ZA201007116B - Biofuel production - Google Patents

Description

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BIOFUEL PRODUCTION

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EIELD OF THE INVENTION

This invention relates to the production of biofuels using microbial consortium derived from the gut of dung beetles.

BACKGROUND TO THE INVENTION

The order Coleoptera (beetles) is the most diverse on earth. The scarab beetle, Euoniticellus intermedius, is highly effective in new habitats and often more successful than native beetles. E. intermedius makes tunnels beneath dung pats where they feed and reproduce. Aduits obtain nutrients from the microbe - rich liquid portion of dung feed and do not actually consume the dung '. The dung beetle larvae consume most of the dung in brood balls where they are laid.

Dung beetles have important agricultural benefits and are often introduced in the environment to alleviate ecological damage. A recent meeting of some 1500 conservation biologists at

Columbia University found that loss of dung beetles could hurt the ecosystem®. Some of the benefits include nutrient recycling, improvements to soil tilth and pest control. E. intermedius beetles are the most beneficial to pasture health and in agriculture as they enhance soll conditions by increasing percolation.

The value of insect ecological services in the United States of America was estimated at $60 billion in 2006 with the dung beetles being the most significant contributors. Scarab beetles contributed services such as dung burial, reducing forage fouling, nitrogen volatilization and control of cattle parasites and pest flies”.

Mostly behavioural, ecological and taxonomic studies of E. intermedius have been conducted so far. The first significant molecular biology study of E.

intermedius has been performed by researchers at the Applicant’. These beetles are of scientific interest because of their microbe-rich habitat, as they may have a potent immune system. Their immune system could provide useful strategies for treatment of infections diseases affecting humans and animals. The study of beetles could also help in managing ecosystems and in enhancing agricultural practices. Moreover, in the context of bio-based energy and materials production, dung beetles’ (and other specialized beetles for that matter) gut microbial consortia can potentially be used in industrial bioprocessing.

Microbial consortia are ubiquitous in nature and are implicated in processes of great importance to humans, from environmental remediation and wastewater treatment to assistance in food digestion and, recently, the production of drugs and fuels. Microbial consortia can perform complicated functions that individual populations cannot and they can be more robust to environmental fluctuations. Microbial consortia can either be associated with higher organisms, i.e. in the form of (endo)symbiosis/(endo)parasitism or in the open environment, i.e. enriched in ecological niches. In the practice of environmental remediation and wastewater treatment, for example, microbial consortia are found in the open environments® and undergo enrichment and selection in the bioprocess itself. In contrast, a very prominent example for the application of consortia associated with a higher organism is cheese production, where “pre-selected” microorganisms from calf rennet are employed to improve the process. Pre-selected, enriched and, therefore, consortia that can perform a specialized bioprocess are termed a “starter culture”. In bioprocessing, such “starter cultures” are employed to render the process stable and reproducible. This concept is also increasingly used in environmental remediation, where the addition of “starter cultures” to waste (water) treatment or other environmental detoxification processes is termed “bioaugmentation”.

The concept of employing symbiotic consortia in industry is as old as cheese production (above example). Commercially available starter cultures also include but are not limited to oil and fat spill treatments, composting, cheese and yoghurt production, ... More recent examples, however, show that the industry is still looking at consortia for the application in various other processes, such as the use of mycorrhizae as fertilizers® or as plant growth promoters’. In addition, the advent of biofuels production has sparked a quest for suitable microorganisms, i.e.

consortia found in organisms that feed on lignocellulose such as the termite? or the locust’. Such consortia can be used in the production of biofuels from cellulose.

OBJECT OF THE INVENTION it is the object of this invention to provide an efficient and safe microbial consortium for the production of chemicals from biomass. :

SUMMARY OF THE INVENTION

In accordance with this invention there is provided a process for producing a hydrocarbon fuel from a biomass feedstock, said process comprising introducing an at least partially liquefied biomass feedstock into a reactor vessel, inoculating the feedstock with at least one microbial consortium obtained from insects of the order Coleoptera, allowing the microbial consortium or consortia to, at least partly, digest the feedstock thus producing at least one hydrocarbon fuel, and separating the hydrocarbon fuel from the partly digested feedstock for storage or immediate use.

