Microbial Biodiesel

Production By

K.Madhu Jegathish

BTE-12-021

Introduction to biodiesel

History Rudolf diesel was the inverter of

biodiesel, estimated nearly 100 years ago. It was developed in the year 1890s.

What is Biodiesel?• Alternative fuel for diesel engines• Made from vegetable oil, animal fat, microbes • Lower emissions, High flash point (>300°F), Safer• Biodegradable, Essentially non-toxic.

Fatty AcidAlcoholGlycerin

Vegetable Oil

BiodieselFA

FAFA

FA

Biodiesel Samples

Characteristics Of Biodiesel

Liquid varying in colorImmiscible in waterHigh boiling point of 360–640°F

(182–338°C)Low vapor pressure: < 2 mmHgSpecific gravity between 0.86 &

0.90Vapor density > 1Less hazardous in terms of

flammability

Biodiesel As Lubricant And Solvent

Biodiesel can be used straight as a

machinery lubricant.

Biodiesel’s solvent properties may be

used to clean dirty or greasy engine or other

machine parts.

Advantages of Biodiesel

Renewable energy source

Less polluting

Utilizes excess production of soybeans for manufacture

Can distribute through existing

diesel fuel pumps

Can use in existing oil

heating systems and diesel engines Can be mixed

with petroleum diesel at any concentration

and time

Disadvantages Of Biodiesel

More expensive

Could harm rubber hoses in engines

Requires energy to:

Produce biodiesel from soy crops & sow, fertilize and

harvestRequires frequent filter

changingRequires improvement in distribution infrastructure

Relative Greenhouse Gas Emissions

0 20 40 60 80 100 120 140 160

Gasoline

CNG

LPG

Diesel

Ethanol 85%

B20

Diesel Hybrid

Electric

B100B100 = 100% BiodieselB20 = 20% BD + 80% PD

Relative emissions: Diesel and Biodiesel

0 20 40 60 80 100 120

Total Unburned HCs

CO

Particulate Matter

**NOx

Sulfates

PAHs

n-PAHs

Mutagenicity

CO2

Percent

B100 **B20Diesel

Biodiesel Production Process

Trans esterification

CH2OOR1 catalyst CH2OH| |CHOOR2 + 3CH3OH 3CH3OORx + CHOH| |CH2OOR3 CH2OHTriglyceride 3 Methanols Biodiesel Glycerin

R1, R2, and R3 are fatty acid alkyl groups.

Biodiesel production by alkali process

Enzymatic Biodiesel Production

Trans esterification

Washing

Biodiesel Reaction

Vegetable Oil or

Animal Fat (100 lbs.)

+Methanol or

Ethanol(10 lbs.)

Biodiesel

(100 lbs.)+

Glycerin(10 lbs.)

In the presence of a catalyst

Combining

Yields

NRRaje Feb 06

Conventional feed stocks

Rapeseed, the major source (>80%)

Sunflower oil (10%, Italy and Southern France)

Soybean oil (USA)

Palm oil (Malaysia)

Linseed, olive oils (Spain)

Cottonseed oil (Greece)

Beef tallow (Ireland), lard, Jatropha (Nicaragua),)

Microorganisms available for biodiesel production

Microalgae Bacteria Fungi Yeast

Biodiesel production using microbial lipids, which is named as single cell oils (SCO), has attracted great attention in the whole world.

Lipids from all cannot be converted into biodiesel mainly due to less yield

Oleaginous microorganisms are able to accumulate lipids above the 20% of their biomass, on dry basis.

Stages of Lipid accumulation

...

Cells reach limit of obesity → stop accumulating.

Lipid accumulation → cells expand

Cells convert C → storage lipid (intracellular)

Exhaustion of N, cells stop divide

Microorganisms grow and multiply until a certain time

Prepare medium: high Carbon, low Nitrogen

Biodiesel from microorganism

• Can grow rapidly • Live in harsh conditions due to their unicellular or simple multicellular structure. Of the 33,000 known species of algae at least forty

are considered oleaginous

Good candidates for biodiesel production,

higher photosynthetic efficiency higher biomass production and faster growth compared to other energy crops

MICROALGAE

continue.. • Can be induced to accumulate substantial quantities of lipids thus contributing to a high oil yield.

• Some of the common algae

chlorella, crypthecodinium, cylindrotheca, dunaliella, isochrysis, nannochloris, nannochloropsis, neochloris, nitzschia, phaeodactylum, porphyridium, schizochytrium, tetraselmis, etc,….

average lipid content- (1-70%) maximum- 90%

How to obtain biodiesel from algae?

