be promoted at the local level. This will bridge the gap and, more crucially,
ease pressure on crude oil to replace other fuels as the primary fuel for managing equipment (Schumacher et
al., 1995). The goal of this project is to create biodiesel from jatropha oil, study it, and compare the properties
to those of diesel and petrol.
Conclusion

The current study shows that although fungi create biodiesel with a low carbon content, it is both costeffective
and environmentally friendly. To manufacture fatty acids including short-chain alcohols suitable for
transesterification, microorganisms must be created. De novo combination of fatty acid ester-based biodiesel
(FAEEs) by microorganisms has not yet been discovered in nature. Although plant oils have played an
important function in the creation of biodiesel, using them directly in diesel engines, particularly direct
injection engines, has been loaded with issues. Spirogyra, an algae species, has been effectively employed as
an unprocessed product for the manufacturing of biodiesel. Transesterification is required to minimize the
fungal isolate's values because they are beyond the threshold level and cause engine blockage. The primary
issue is that most of the saturated hydrocarbons in plant oil have big triglyceride molecules and have a high
molecular mass. If the vegetation oils are chemically altered to yield biodiesel, this problem can be overcome.

Transesterification is required to minimize the fungal isolate's values since they are beyond the threshold
level and cause engine blockage. In order to meet the demands of the ASTM (American Society for Testing
and Materials) D6751 standards, the study concluded that the consumption of fungal lipids from (PR) as
biodiesel should go though some pretreatment and transesterification.

References
1) Ahmed. S. u., K.S. sudheer, P. ashok, K. sanjit and B.N.Prasad. 2006. Effects of various process
parameters on the production of linolenic acid in submerged fermentation. Food technol. Biotechnol.,
44(2): 283-287
2) Bajpai, P.K., P. Bajpai and O.P. ward. 1991. Arachidonic acid production by fungi. Appl. Environ.
Microboil., 57: 1255-1258.
3) Baljinder Singh* Baljinder Singh, PhD, Assistant Professor, Department of Biotechnology, Panjab
University, Chandigarh 160014, India
4) Bligh E.G. and N.J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can J.
Biochem. Physiol., 37: 911-917.
5) C. L. Peterson, J. C. ompson, J. S. Taberski, D. L. Reece, and G. Fleischman, “Long-range on-road
test with twenty-percent rapeseed biodiesel,” Applied Engineering in Agriculture, vol. 15, no. 2, pp.
91–101, 1999.
6) Cerik, M., P. Andrani and J. Sajbidor. 1996. Effect of extraction methods on lipid yield and fatty acid
composition of lipid classes containing λ-linolenic acid extracted from fungi. JAOCS, 73(3): 357-365
7) Chisti, Y. Biodiesel from microalgae. Biotechnology Adv., 2007; 25: 249–306.
8) Chuan-chao Dai, Jie Tao, Feng Xie, Yi-jun Dai and Mo Zhao Biodiesel generation from oleaginous
yeast Rhodotorula glutinis with xylose assimilating capacity, 2007
9) Courchesne NMD, Parisien A, Wang B, Lan CQ. Enhancement of lipid production using biochemical,
genetic and transcription factor engineering approaches. J Biotechnol 2009;
 141:31-41; PMID:19428728; http://dx.doi.org/10.1016/j.jbiotec.2009.02.018

10)Duan YK, Zhu Z, Cai K, Tan XM, Lu XF. De novo biosynthesis of biodiesel by Escherichia
coli in optimized fed-batch cultivation. PLoS One 2011; 6:e20265; PMID:21629774;
http://dx.doi.org/10.1371/journal. pone.0020265.
11) Elbahloul Y, Steinbüchel A. Pilot-scale production of fatty acid ethyl esters by an engineered
Escherichia coli strain harboring the p(Microdiesel) plasmid. Appl Environ Microbiol 2010;
76:4560-5; PMID:20453138; http://dx.doi.org/10.1128/AEM.00515-10.
12) Frances Duncan, 2005. Applied Microbiology Laboratory Manual By Frances Duncan With
contributions from Valerie Walker and Neil Clark Fourth Edition.