There is further provided for the Coleoptera from which the microbial consortia are obtained are dung beetles, preferably copraphagous beetles, and further preferably beetles of the species

Euoniticellus intermedius. For the microbuial consortia to be obtained from adult, alternatively larval beetles, preferably third instar larvae.

There is also provides for the microbial consortia obtained from E. intermedius to be supplemented with additional microbial consortia to produce a variety of desired hydrocarbon compounds such as, for example, yeasts where ethanol is a desired product.

There is also provides for the biomass feedstock to be a nitrogen rich feedstock, preferably animal waste, and further preferably animal, preferably herbivore and further preferably ruminant, dung . For the feedstock to be at least partially liquefied before its introduction into the

£ 2010707118 reactor vessel by the addition of a liquid, preferably water and or nutrients which, in use, stimulate initial growth of the microbial consortia, to the raw feedstock, alternatively to a partially comminuted feedstock, further alternatively to a chopped feedstock. Further, where the feedstock is sufficiently liquefied such as, for example, in the case of ruminant dung sourced from a feed lot, the liquefaction step may be omitted.

There is further provided for the feedstock to be inoculated with at least one microbial consortium prior to its introduction into the reactor vessel. Alternatively there is provided for the feedstock to be inoculated with at least one microbial consortium after its introduction into the reactor vessel.

There is further provides for the hydrocarbon fuel to be a gas, preferably methane, which is drawn from the top of the reactor vessel for storage and later use or for immediate use.

Alternatively there is provides for the hydrocarbon fuel to be a liquid, preferably an alcohol or a related compound, which is drawn from liquid in the reactor vessel. Further alternatively there is provides for the hydrocarbon fuel to be a gas, preferably methane, and a liquid, preferably an alcohol or a related compound, which are drawn from the top and from liquid in the reactor vessel respectively.

There also provided for the feedstock and microbial consortia to be heated in the reactor vessel to a temperature of between 25°C and 35°C, preferably 30°C, and for energy required to heat the feedstock to be obtained from biofuel produced in the reactor vessel.

The invention extends to a biofuel reactor for producing a hydrocarbon fuel from biomass according to the above process, said biofuel reactor comprising a reactor vessel having an inlet for receiving, in use, a biomass feedstock, and at least one outlet for biofuel produced, in use, in the reactor.

There is further provided for the reactor vessel to be generally circular cylindrical having a feedstock inlet towards its base, a gaseous fuel outlet towards its top and a liquid fuel outlet between the feedstock inlet and the gaseous fuel outlet. The reactor vessel includes an agitator, preferably a set of paddles, located towards the base which, in use, stirs the reactor vessel contents thus inhibiting collection of sediment and/or sludge at the base of the reactor vessel.

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There is further provided for the reactor vessel to have a microbial consortia inoculant inlet and, preferably, an inoculant inlet which includes a feedback for material drawn from the reactor vessel as well as for fresh inoculant.

There is also provided for the reactor vessel to include a means for heating the contents of the vessel to a temperature of between 25°C and 35°C, preferably to 30°C, and for the means for heating the contents of the reactor vessel to be an internal heating element which is located within the reactor vessel, alternatively an external heating system. There is also provided for the heating means to be an electric element, alternatively a series of steam charged hollow tubes.

BRIEF DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The above and additional features of the invention will be described below by way of example only and with reference to the accompanying figures in which:

Figure 1: is a series of gas chromatography results for methane production. Results are expressed in moles of methane produced per liter of media used. Carbon sources were 0.225g/l D-glucose and 0.855 g/l D-lactose for basal medium AM1. Carbon sources for basal medium AM2 was 0.405 g/l microcrystalline cellulose, AM3 was 0.225 g/l D-glucose, 0.855 g/l D-lactose and 0.17 g/l sodium formate, AM4 was 0.225 g/l D-glucose, 0.855 g/l D-lactose and 0.21 g/l sodium acetate. No methane was detected in the control experiments (not shown);

Figure 2: are HPLC results for acetic acid production by gut consortia of Euoniticellus intermedius larvae after 4 weeks of incubation;

Figure 3: is a series of photographic images of filter paper degradation by Euoniticellus intermedius midgut consortia. A is the midgut consortia enriched in TSB (TSB M),

B is midgut consortia enriched in M1A (M1A M), C is midgut consortia originally from the larvae which had not been enriched (DB M), D is midgut consortia enriched in M1B (M1B M), E is midgut consortia enriched in NB (NB M) and F is the control (non inoculated culture). Control filter paper is still completely intact.

