WO2010081335A1

WO2010081335A1 - A method of producing biofuel using sugarcane as feedstock

Info

Publication number: WO2010081335A1
Application number: PCT/CN2009/074298
Authority: WO
Inventors: Qingyu Wu; Yun Cheng
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2009-01-16
Filing date: 2009-09-28
Publication date: 2010-07-22
Anticipated expiration: 2011-07-16
Also published as: CN101475823A; CN101475823B

Abstract

The subject invention provides methods for producing biofuel from microorganism metabolite by using sugarcane as feedstock. In a preferred embodiment, alga was cultivated by using sugarcane- originated carbon 5 source as feedstock, resulting algal oil accumulation in algal cells at high content. And biodiesel was produced from said algal oil by transesterification.

Description

A method of producing biofuel using sugarcane as feedstock

Cross-Reference to a Related Application

This application claims the benefit of China Application Serial No. 200910000464.1 , filed on January 16, 2009, all of which are hereby incorporated by reference in their entirety, including any figures and/or tables.

Field of the invention The present invention refers to renewable energy. In particular, the present invention refers to biofuel production, and more particularly biodiesel production.

Background of the invention Biofuel can be broadly defined as solid, liquid or gas fuel derived from biomass, in which liquid biofuel is the most common form for transportation use. The growths of automotive transport and declining petroleum supply have strongly promoted the commercialization of liquid biofuel. In the recent decades, biofuel was made from food-based sources, such as bio-alcohols (ethanol, propanol and butanol etc.) from fermentation of corn starches (Smith AM. Prospects for increasing starch and sucrose yields for bioethanol production. Plant J. 2008, 54 : 546-58) and biodiesel from oil plants and animal fats (M. Canakci and H. Sanli. Biodiesel production from various feedstocks and their effects on the fuel properties. J Ind Microbiol Biotechnol. 2008, 35 :431-41). However using food-based crop for biofuel production is not practical, currently available lands are limited, and most of which are used to satisfy food supply. Therefore food-based feedstock can meet but a limited need in i liquid fuel. Nevertheless biofuels that are not food-based are likely to be of greater importance in the long run.

Microorganism-originated biofuels, such as microbial-, yeast- and especially algae-originated fuel, have been well-known as novel generation of biofuels that are not food-based. Microorganism-originated fuel has advantages in avoiding threatening food supplies and biodiversity. For example, the recent decade witnessed a surge of interest in microorganism-originated oil, lipid and/or fatty acids, especially algal oil, as a promising supplement of oil source for biodiesel production (Meng X et al. Biodiesel production from oleaginous microorganisms. Renew Energy. 2009, 34 : 1 -5). However, microorganism-originated fuel hasn't been widely accepted by energy market because of the technical and economic obstacles exist in their commercialization. The cost of cultivating microorganism is still high, and sometimes it is hard to extract or collect interest components from microorganism for subsequent process of fuel preparation.

Some species of microorganism including oleaginous yeasts (such as Lipomyces starkeyi, Rhodotorula glutinis and Candida curvata), bacillus (such as Rhodococcus opacus and Acinetobacter calcoaceticus), fungi (such as Mortierella isabellina and Mortierella vinacea) and algae (such as Schizochytrium sp. , Nitzschia sp. and Botryococcus braunii) have been reported to accumulate metabolite (eg. lipids, oil, methyl-esters or soaps) which can be used as sole carbon and energy sources. For example, the present inventor proved that triglyceride and fatty acids are usually the dominating components of algal oil, from which biodiesel can be prepared by transesterification, yielding monoalkyl esters of fatty acids and alcohols.

Some microorganism can grow with inorganic carbon source and sunlight as energy supply. However, both biomass productivity and oil content of photoautotrophic cultures are extremely low, the photoautotrophic technology mainly suffers from limited supply of light and lower energy conversion efficiencies. Unfortunately, microorganism during photoautotrophic cultivation can hardly provide enough useful components that can be prepared into biofuel. The present inventor marvelously discovered that some species of microorganism (such as alga) accumulate very high proportion of oil when specific organic carbon source (such as glucose) is provided to the microorganism. The most important thing is that the accumulated oil can be prepared into high quality biofuel (such as biodiesel). Therefore, the described cultivation strategy allows microorganism to accumulate much higher proportion of oil within less time and the scale-up is much easier. Thus it offers a potential pathway to produce oil for diesel production in large scale. However, the cost of producing biodiesel from microorganism-originated oil is much higher than that of diesel derived from petroleum, and the cost of carbon source feeding represents dormitory part of the cost of medium for heterotrophic cultivation. Therefore economic considerations need to explore cheap and easily available alternate feedstock. Sugarcane is a highly efficient C4 crop and can store about 1 % of the radiation on biomass per year (Lunn JE and Furbank RT. Sucrose biosynthesis in C-4 plants. New Phytol. 1999, 143 :221 -37). C4 crop refers to a kind of plants in which the CO₂ is first fixed into a compound containing four carbon atoms before entering the Calvin cycle of photosynthesis. It is estimated that 750 1 of raw sugarcane juice and 250 kg of wet bagasse can be harvested from 1 ton of sugarcane stalks. The juice contains 18.5% sucrose and 1 .7% of other fermentable sugars such as glucose and fructose, indicating a yield of about 150 kg of sugars per ton of stalks. Recently sugarcane has received increasing attention for its application in ethanol production. The most well-known example is The Brazilian National Alcohol Program (Programa Nacional do Alcool), 50% of sugarcane production in Brazil is used to satisfy alcohol consumption (Moreira JR and Goldemberg J. The Alcohol Program. Energ Policy . 1999, 27 :229-245). However, in so far as is known a strategy for biofuel (except bioethanol) production by using sugarcane as feedstock has never been reported.