13) Handke P, Lynch SA, Gill RT. Application and engineering of fatty acid biosynthesis in
Escherichia coli for advanced fuels and chemicals. Metab Eng 2011; 13:28- 37; PMID:21056114;
http://dx.doi.org/10.1016/j. ymben.2010.10.007.
14) Holder JW, Ulrich JC, DeBono AC, Godfrey PA, Desjardins CA, Zucker J, et al. Comparative and
functional genomics of Rhodococcus opacus PD630 for biofuels development. PLoS Genet
2011; 7:e1002219; PMID:21931557; http://dx.doi.org/10.1371/journal. pgen.1002219.
15) Hong KK, Nielsen J. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory
platform for future biorefineries. Cell Mol Life Sci 2012; 69:2671- 90; PMID:22388689;
http://dx.doi.org/10.1007/ s00018-012-0945-1.
16) Huffer S, Roche CM, Blanch HW, Clark DS. Escherichia coli for biofuel production: bridging the
gap from promise to practice. Trends Biotechnol 2012; 30:538-45; PMID:22921756; http://dx.doi.
org/10.1016/j.tibtech.2012.07.002.
17) Ihsanullah1,3*, Sumaira Shah2 , Muhammad Ayaz3 , Iftikhar Ahmed3 , Murad Ali3, Naveed
Ahmad4 and Irshad Ahmad5. Production of Biodiesel from Algae. JOURNAL OF PURE AND
APPLIED MICROBIOLOGY, March 2015. Vol. 9(1), p. 79-85.
18) Ingram LO, Conway T, Clark DP, Sewell GW, Preston JF. Genetic engineering of ethanol
production in Escherichia coli. Appl Environ Microbiol 1987; 53:2420-5; PMID:3322191.
19) Kalscheuer R, Stölting T, Steinbüchel A. Microdiesel: Escherichia coli engineered for fuel
production. Microbiology 2006; 152:2529-36; PMID:16946248;
http://dx.doi.org/10.1099/mic.0.29028-0.
20) Kosa M, Ragauskas AJ. Lipids from heterotrophic microbes: advances in metabolism research.
Trends Biotechnol 2011; 29:53-61; PMID:21146236; http://
dx.doi.org/10.1016/j.tibtech.2010.11.002.
21) Lapinskiene, A., Martinkus, P., Rebzdaite, V. Eco-toxicological studies of diesel and biodiesel
fuels in aerated soil. Environ. Pollut., 2006; 142
22) Lee, D.H., Algal biodiesel economy and competition among bio-fuels. Bioresource Technol.,
2011; 102: 43–49.
23) Li Q, Du W, Liu D. Perspectives of microbial oils for biodiesel production. Appl Microbiol
Biotechnol 2008; 80:749-56; PMID:18690426; http://dx.doi. org/10.1007/s00253-008-1625-
9
24) Lin Y, Tanaka S. Ethanol fermentation from biomass resources: current state and prospects. Appl
Microbiol Biotechnol 2006; 69:627-42; PMID:16331454; http:// dx.doi.org/10.1007/s00253-005-
0229-x.
25)Lindberg, A. M., G. Molin. 1993. Effect of temperature and glucose supply on the production
of poly unmsaturated fatty acids by the fungus Mortierella alpine CBS 343.66 in fermentor
cultures. Appl. Microbiol. Biotechnol., 39: 450-455.
26) Loira N, Dulermo T, Nicaud JM, Sherman DJ. A genome-scale metabolic model of the
lipidaccumulating yeast Yarrowia lipolytica. BMC Syst Biol 2012; 6:35; PMID:22558935;
http://dx.doi. org/10.1186/1752-0509-6-35.
27) Miller, J. (1972).Notes on sugar determination. J. Biol. Chem., 200: 245-247
28) Nithya Devi .M1 * and Velayutham .P2, 2011. Biodiesel Production from Fungi. Indian
Journal Of Natural Sciences ISSN: 0976 – 0997 Vol1 / Issue 5 / April 2011
29) Peralta-Yahya PP, Keasling JD. Advanced biofuel production in microbes. Biotechnol J 2010;
5:147- 62; PMID:20084640; http://dx.doi.org/10.1002/ biot.200900220.