Incubation was done for 4 weeks at 30°C in the dark.

Figure 4: is a series of photographic images of filter paper degradation by Euoniticellus intermedius hindgut consortia. A is the hindgut consortia enriched in NB (NB H), B :

is hindgut consortia enriched in M1A (M1A H), C is hindgut consortia enriched in

TSB (TSH H), D is hindgut consortia enriched in M1B (M1B H), E is hindgut consortia originally from the larvae which has not been enriched (DB H) and F is the control (non inoculated culture). Control filter paper is still completely intact.

Incubation was done for 4 weeks at 30°C in the dark;

Figure 5: are HPLC results for D-glucose production by gut consortia of Euoniticellus intermedius larvae after 4 weeks of incubation; and

Figure 6: is a simplified sketch of a methane biofuel reactor according to one embodiment of the invention.

In the production of biogas from dung, processes rely on the productivity of consortia found in the substrate itself. The use of symbiotic consortia other than found in the dung itself, e.g. water buffalo dung, in the processing of dung for the production of biogas has not been attempted.

The application of consortia found in the gut of the dung beetle as well as their larvae as starter culture in the production of biofuels and commodity/specialty chemicals from nitrogen-rich substrates such as dung is proposed. Coprophagous beetles as well as their larvae have adapted to feeding on dung. it can be assumed that the microbial consortia in their guts have evolved to be adapted to their food and are highly enriched and specialized for the utilization of dung. In general, the employment of specialized starter cultures has proven to stabilize bioprocesses to environmental fluctuations and, therefore, make the process more productive. In terms of biogas production from dung, this means increase the robustness of the bioprocess to pH and temperature fluctuation, higher turnover rates and ultimately higher biogas yields. In addition, it is very likely that these consortia convert dung into chemicals other than biogas, which can be used for materials and commodities. In practice, such a starter culture is produced, packed and sold to waste and dung treatment plants, biogas production plants, or can be marketed as part of a (domestic) biogas production facility for cattle farms. The following sections describe (A) the methane production and (B) the cellulose degradation capacities of the starter culture as well as (C) the identity of the microorganisms that constitute the starter culture (A) Methane production by microbial consortia

Biogas is produced by anaerobic digestion of organic material. In anaerobic digestion, there are several interdependent steps, complex sequential and parallel biological reactions in the absence of oxygen, during which the products from one group of microorganisms, in a consortia, serve as the substrate for the next, resulting in transformation of organic matter mainly into a mixture of methane and carbon dioxide (Parawira, 2004; Kelleher et al., 2000; Marty et al., 2001;

Demirbas, 2008).

The digestive process is in three stages done by consortia of microbes; in the first stage, a group of microorganisms convert organic matter into a form that can be used by the second group, acetogens, which form organic acids. Methanogens then utilize the acids in the third stage producing biogas as a by-product (Demirbas, 2008).

Methanogens are strict anaerobic microorganisms which are methane producing that belong to the kingdom Archaebacteria (Abbanat et al, 1989; Zinder, 1998). Methanogens are terminal microorganisms in microbial consortia food chain comprised of at least three interacting metabolic groups of strictly anaerobic bacteria that together convert complex organic matter to carbon dioxide and methane (Abbanat ef al., 1989; Zinder, 1998). The methanogens metabolize acetic, formic acid or hydrogen produced by acidogens to produce methane (Abbanat et al, 1989; Zinder, 1998; Wang et al., 2009).

Termites, cockroaches, and scarab beetles are the only insects known to emit methane (Brune, 2009). Methanogenesis occurs in the enlarged hindgut compartment and is fueled by hydrogen and reduced one-carbon compounds that are formed during the fermentative breakdown of plant fiber and humus (Brune, 2009). However, in scarab beetle like Pachnoda epphippiata, it has been shown that midgut fermentations are coupled to methanogenesis in the hindgut by formate transported via the hemolymph (Lemke et al., 2003) and possible acetate.