Summary of the invention

The present invention refers to a method of producing biofuel from microorganism-originated metabolite by using sugarcane as feedstock. In particular, the present invention provides method for producing biofuel from microorganism-originated oil, lipid and/or fatty acids by using sugarcane as feedstock. More particularly, the present invention provides method for producing biodiesel from alga-originated oil, lipid and/or fatty acids by using sugarcane as feedstock.

Accordingly, the present invention provides method of producing biofuel, comprising: (a) providing fermentable carbon source of sugarcane;

(b) cultivating microorganism in medium containing the carbon source described in (a);

(c) extracting or collecting interest microorganism-originated metabolite from microorganism; (d) preparing biofuel from microorganism-originated metabolite.

Preferably, the present invention provides method of producing biodiesel, comprising:

(a) providing fermentable carbon source of sugarcane; (b) cultivating alga in medium containing the carbon source described in (a);

(c) extracting or collecting alga-originated metabolite from alga;

(d) preparing biodiesel from algal metabolite. The subject invention is particularly applicable to biofuel production, especially biodiesel production. The present invention provided a preferred embodiment of producing biodiesel from algal oil by transesterification using sugarcane-originated carbon source as feedstock. However, the spirit and purview, by no means, is restricted into particular embodiment. The following non-limiting list of microorganism can be treated using sugarcane-originated carbon source provided in the present invention: bacteria, fungi, archaea, and protists, microscopic plants, alga and microscopic animals. The following non-limiting list of microorganism-originated metabolite can be produced from sugarcane-originated carbon source provided in the present invention: alcohols, fatty acids, oils, lipids, hydrocarbons and the derivatives thereof. The following non-limiting list of techniques can be applied in preparing biofuel from microorganism-originated metabolite: extraction, fractionation, distillation, addition reaction, neutralization, hydrogenation, dehydrogenization, oxidation, reduction, substitution, esterification, transesterification and hydrolysis.

Brief description of drawing

Figure 1 shows the comparison of glucose and sugarcane juice-originated carbon source for microalgal heterotrophic cultivation.

Figure I a: Cell density (-♦"-) and residual sugar (™O™) in the medium with

20 g I^"1 glucose as comparison with cell density (••♦—) and residual sugar

( -0") in the medium without carbon source (basic medium). Figure Ib: Cell density (-♦"-) and residual sugar (H>~) in the medium with 20 g I^{" 1} sugarcane juice-originated carbon source as comparison with cell density (-♦— ) and residual sugar (■ ■€>■■) in the medium with 20 g I^{" 1} sugarcane juice without hydrolysis. Figure 2 shows the comparison of glucose and bagasse-originated carbon source for microalgal heterotrophic cultivation. Figure 2a: Cell density (■) and residual sugar (•) in the medium with 1 1 g I^{" 1} glucose. Figure 2b: Cell density (■) and residual sugar (•) in the medium with 11 g I^{" 1} bagasse-originated carbon source. Figure 3 shows heterotrophic fermentation in 5-1 bioreactor using glucose (a) and juice-originated carbon source (b) as feedstock, respectively. (•) Biomass yield, (o) Concentration of residual sugar. Arrows indicated the points of feeding.

Figure 4 shows microalgal heterotrophic cultivation in juice-originated carbon source media supplemented with and without other nutritional elements. Cell density (•) and residual sugar (o) in the medium containing 20 g I^{" 1} sugarcane juice-originated carbon source and other components, as comparison with cell density ( A ) and residual sugar (Δ) in the medium with 20 g I^{" 1} juice-originated carbon source only.

Figure 5 shows microalgal heterotrophic cultivation in non-sterilized medium containing 30 g I^{" 1} juice-originated carbon source. Cell density (•); residual sugar (D) .