30) Radakovits R, Jinkerson RE, Darzins A, Posewitz MC. Genetic engineering of algae for
enhanced biofuel production. Eukaryot Cell 2010; 9:486- 501; PMID:20139239;
http://dx.doi.org/10.1128/ EC.00364-09.
31) Rawat,I., Kumar, R. R., Mutanda,T. and Bux,F. Biodiesel from microalgae: A critical
evaluation from laboratory to large scale production. Appl. Energ., 2013; 103,:444–467.
32) Rogers PL, Jeon YJ, Lee KJ, Lawford HG. Zymomonas mobilis for fuel ethanol and higher
value products. Adv Biochem Eng Biotechnol 2007; 108:263-88; PMID:17522816;
http://dx.doi. org/10.1007/10_2007_060.
33) Saxena S, Brody AL, Schwartz JM, Baxter LR (1998) Neuroimaging and fronto-subcortical
circuitry in obsessive-compulsive disorder. Br J Psychiatry 35(Suppl):26–37
34) Seo JS, Chong H, Park HS, Yoon KO, Jung C, Kim JJ, et al. The genome sequence of the
ethanologenic bacterium Zymomonas mobilis ZM4. Nat Biotechnol 2005; 23:63-8;
PMID:15592456; http://dx.doi.org/10.1038/ nbt1045.
35) Shi SB, Valle-Rodriguez JO, Khoomrung S, Siewers V, Nielsen J. Functional expression and
characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for
biodiesel production. Biotechnology for Biofuels 2012; 5.
36) Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, et al. Microbial production of
fatty-acidderived fuels and chemicals from plant biomass. Nature 2010; 463:559-62;
PMID:20111002; http://dx.doi. org/10.1038/nature08721.
37) Sumner, J. L., E.D. Morgan and H. C. Evans. 1969. The effect of growth temperature on the
fatty acid composition of fungi of the order Mucorales. Can. J. Microbiol., 15: 515-520
38) Tehlivets O, Scheuringer K, Kohlwein SD. Fatty acid synthesis and elongation in yeast.
Biochim Biophys Acta 2007; 1771:255-70; PMID:16950653; http://
dx.doi.org/10.1016/j.bbalip.2006.07.004.
39) W. Marshall, L. G. Schumacher, and S. A. Howell, “Engine Exhaust Emissions Evaluation of
a Cummins L10E When Fueled with a Biodiesel Blend,” SAE Paper No. 952363, SAE,
Warrendale, Pa, USA, 1995.
40) Wang B, Lin H, Zhan J, Yang Y, Zhou Q, Zhao Y. Biodiesel synthesis by a one-step method in
a genetically engineered Escherichia coli using rice straw hydrolysate and restaurant oil wastes
as raw materials. J Appl Microbiol 2012; 113:531-40; PMID:22681508;
http://dx.doi.org/10.1111/j.1365-2672.2012.05357.x.
41)Weber C, Farwick A, Benisch F, Brat D, Dietz H, Subtil T, et al. Trends and challenges in the
microbial production of lignocellulosic bioalcohol fuels. Appl Microbiol Biotechnol 2010;
87:1303-15; PMID:20535464; http://dx.doi.org/10.1007/s00253-010-2707-z.
42) Yamada, H., S. Shimizu and Y. shinmen. 1987. Production of arachidonic acid by Mortierella
elongate IS-5. Agric. Biol. Chem. 51: 785-790.
43) Yanase H, Miyawaki H, Sakurai M, Kawakami A, Matsumoto M, Haga K, et al. Ethanol
production from wood hydrolysate using genetically engineered Zymomonas mobilis. Appl
Microbiol Biotechnol 2012; 94:1667-78; PMID:22573268; http://dx.doi. org/10.1007/s00253-
012-4094-0.
44) Yu KO, Jung J, Kim SW, Park CH, Han SO. Synthesis of FAEEs from glycerol in engineered
Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression
of an unspecific bacterial acyltransferase. Biotechnol Bioeng 2012; 109:110-5;
PMID:21858787; http://dx.doi.org/10.1002/ bit.23311.
45) Yusuf NNAN, Kamarudin SK, Yaakub Z. Overview on the current trends in biodiesel
production. Energy Convers Manage 2011; 52:2741-51; http://dx.doi.
org/10.1016/j.enconman.2010.12.004