From Figure 1 it can seen that the greatest amount of methane was produced by microbial consortium grown in basal medium AM1 (carbon source was D-glucose and D-lactose), specifically the hindgut consortium with an average of 0.034 moles/| of the medium AM1 after 8 weeks of incubation. Methane was detected in midgut consortium in AM1 with an average of 0.004 moles/| of methane after 8 weeks of incubation (as shown in Figure 1) this could be attributed to transient methanogens not necessarily a part of the midgut community. It should be pointed out that only one sample out of three for AM1 M produced methane.

(B) Cellulose degradation by microbial consortia

Insects are the most successful class of terrestrial animals, with respect to both species richness and biomass (Brune, 2009). One important reason that has been attributed to this success has been their ability to feed on a variety of diets (Harada and Ishikawa, 1993) which includes fiber- rich foods (Brune. 2009). These feeding habits are closely related to endo-symbionts harbored by the insects with an estimated 10% of all insect species believed to contain microorganisms within their cells (Harada and Ishikawa, 1993). These intracellular symbionts are harbored by epithelial cells of the gut or some special cells present closely around the gut (Buchner, 1965;

Ishikawa, 1989).

Research work by many scientists has shown that different insects have gut microbial consortia capable of digesting cellulose or hemicelluloses material into many different metabolites amongst them being organic acids, ethanol and biogas (Egert et al., 2003; Egert et al., 2005; Lemke et al., 2003; Tholen et al., 1997; Tholen et al., 2007; Italo et al., 2005; Wagner and Brune, 1999; Bignell, 1994; Brennan et al., 2004; Tokuda and Watanabe, 2007). These gut microbial consortia play a vital role in the survival of these insects with some work having had shown that loss of the of the gut consortia often results in abnormal development and reduced survival of the insect host (Eutick et al., 1978; Fakatsu and Hosokawa, 2002).

From an examination of tubes C and D of Figure 3 and from tube D or Figure 4 it is clearly visible that there was degradation of the filter paper by the mid and hindgut microbial consortium under aerobic conditions. Non enriched midgut consortium, DBM, produced the greatest amount of degradation followed by midgut consortium enriched in basal medium M1B and lastly hindgut consortium enriched in M1B. Lemke et al., (2003) report the complete dissolution of filter paper disks by the consortium under oxic conditions. Cellulose degradation amongst the larvae has long been debated as to whether the host produces the enzymes that degrade the cellulose or the symbionts do the degradation or alternatively play a supporting role.

The results shown in Figures 3 and 5 indicate that the symbionts in the midgut and hindgut of the Euoniticellus intermedius third instar larvae do degrade the cellulose and therefore could be degrading the cellulose feed for the host or possible playing a supporting role with the host possible producing more cellulose degrading enzymes.

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M1B H and M1B M show the greatest amount of filter paper degradation for all the enrichments probably due to the fact that the consortium have been enriched on a cellulose carbon substrate : basal medium M1B. This then meant that consortium does not go through a longer lag phase in the basal medium M1C compared to the other enrichments

Visible filter paper degradation noted for DBM in Figure 3 would have suggested a possible high concentration of glucose, however this proved not to be the case as seen on HPLC results for D- glucose on Figure 5. Instead it was TSB H (hindgut consortium enriched in Tryptone soy broth) that produced the highest amount of glucose with 0.34 g/l. Lack of glucose from DBM could be attributed to the fact that sampling for HPLC analysis was done after 4weeks of incubation and possible by that time all the glucose produced would had been utilized by the microorganisms.

For the other samples that glucose was picked up from could be only starting to degrade the filter paper after going through the lag phase. (C) Microbial gut consortia identification 16s rRNA sequences obtained from microorganisms led to the identification of the following microorganisms:

Bacillus. cereus Q1 :

Uncultured bacterial clone DTE1

Uncultured Bacillus sp. © Escherichia coli/Shigella flexneri

Gordornia bronchiallis

CoE ° Comamonas testosteroni aureginosa [eee .

Pseudomonas putida strain ATCC

Uncultured Pseudonocardia

Pseudomonas putida

Ochrobactrum anthropi

Campylobacter showae

Dysgonomonas wimpennyi

MATERIALS AND METHODS

Dissection of dung beetle larvae

The dissection method used by Lemke et al., (2003) was used with some modifications.