Figure 6 is a schematic description that shows the progress of producing biofuel from microorgnism-originated metabolite by using carbon source of sugarcane as feedstock, comprising: (a) providing fermentable carbon source of sugarcane; (b) cultivating microorganism in medium containing the carbon source described in (a); (c) extracting or collecting interest microorganism-originated metabolite from microorganism; (d) preparing biofuel from microorganism- originated metabolite.

Detailed description of the invention

The present invention provides method of producing biofuel from microorganism-originated metabolite by using sugarcane as feedstock.

More particularly, the present invention provides method for producing biodiesel from alga-originated oil, lipid and/or fatty acids by using sugarcane as feedstock.

Accordingly, the present invention provides method of producing biofuel, comprising:

(a) providing fermentable carbon source of sugarcane;

(c) extracting or collecting interest microorganism-originated metabolite from microorganism;

(d) preparing biofuel from microorganism-originated metabolite. Preferably, the present invention provides method of producing biodiesel, comprising:

(a) providing fermentable carbon source of sugarcane;

(b) cultivating alga in medium containing the carbon source described in (a);

(c) extracting or collecting alga-originated metabolite from alga; (d) preparing biodiesel from algal metabolite.

Terms:

As used herein, the term "biofuel" as used herein, refers to solid, liquid or gaseous fuel obtained from renewable biological material. Biofuel is different from fossil fuels, which are derived from long dead biological material. Also, various plants and plant-derived materials are used for biofuel manufacturing. As used herein, the term "biodiesel" refers to non-petroleum-based diesel fuel consisting of alkyl (eg. methyl, propyl or ethyl) esters. Biodiesel is made by chemically-reacting lipids, typically vegetable oil or animal fat, and alcohol. It can be used alone or blended with conventional petrodiesel in unmodified diesel-engine vehicles (Specification for Biodiesel (B l OO)-ASTM D6751 ).

As used herein, the term "microorganism" refers to an organism that is microscopic. Microorganisms are very diverse, they include bacteria, fungi, archaea, and protists, microscopic plants, algae and microscopic animals. In this invention, microorganism is cultivated in medium containing carbon source of sugarcane. Therefore, microorganism refers to microorganism that has the ability of using organic substrates to maintain life cycle and to generate interest metabolite that can be processed into biofuel or directly served as biofuel, unless otherwise noted. As used herein, the term "lipid" is broadly defined as any fat-soluble molecule, such as fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids and others.

As used herein, the term "oil" includes compound classes with otherwise unrelated chemical structures, properties and uses, including vegetable oils, petrochemical oils, volatile essential oils and microorganism-originated oils. Oil is a non-polar substance.

As used herein, the term "triglyceride" (also known as triacylglycerol, TAG or triacylglyceride) is a glyceride in which the glycerol is esterified with three fatty acids. It is the main constituent of vegetable oil, animal fats and microorganism-originated oil.

As used herein, the term "sugarcane juice" refers to the liquid extracted from the crushed stalks of sugarcane.

As used herein, the term "bagasse" refers to the dry, fibrous residue remaining after the extraction of juice from the crushed stalks of sugarcane.

As used herein, the term "transesterification" refers to a process of exchanging the alcohol group of an ester compound with another alcohol.

Almost all biodiesel is produced using transesterification technique as it is the most economical process. The process involves reacting oils or animal fats catalytically with short-chain aliphatic alcohols (typically methanol or ethanol). These reactions are often catalyzed by the addition of acid, base or enzyme.

As used herein, the term "heterotrophic" refers to an organism or condition that uses organic substrates to get its chemical energy for its life cycle.

As used herein, the term "fermentable carbon source" refers to the organic carbon source that can be directly or indirectly utilized by heterotrophic microorganism.

As used herein, the term "reducing sugar" refers to any sugar that, in basic solution, forms some aldehyde or ketone. This allows the sugar to act as a reducing agent and react with, for example, 3,5-dinitrosalicylic acid (DNS), Benedict's reagent and Fehling's solution. Reducing sugars include glucose, fructose, lactose, arabinose, maltose and glyceraldehyde. Significantly, sucrose and cellulose are not reducing sugars. According to one aspect of the present invention, the present invention provides method of providing fermentable carbon source to microorganism. Carbon source is a nutrient that provides carbon skeletons needed for synthesis of new organic molecules, which can be classified into organic and inorganic carbon sources. Carbon dioxide is known as inorganic carbon source. The most common organic carbon source for heterotrophic microorganism includes, but not limited to, pentose, hexose, acetate, starch, sucrose, etc. The carbon sources in sugarcane stalks are mainly juice-originated carbon source (sucrose, fructose and glucose etc.) and bagasse-originated carbon source (cellulose etc.), which are ideal for preparing fermentation media. Many species have the ability of utilizing organic carbon source, such as but not limited to bacteria, fungi, archaea, and protists, microscopic plants, algae and microscopic animals. The recombinant or mutant species are also included in the spirit of the present invention.