Picking up the best algaeGrowing the algaeExtractionTrans esterificationBiodiesel

Choosing an Algae

Important characteristics of AlgaeHigh % of total

biomass is oilMaintains a high %

of oil even under stress

Compatible with our regional climate

Where To Grow It

Extensions onto our water treatment plantsClean up our waste and generate fuel

Agriculture runoffWater ponds

Algae could well grow on salty water as well as soft water

How Is Algae Grown?Open system

Tubular PBR

Flat plate PBR

Closed systems(photo bioreactor)

ExtractionSolvent extractionMechanical extraction

Super critical fluid extraction

Mechanical Extraction

Solvent extraction

Supercritical fluid extraction

DIFFERENCE B/W BIODIESEL FROM ALGAE AND OTHER

PLANT/VEGETABLE OILS • The yield• According to some estimation, the yield (per acre) of oil from algae

is over 200 times the yield from the best-performing plant/vegetable oils.

ADVANTAGES OF MICROALGAE AS A SOURCE OF BIODIESEL

• High Yield -low cost of production

• Algae can grow –In places away from farm land (No destruction to food chain) –Sewages –Near to power plants (takes CO2from smokestacks and yields oil)

• Oil Productivity –Greater than best producing oil crops

• Higher grade protein→Animal Feed

• Balanced N : P ratio→Organic Fertilizers

Biodiesel from FungusOleaginous fungi has also been considered as

potential oil sources for biodiesel production because they accumulate large amounts of lipidsAmong these microorganisms, particular attention has been dedicated to various oleaginous zygomycetes species, such as Mortierella isabelina and Cunninghamella echinulata, which may accumulate up to 86% and 57% of lipids in the dry biomass, respectively

Biodiesel from Fungus

Fungal Bio Mass Mucor circinelloides

Three Different Solvent Systems: Chloroform:Methanol (C:M), Chloroform:Methanol:Water (C:M:W)

n-hexaneAcid Catalyst ( BF3, H2SO4 and HCl )

Materials

Biodiesel from Fungal Bio MassExtraction of lipids Solvent +Dried

biomass Mixture

Ultrasonication

Centrifugation

Rotary Evaporation

Lipids

Biodiesel from Fungal Bio Mass

Rhodosporidium sp., Rhodotorula sp. and Lipomyces sspecies can accumulate intracellular lipids as high as 70% of their biomass dry weight.

• Cryptococcus curvatus oleaginous yeast Accumulate storage lipid up to >60% on a dry weight basis)

Biodiesel from yeasts

Biodiesel from yeasts

Media

Freeze drying

Lipid extraction

Tran esterification

Biodiesel

MediaPre-culture

YM medium was used as pre-culture to cultivate oleaginous yeast cellso glucose as a carbon sourceo Peptoneo yeast extract

Sterlisation - 120 ºC temperature for 20 minutes

Semi-synthetic medium was used as main culture to grow oleaginous yeastso Glucose, xylose or a mixture of both were used as carbon sourceso nitrogen sources (NH4Cl)o phosphate buffer (KH2PO4)o Mineral-element solution (CaCl2×2H2O ,FeSO4×7H2O ,citric acid×H2O)

Medium pH - 5.8

Main culture

Freeze drying

o Collected samples were centrifuged at 10 min and washed with sterile water once, then centrifuged again

o The pellets were transferred into pre-weighed vials and kept at -50 °C until freeze drying

Lipid extractionChloroform- Methanol Method

Adding Choroform: Methanol mixture

Centrifugation

Freeze dried pellets + HCL and incbated at 55° C

Solvent Evaporation

Extracted lipids

Extracted lipids

Trans esterification

biodiesel

methanol

glycerin

catalyst

Biodiesel from BacteriaBacteria can accumulate oil of about 20-

40% of dry biomass Arthrobacter sp. - 40% Acinetobacter calcoaceticus - 38%

Have a superiority in the production of biodiesel due to Highest growth rate (reach huge biomass only need 12–24 h) Easy culture method. Actinomycete group high amount of fatty acids (up to 70% of the cellular dry weight) using glucose under growth-restricted condition

Downside

• Very few are oil producer

• Only a few bacteria accumulate complicated lipoid.

• It is difficult to extract because these lipoid are generated in the outer membrane

• So there is no industrial significance in the actual production of biodiesel by using oleaginous bacteria as raw material

Quality of biodiesel from microbes • Biodiesel fuel, in the form of FAME, is now manufactured in many countries.

• Relevant standard to assess biodiesel are;

ASTM D6751 ( In USA) EN 14214 (In EU, intended for vehicle use) EN 14213 (In EU, for use as heating oil)

Conclusion • At present plant oil is the main feedstock for biodiesel production. • However, at present biodiesel is not competitive with conventional fuels in the whole world due to high cost of production.

• Production of microbial based diesel can be an economical beneficial.

• However, it still needs lots of improvement which could be done using the various biotechnological techniques and methods.

• Developing high lipid content microorganisms for biodiesel production from microbes are promising option in future and opens a possibility for academic research

Thank you