A preparation dish filled with wax was sterilized by being left for 24 hours covered with 70% ethanol. The dish was then placed in a laminar flow cabinet 2 hours prior to dissection to allow the ethanol to vaporize off completely. Steel pins, forceps and scissors were sterilized by autoclaving for 15 minutes at 121°C. Insect Ringer solution was prepared following a recipe described by Hayashi and Kamimura, (2001). Composition per 100ml distilled water, 0.9 g sodium chloride (Merck), 0.02g calcium chloride (Merck), 0.02g potassium chloride (Merck) and 0.02g sodium hydrogen carbonate (Merck). The insect Ringers solution was autoclaved for 15 minutes at 121°C.

Third instar larvae were used for all the experiments and all the dissection was done under the dissection microscope. The larvae were first anesthetized by exposure to a nitrogen, hydrogen and carbon dioxide (71/7/22 vol/vol respectively) (Afrox grade) gas mixture for 15 minutes as described by Lemke et al., (2003). Larvae were then fixed with steel pins, with the larvae laid on its sides, in a preparation dish filled with sterile insect ringer solution. The cuticle was cut along the sidelines and the ventral integument, circular muscles and trachea carefully removed. The head was decapitated followed by a circular cut on the anus.

The intestinal tract was then carefully removed from the body. The midgut and hindgut were then separated at the midgut hindgut muscular junction.

Anaerobic cultivation of gut consortia

Basal media AM5 described by Lemke et al., (2003) with some modifications was used in the cultivation of the dung beetle larvae gut consortia.

The composition of AM5 was as follows, 10mg/l resazurin, 1g/l NaCl, 0.5g/1 KCI, 0.5g/l MgCl, .6H,0, 0.1¢g/l CaCl,.2H,0, 0.3g/l NH,Cl, 0.2g/l KH,PO,, and 0.5¢g/l yeast extract.

After complete dissolution of the salts and yeast extract, the medium was then split into four conical flasks in equal volumes. From there, four different medium were then prepared, AM1,

AM2, AM3 and AM4. The carbon sources for the different medium were, for AM1 0.225g/l D- glucose and 0.855g/l D-lactose, for AM2 0.405g/! cellulose microcrystalline (Merck), for AM3 0.225g/l D-glucose, 0.855g/l D-lactose and 0.17g/l sodium formate and for AM4 0.225g/l D- glucose, 0.855g/l D-lactose and 0.2g/1 sodium acetate.

Reducing solution preparation 1.0.2 NNaOH.......................200.0 ml 2. NaS. 9H,0 ....ccoeeeoeeee.2.5¢g 3. L-Cysteine . HCI.....................2.5 g

The sodium hydroxide solution was brought to a boil and bubbled with carbon dioxide (99.999%,

Afrox) for 15 minutes. The solution was then allowed to cool and then sodium sulfide and cysteine were added.

Hungate tube preparation

Each Hungate tube (15ml size tubes) was first gassed with carbon dioxide (99.999%, Afrox) for 3 minutes so as to dust out oxygen from the sealed tubes. Two sterile needles were used both plucked with cotton wool. One needle was connected to the gas supply line therefore brought in the carbon dioxide whilst the other allowed the air to come out the tube during gassing. After autoclaving the tubes containing the reduced media, the tubes were further dusted for 3 minutes removing any traces of oxygen.

Reduced medium preparation

The medium, AM1, AM2, AM3 AND AM4, were brought to a boil while gassing with carbon dioxide (99.999%, Afrox). The solution was kept boiling for several minutes until time the indicator color turned from blue to reddish-pink at which stage the reducing solution was added (40ml/l) during continuous gassing and boiling. The pink color disappears, indicating reduction.

All medium were corrected to pH 7 using 1 M NaHCO;. Palladium catalyst was then added to the reduced media (50mg/l) which was then left overnight in an anaerobic chamber (Forma anaerobic systems, 1025/1029 USA). The reduced medium were then added to dusted Hungate tubes (4.5 ml/ tube), from 2.3.2, anaerobically under nitrogen, hydrogen and carbon dioxide (71/7/22% vol/vol respectively) gas mixture in an anaerobic chamber (Forma anaerobic systems, 1025/1029 USA). The tubes were then autoclaved for 15 minutes at 121°C. Anaerobically homogenized gut sections were then inoculated into the reduced medium (0.5m! of homogenate to 4.5ml reduced medium) working in the anaerobic chamber. The inoculated tubes were then incubated in the dark at 30°C in an orbital incubator at 100 rotations per minute for 8week.