In one embodiment of the present invention, alga was administrated with the described sugarcane-originated carbon source. In one preferred embodiment of the present invention, Chlorella protothecoides was chosen as the subject administrated with the described sugarcane-originated carbon source, because C. protothecoides has the ability of accumulating oil at high content whenever organic carbon source is provided. However, the known organic carbon source of C. protothecoides is hexose. Thus, sugarcane was processed before fermentation. In order to investigate the utilization of juice-originated and bagasse-originated carbon source, juice and bagasse were obtained and processed respectively. The method used to separate juice from bagasse can be any device, method or technique that are known to those skilled in the field, includes but not limited to centrifugation, crushing, milling, grinding. In a preferred embodiment, the sugarcane stalks was processed by juice extractor device equipped with roller mill apparatus, which is available in market. And the extracted sugarcane juice was collected and filtered through a screen filter. The dominating carbon source in sugarcane juice is sucrose. Sucrose is a disaccharide of glucose and fructose. One convenient way of providing hexose from sucrose is hydrolysis. Hydrolysis can be catalyzed by acid, base and enzyme. In a preferred embodiment, sugarcane juice was supplemented with fructofuranosidase to achieve the hydrolysis of sucrose into glucose and fructose. The dominating carbon source in bagasse is cellulose. Cellulose is a polysaccharide consisting of a linear chain of several hundred to over ten thousand (1 →4) linked D-glucose units. One convenient way of providing hexose from cellulose is hydrolysis. Hydrolysis can be catalyzed by acid, base and enzyme. In a preferred embodiment, bagasse was supplemented with cellulase to achieve the hydrolysis of cellulose into glucose. The preferred embodiments should not be considered as limitation, some other species may utilize the sugarcane-originated carbon source without or with different processing.

According to another aspect of the present invention, the present invention provides method of cultivating microorganism in medium containing the sugarcane originated-carbon source provided according to the present invention. The sugarcane originated-carbon source can be provided to microorganism in any form, by any pathway or strategy, at any amount, which depends on the specific needs of specific microorganism. The sugarcane originated-carbon source can be provided once at a time or by fed-batch. The sugarcane originated-carbon source can be provided in the form of liquid media or solid media. The sugarcane originated-carbon source can be provided at a concentration allowing microorganism to accumulate metabolite suitable for biofuel preparation. The media containing sugarcane originated-carbon source can be sterilized or non-sterilized. The media containing sugarcane originated-carbon source can be supplemented with or without other components, which depends on the needs of specific microorganism. Various devices and conditions for cultivating microorganism are well known and readily used by those skilled in the art and include, but not limited to, bioreactor, flask, incubator, pH, dissolved oxygen, temperature, stirrer, inoculation amount, shaking speed, ventilation or light etc. In some embodiments, alga was cultivated in sterilized liquid medium containing sugarcane (juice or bagasse) originated-carbon source. In one embodiment, alga was cultivated in non-sterilized liquid medium containing sugarcane originated-carbon source. In a preferred embodiment, nitrogen source (such yeast extract) and/or ion source (such as phosphate, magnesium salt, ferric salt) and/or vitamin (such as VB i) and/or trace element were also added in the media containing sugarcane originated-carbon source. In another preferred embodiment, nothing was added in the media containing sugarcane originated-carbon source. In a preferred embodiment, alga was cultivated in flask with shaken, and in another preferred embodiment, alga was cultivated in bioreactor. In a preferred embodiment, alga was cultivated in media containing sugarcane originated-carbon source with an initial concentration of 1 -60 g I^"1, preferably at 5-50 g I^"1, more preferably at 10-30 g I^"1.

According to another aspect of the present invention, the present invention provides method of extracting or collecting interest microorganism-originated metabolite by using sugarcane-originated carbon source as feedstock. The following non-limiting list of microorganism-originated metabolite can be produced from sugarcane-originated carbon source provided in the present invention: alcohols, fatty acids, oils, lipids, hydrocarbons and the derivatives thereof. The devices and methods for extracting or collecting interest metabolite depend on the character of interest metabolite, the condition of subject microorganism and the needs of subsequent process. Various devices and methods for extracting or collecting interest microorganism-originated metabolite are well known to those skilled in the art. In a preferred embodiment, the interest microorganism-originated metabolite is algal oil, in which triglyceride and fatty acids are the dominating components. The following non-limiting list techniques can be applied in extracting or collecting agal oil: oil expeller, extraction, fractionation, distillation, chromatography. Algal oil can be easily extracted by organic reagent such as but not limited to acetone, n-hexane, chloroform, methane, acetonitrile etc. In a preferred embodiment, algal oil was extracted from the algal cells by Soxhlet apparatus with n-hexane as solvent. After removing n-hexane by a rotary evaporator, oil was obtained.

According to another aspect of the present invention, the present invention provides method of preparing biofuel from microorganism-originated metabolite obtained according to the present invention. The following non-limiting list of techniques can be applied in preparing biofuel from microorganism-originated metabolite: extraction, fractionation, distillation, addition reaction, neutralization, hydrogenation, dehydrogenization, oxidation, reduction, substitution, esterification, transesterification and hydrolysis. The method of preparing biofuel from microorganism-originated metabolite depends on the character of interest metabolite. In a prepared embodiment, the inventor unexpectedly found that specific metabolite, preferably oil, was accumulated in algal cells when the described sugarcane-originated carbon source was administrated to alga. The inventor also unexpectedly found that the specific metabolite, preferably algal oil, was extremely suitable for biodiesel preparation. In a prepared embodiment, the extracted algal oil was processed into biodiesel by transesterification. The process involves reacting oils catalytically with short-chain aliphatic alcohols (typically methanol or ethanol). These reactions are often catalyzed by the addition of acid, base or enzyme. In a prepared embodiment, the said algal oil reacted with methanol by lipase catalysis.

Examples :

Example 1. Microorganisms, maintenance and inoculum

Chlorella protothecoides was cultivated in basic medium supplemented with 10 g l^glucose and 3 g I^"1 yeast extract (YE). Basic medium contains: 0.7 g I^"1 KH₂PO₄ , 0.3 g T¹K₂HPO₄ , 0.3 g I^"1 MgSO₄ ^»7H₂O, 0.3 mg I^"1 FeSO₄ ^»7H₂O and 0.01 mg I^"1 VBi . Heterotrophic cells in exponential phase were used to inoculate fresh media. Example 2. Preparation of fermentable carbon source originated from sugarcane iuice Sugarcane juice was extracted from the sugarcane stalks by juice extractor equipped with roller mill apparatus; the extracted sugarcane juice was collected and filtered through a screen filter; the extracted juice was supplemented with fructofuranosidase (Valisase R Ivertase ANL, 15000 Valley Summer Unit g^{" 1}) and hydrolyzed according to the instruction provided by manufacturer. The concentration of reducing sugar (hydrolysate) was monitored at regular intervals by dinitrosalicyclic acid (DNS) method to determine the complete digestion. Example 3. Cultivation in shake flask with sugarcane juice-originated carbon source as feedstock

In comparison experiment, basic medium supplemented with 3 g I^"1 YE and basic medium supplemented with 20 g I^"1 glucose and 3 g I^"1 YE were respectively used as negative and positive control to investigate the cell growth and oil accumulation of algal cells in basic medium supplemented with 20 g I ¹ sugarcane juice-originated carbon source and 3 g I^"1 YE. All media and cultivation apparatus were sterilized with steam at 112°C, 0.12 Mpa for 30 min. C. protothecoides in exponential phase was inoculated into media at equivalent initial cell density. Heterotrophic cultivation was carried out in 500 ml flasks containing 200 ml medium at 28 ± 1 °C with continuous shaking (220 rpm). Samples were taken at regular intervals to determine the cell density and sugar concentration, curves of cell growth and sugar consumption were recorded (Figure 1 ). Example 4. Preparation of fermentable carbon source originated from bagasse

The bagasse was collected after the extraction of juice from the crushed stalks of sugarcane. The bagasse was washed with water for 3 times to remove the residual juice, and then air-dried to constant weight. The bagasse was processed into dry powder by mill before later use. 50 g of the bagasse powder was added into 1000 ml of 50 mM citrate buffer (pH 4.8), and then supplemented with 6.5 ml cellulase (DENICELL 101L, India) to make a reaction mixture. The mixture was incubated at 50⁰C, 140 rpm for 24-36 hours, and then the mixture was inactivated at 100⁰C for 5 min. The supernate was collected by centrifugation (100Og, 10 min) for cultivating alga. The concentration of reducing sugar in supernate was measured by DNS method.

Example 5. Cultivation in shake flask with carbon source originated from bagasse as feedstock In comparison experiment, basic medium supplemented with H g l^glucose and 3 g I^"1 YE (Figure 2 a) were respectively used as positive control to investigate the cell growth and oil accumulation of algal cells in basic medium supplemented with 11 g I^"1 bagasse-originated carbon source and 3 g I^"1 YE (Figure 2 b). All media and cultivation apparatus were sterilized with steam at 112°C, 0.12 Mpa for 30 min. C. protothecoides in exponential phase was inoculated into media at equivalent initial cell density. Heterotrophic cultivation was carried out in 500 ml flasks containing 200 ml medium at 28 ± 1 °C with continuous shaking (220 rpm). Samples were taken at regular intervals to determine the cell density and sugar concentration, curves of cell growth and sugar consumption were recorded.