Methane analysis

After 8weeks of incubation all samples were analyzed for methane and hydrogen production using gas chromatography (Clarus 500, Perkin Elmer USA) equipped with a thermal conductivity detector (temperature set at 200°C), using argon as the carrier gas at flow rate of 30ml/minute. 50 pl of gas sample was injected from each Hungate tube into the gas chromatography. }

Medium inoculation and culturing

Euoniticellus intermedius larvae were dissected as following the protocol described in section above. The midgut and hindgut were separately homogenized in 8mi sterile phosphate buffered saline solution (8g/l sodium chloride, 0.2g/l potassium chloride, 1.44g/l Na,HPO, and 0.24g/l

KH,PO,). 0.5ml of homogenate was then inoculated into culture tubes containing 4.5ml of the 4 different medium in triplicate for each gut section for each medium. Control media were set up by incubating the media without inoculation. The tubes were then incubated in the dark at 30°C in a rotary shaker (Shaking incubator, 5082U, Labcon) at 100 rotations per minute for 4 weeks.

Plate counts and pH analysis

After 4 weeks of incubation (enrichment of microbes), plate counts were done for the inoculated culture medium using the pour plate method using solid Tryptone soy broth at 30g/l with Agar bacteriological (Merck) at 12g/l. From each culture bottle 1 ml was serially diluted in sterile saline solution (8.5g/l sodium chloride) and only the 10 dilution being plated. The plates were then incubated for 24 hours at 30°C with the plate counts being done after the 24 hour period using a colony counter (3329, Dark field Quebec, USA) and pH (pH meter — D-82362, Inolab pH level 1) analysis was then done for all the tubes after pour plating had been done.

Cellulose degradation

Basal media M1C was prepared using a similar recipe to that for M1A with the only difference being that Whatman filter paper strips (5.5cm x 1cm) were used as carbon source and not D- glucose and D-lactose. A pair of Whatman filter paper strips was added per tube containing 10 ml of the salts solution. The media was then autoclaved at 121°C for 15 minutes. The tubes were then inoculated with a loop full of inoculums from plates after doing the plate counts.

Midgut and hindguts were inoculated in separate culture tubes in duplicate for each gut section.

Control experiment was an uninoculated M1C. Four M1C culture tubes (containing 9ml medium) were inoculated with each 1ml of the homogenized gut sections without being enriched first. Two tubes were inoculated with midgut homogenate (DB M) while the other two were inoculated with hindgut homogenate (DB H). All culture tubes were then incubated in the dark at 30°C in an orbital incubator (Shaking incubator, 5082U, Labcon) at 100 rotations per minute for 4 weeks.

Glucose, acetic acid and ethanol production analysis

After 4 weeks of incubation of tubes liquid samples of 1ml were taken from the tubes and centrifuged (Mini spin, Epperndorf AG, Germany) at 10,000 rpm for 15 min, and the supernatant was passed through a 0.45um membrane filter for the analysis of glucose, acetic acid and ethanol using High performance liquid chromatography (HPLC). HPLC used was the Agilent

Technologies 1200 series, using a Biorad Fermentation Monitoring column (Particle size Sum,

150 x 7.8mm), fitted with a refractive index column detector (Temperature at 40°C). Mobile phase was 0.001M H2SO4 and HPLC grade water with a flow rate of 0.8ml/min.

DNA extraction

DNA was extracted from the midgut and hindgut sample using the ZR bacterial DNA kit (Zymo research, USA). Extracted and purified DNA was separated on a 1% agarose gel stained with ethidium bromide and visualized using uitra violet light.

PCR amplification, sequencing and identification

PCR reactions were performed on the DNA using a eubacterial specific primer set the 16s rDNA.

PCR reactions were done using a GeneAmp PCR System 2400 (AppliedBiosystems, USA).

PCR samples were separated on a 1% agarose gel, stained with Ethidium Bromide and visualized using ultra violet light. The PCR products of every sample were run on an ABI 3010xl

Genetic analyser to obtain an the sequence. B LAST analysis revealed the identity of the

Bacteria.