Example 6. Fermentation of alga in 5-1 bioreactor by using sugarcane-originated carbon source Algal heterotrophic fermentation in bioreactors, instead of cultivation in shake flask, is commonly used in practical production. Therefore algal fermentations in 5-1 bioreactors (Minifors, INFORS AG CH-4103, Bottmingen, Switzerland) containing 3-1 medium were conducted with 3 g I^"1 YE and 30 g I^"1 glucose (Figure 3 a) or juice-originated carbon source (Figure 3 b) as the starting condition. Carbon source and YE were batch-fed whenever the carbon source was exhausted. 0.5 M KOH solution was batch-fed to keep pH at 6.3 ± 0.3 ; temperature was controlled at 28 ± 1 ⁰C; dissolved oxygen concentration was controlled between 20-50% air saturation by airflow and agitation speed. The cell density and consumption of carbon source were determined and recorded at regular interval.

Example 7. Cultivation in sugarcane juice-originated carbon source media supplemented with and without other nutritional elements The sugarcane juice prepared in example 2 was adjusted to 20 g I^"1 (the concentration of reducing sugar) and served as medium (a). Medium (b) was prepared by adding 3 g I^"1 YE, 0.7 g I^"1 KH₂PO₄, 0.3 g 1^"1K₂HPO₄, 0.3 g I^"1 MgSO₄«7H₂O, 0.3 mg I^"1 FeSO₄«7H₂O and 0.01 mg I^"1 VB₁ to medium (a). All media and cultivation apparatus were sterilized with steam at 112°C, 0.12 Mpa for 30 min. C. protothecoides in exponential phase was inoculated into media at equivalent initial cell density. Heterotrophic cultivation was carried out in 500 ml flasks containing 200 ml medium at 28 ± 1 °C with continuous shaking (220 rpm). Samples were taken at regular intervals to determine the cell density and sugar concentration, curves of cell growth and sugar consumption were recorded (Figure 4).

Example 8. Cultivation in non-sterilized medium containing sugarcane-originated carbon source The basic medium described in example 1 was supplemented with 30 g I^"1 sugarcane juice-originated carbon source prepared according to the method in example 2. All media and cultivation apparatus were not sterilized before using. C. protothecoides in exponential phase was inoculated into media at a relative higher inoculation concentration (about 4 g T¹). Heterotrophic cultivation was carried out in 500 ml flasks containing 200 ml medium at 28 ± 1 °C with continuous shaking (220 rpm). Samples were taken at regular intervals to determine the cell density and sugar concentration, curves of cell growth and sugar consumption were recorded (Figure 5). Example 9. Monitor of cell growth, reducing sugar consumption and oil content

Cell growth was monitored by optical density measurements at 540 nm using UV/visible spectrophotometer (Pharmacia Biotech Ultrospec 2000, Sweden). Algal cells were harvested and weighed after lyophilization. Concentration of reducing sugar was analyzed by DNS method. Oil content was analyzed by TD-NMR on Bruker Minispec

MQ20 NMR Analyzer (Bruker, Rheinstetten, Germany) with a 35-mm absolute probe, at resonance frequency of 19.95 MHz (Gao C et al. Rapid quantitation of lipid in microalgae by time-domain nuclear magnetic resonance. J Microbiol Methods. 2008, 75 :437-40).

Example 10. Cell collection, oil extraction

Cells in flask and/or bioreactor cultures were collected by centrifugation and lyophilized to constant weight. Total oil in algal cells was extracted by standard Soxhlet extraction. After removing n-hexane by a rotary evaporator (N-1000, Eyela, Japan), oil was obtained and weight.

Example 11. Biodiesel preparation from algal oil Alga-based biodiesel was prepared by transesterification of algal oil by using sugarcane-originated carbon source.

The lipase catalyzed transesterification was performed in shaking flasks and heated to the reaction temperature on a constant temperature shaker, with the rotation rate of 180 rpm. The reaction conditions were 30% immobilized lipase (w/w, 12,000 U), 10% water content (w/w) based on lipids quantity and 3 : 1 molar ratio of methanol to oil, at the temperature of 38 ⁰C and the pH value of 7.0

To determine the reaction rate and conversion from heterotrophic lipids to biodiesel, the reaction mixture was sampled every 2 h and analyzed by gas chromatograph (GC), through which the concentration of triglycerides, diglycerides, monoglyceride and fatty acid methyl esters could be determined. The conversion rate was calculated by the proportion of fatty acid methyl esters in the mixture. Example 12. Analysis of biodiesel

The properties of biodiesel such as density, viscosity, flash point, cold filter plugging point, solidifying point and heating value were also measured. The composition of biodiesel produced from extracted algal oil was analyzed on GC-linked mass spectrometry (GC-MS), dual-stage quadrupoles GC apparatus (Thermo, USA) was equipped with a Varian VF-5ms column (30m^χ 0.25mm ID DF=O .25 μm) and the GC was manipulated with a flow rate of 10 ml min^{" 1}.

Result

The preparation of fermentable carbon source from sugarcane juice and bagasse

To obtain carbon source available to microalgal utilization, sugarcane juice and bagasse was enzymatically hydrolyzed. The concentration of reducing sugar reached maximum level (for juice : 199.4 ± 8.7 g T¹ , for baggase : 15.6 ± 1 .0 g I^{" 1}) within one hours. Cell growth and oil accumulation in C. protothecoides by using sugarcane iuice-originated carbon source as feedstock Media without carbon source and media with glucose were used as comparison to evaluate the effects of media with juice-originated carbon source on heterotrophic cultivation of C. protothecoides (Figure I a). It was observed that sugarcane juice (Figure I b) could not be utilized directly by C. protothecoides before hydrolysis treatment. However, juice-originated carbon source can be used as carbon source appropriate to algal utilization. It is showed that the biomass, oil content and conversion ratio obtained in media containing juice-originated carbon source reached equivalent levels to those of glucose media (Table 1 ). Table 1. The effects of different carbon source feedings on heterotrophic cultivation (in 500 ml flasks containing 200 ml media).

Feedings Oil content Conversion ratio (%

(2O g I^"1) (%: Biomass/Sugar Oil/Sugar

Glucose 46.7±1.1 30.8 14.4

Juice-originated ^ ^ ^ ^ carbon source

Cell growth and oil accumulation in C. protothecoides by using sugarcane bagasse-originated carbon source as feedstock Media with glucose (H g I^{" 1}) (Figure 2a) were used as comparison to evaluate the effects of media with bagasse-originated carbon source (H g I^{" 1}) (Figure 2b) on heterotrophic cultivation of C. protothecoides. It was observed that sugarcane bagasse-originated carbon source can be used as carbon source appropriate to algal utilization. Table 2. The effects of different carbon source feedings on heterotrophic cultivation in flask (200 ml media).

Feedings Oil content Conversion ratio (%

(H g r¹) ^(O/- Biomass/Sugar Oil/Sugar

Glucose 49.1±2.0 30.5 15.0

Baggase-originated ^ _{g±3 η} ^ _η ^ _g carbon source

Fermentation of algal cell

Within 7-day cultivation, it is interesting to note that C. protothecoides grown faster and provided higher yield of biomass and oil with juice-originated carbon source than that with glucose feeding (Fig. 3). The algae with juice-originated carbon source consumed less sugar but yielded more biomass and oil than that with glucose feeding (Table 3). The conversion ratio of sugar-to-biomass and sugar-to-oil with juice-originated carbon source were higher than that with glucose feeding by 15.2 % and 8.8 %, respectively (Table 3). It is reasonable as there are many nutritions in sugarcane juice benefit to cell growth and oil accumulation.

Table 3. The effects of juice-originated carbon source feedings on oil production and carbon utilization in 5-1 bioreactor.

Consumed Biomass Oil yield Conversion ratio (%

Feedings sugar (g) yield (g) (g) _{Biomass/Sugar Qj}l/ Sugar

Glucose 304.0 107.8 51.8 35.5 17.0 juice-originated ^_{5 4} ^ ₃ ^₇ ^ ₉ ^ ₅ carbon source

Cultivation in sugarcane iuice-originated carbon source media supplemented with and without other nutritional elements

The present inventor proved that media containing sugarcane-originated carbon source can be used directly, and there is no need to add other nutritional elements (example 7). Therefore the cost of cultivation was reduced. In Figure 4, cells in sole sugarcane-originated carbon source grew faster and consumed more carbon source during the first two days, but reach a lower level of maximum cell density than that with supplementation, less nitrogen in the source feed may account for the lower level. Nevertheless, the result indicated that sugarcane-originated carbon source can be used directly as feedstock for microalgal cultivation.

Cultivation in non-sterilized medium containing sugarcane- originated carbon source The present inventor further proved that there is no need to sterilize the media before using (example 8). Therefore the cost of cultivation was reduced, the present invention also indicate a possibility of cultivating microorganism in open system. However, a lower level of maximum cell density was obtained (Figure 5).

Analysis of biodiesel produced from algal oil by transesterification

The main fatty acids methyl esters detected in biodiesel from both juice-originated and bagasse-originated carbon source include 9-Octadecenoic acid methyl ester, 9, 12-Octadecadienoic acid methyl ester and hexadecenoic acid methyl ester. Other minor methyl esters were also detected (Table 4). This result illustrates that the main components of biodiesel from both juice and bagasse feeding are similar.

Table 4. The components of biodiesel produced from sugarcane- originated carbon souce feeding.

Relative content of fatty

9-Octadecenoic acid methyl ester (C₁₉H₃₆O₂) 53.75 52.56

9, 12-Octadecadienoic acid methyl ester (C₁₉H₃₄O₂) 19.48 10.33

Hexadecenoic acid methyl ester (C₁₇H₃₄O₂) 11.34 12.49

Octadecanoic acid methyl ester (C₁₉H₃₈O₂) 4.88 5.40

Heptadecanoic acid methyl ester (C₁₈H₃₆O₂) 1.48 0.38 9-Octadecenoic acid (z)-,2-hydroxyl-l-(hydroxym ethyl) ethyl ,_* ester (C₂₁H₄₀O₄)

8-Octadecenoic acid methyl ester (C₁₉H₃₆O₂) nd 3.86

Oxiraneoctanoic acid, 3 -octyl-, methyl ester, cis- (C₁₉H₃₆O₃) nd 2.1

Methyl tetradecanoate (C₁₅H₃₀O₂) nd 0.46

7- Hexadecenoic acid methyl ester (C₁₇H₃₂O₂) nd 0.41

10-Nodadecenoic acid methyl ester (C₂₀H₃₈O₂) 0.75 nd

Eicosanoic acid methyl ester (C₂₁H₄₂O₂) 0.51 nd

Cyclopropaneoctanoic acid 2-hexyl-methyl ester (C₁₈H₃₄O₂) 0.37 nd

Heptadecanoic acid 16-methyl-methyl ester (C₁₉H₃₈O₂) 0.24 nd

11 -Hexadecenoic acid methyl ester (C₁₇H₃₂O₂) 0.18 nd nd = not detected

Biodiesel obtained from heterotrophic algal oil by transesterification was characterized by a density of 0.864 kg-L^"1, a higher heating value of 41 MJ-kg^"1 and a viscosity of 5.2 ^χ l O^~4 Pa-s (at 40⁰C) (Table 5).

Table 5. Comparison of properties of biodiesel from microalgal oil and diesel fuel and ASTM biodiesel standard.

Properties Biodiesel from Diesel fuel^a ASTM biodiesel algal oil standard

Density (kg-L^"1) 0.864 0.838 0.86 - 0.90

Visicosity (mmV¹, cSt at 40⁰C) 5.2 1.9 - 4.1 3.5 - 5.0

Flash point (⁰C) 115 75 Min 100

Solidifying point (⁰C) -12 -50 - 10 -

Cold filter plugging point (⁰C) -11 -3.0 (Max -6.7) Summer max 0

Winter max < -15

Acid value (mg KOH-g^"1) 0.374 Max 0.5 Max 0.5

Heating value (MJ- kg^"1) 41 40 - 45 -

H/C ratio 1.81 1.81 - ^aThe data about diesel fuel was taken from published literature as indicated in the text.

The results suggest that the novel process was a feasible and effective method for the production of high quality biodiesel from algal oil.

All patents, patent applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Claims

1. A method of producing biofuel from microorganism- originated metabolite by using sugarcane as feedstock, comprising: (a) providing fermentable carbon source of sugarcane;

(c) extracting or collecting interest microorganism-originated metabolite from microorganism; (d) producing biofuel from microorganism-originated metabolite.

2. The method according to Claim 1 , wherein said biofuel is biogas, bioethanol, biodiesel or biomass, and more preferably is biodiesel.

3. The method according to Claim 1 , wherein said microorganism is selected from the group consisting of bacteria, fungi, archaea, protists, alga, microscopic plants and microscopic animals; and more preferably is selected from the group consisting of alga and microscopic plants; and most preferably is alga.

4. The method according to Claim 1 , wherein said microorganism- originated metabolite is the interest components that can be processed into biofuel or directly served as biofuel; and more preferably said microorganism-originated metabolite is microorganism-originated oil, lipid and fatty acids; and most preferably said microorganism-originated metabolite is algal oil, lipid and fatty acids.

5. The method according to Claim 1 , wherein said fermentable carbon source of sugarcane originates from sugarcane juice and bagasse; and more preferably originates from sugarcane juice.

6. The method according to Claim 1 or 5, wherein said fermentable carbon source is processed by hydrolysis before being provided to said microorganism.

7. The method according to Claim 6, wherein said hydrolysis is catalyzed by acid, alkali or enzyme.

8. The method according to Claim 1 , 2 or 4, wherein said biofuel is produced from said microorganism-originated metabolite by transesterification.

9. The method according to Claim 7, wherein said transesterification is catalyzed by acid, alkali or enzyme.

10. A method of producing biofuel from alga-originated metabolite by using sugarcane as feedstock, comprising:

(a) providing fermentable carbon source of sugarcane;

(b) cultivating alga in medium containing the carbon source described in (a);

(c) extracting or collecting interest alga-originated metabolite from alga;

(d) preparing biodiesel from algal-originated metabolite.