Referring now to Figure 6, a simplified sketch of a methane biofuel reactor (1) illustrating the above and additional features of the invention is shown. The reactor (1) has a reactor vessel (2) having an inlet (3) for a liquefied animal dung based feedstock which is carried to the inlet (3) by a conduit 1(4) leading from a liquefaction plant (not shown). The reactor vessel (2) has a sediment or sludge stirrer (5) located at its base (6).. The stirrer (5) has a pair of paddles (7) located in the reactor vessel (2) which are attached to an electric motor (8) by a rigid shaft (9).

The motor (8) causes the shaft (9) and, consequently, the paddles (7) to rotate, in use and stir any sediment or sludge precipitating towards and collecting in the base (6) of the reactor vessel.

Biofuels production

The reactor vessel (2) also has a first outlet (10) for a gas which outlet is located at the top (11) of the reactor vessel (2) and a second outlet (12) for liquid which outlet is located in a side wall (13) of the reactor vessel towards its top (11).

In addition, the reactor vessel (2) has an inoculum inlet (14) which is also in the form of a feedback loop.

In use, animal dung or dung is, at least partly liquefied in a liquefaction plant (not shown) and the liquefied dung is pumped into the reactor vessel (2) through the conduit (4) to charge the vessel (2). As the vessel (2) is charged the stirrer (5) is activated to mix the liquid and to inhibit precipitation of solids therein. When the reactor vessel (2) is charged an inoculum of microbial consortia obtained from the dung or scarab beetle, Euonithicellus intermedius, as described above is introduced into the reactor vessel (2) through the inoculum inlet (14) and the reaction mixture is heated to about 30°C.

As the microbial consortia digest the dung feedstock methane is produced which bubbles to the top (11) of the reactor vessel (2) and is piped from the first outlet (10) through a conduit (15) for use as fuel or for conversion to a liquefied hydrocarbon via a Fisher Tropsh process. Excess or spent liquid in the reactor vessel (2) is drawn off from the second outlet (12) and its associated conduit (16) to be discarded or for further processing to extract useful products like ethanol.

Additional feed is introduced into the reactor vessel (2) to replace the drawn off liquid. From time to time the reaction is stopped and any sludge in the bottom (6) of the reactor vessel (2) is removed through a hatch (not shown).

The microbial consortia are replaced from time to time as needed by introducing fresh through the inoculums inlet (14) and/or by drawing off liquid containing the consortia used from the top of the reactor vessel through an outlet (17) and reintroducing this drawn off inoculums into the base of the reactor vessel (2). It is envisaged that the reaction could, in this way, be self- sustaining for as long as any sludge build up in the reactor vessel (2) was contained.

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Claims (43)

TN : i CL CLAIMS

1. A process for producing a hydrocarbon fuel from a biomass feedstock comprising introducing an at least partially liquefied biomass feedstock into a reactor vessel, inoculating the feedstock with at least one microbial consortium obtained from insects of the order Coleoptera, allowing the microbial consortium or consortia to, at least partly, digest the feedstock thus producing at least one hydrocarbon fuel, and separating the hydrocarbon fuel from the partly digested feedstock for storage or immediate use.

2. A process as claimed in claim 1 in which the Coleoptera from which the microbial consortia are obtained are dung beetles.

3. A process as claimed in claim 2 in which the beetles are copraphagous beetles.

4. A process as claimed in claim 3 in which the beetles are of the species Euwoniticellus intermedius.

5. A process as claimed in any one of the preceding claims in which the microbial consortia are obtained from adult beetles.

6. A process as claimed in any one of claims 1 to 4 in which the microbial consortia are obtained from larval beetles.

7. A process as claimed in claim 6 in which the microbial consortia are obtained from third instar beetle larvae.

8. A process as claimed in any one of claims 4 to 7 in which the microbial consortia obtained from E. intermedius is supplemented with additional microbial consortia to produce a variety of desired hydrocarbon compounds such as, for example, yeasts where ethanol is a desired product.

9. A process as claimed in any one of the preceding claims in which the biomass feedstock is a nitrogen rich feedstock.

10. A process as claimed in claim 9 in which the biomass feedstock is animal waste.

11. A process as claimed in claim 10 in which the biomass feedstock is animal dung.

12. A process as claimed in claim 11 in which the biomass feedstock is herbivore dung.

13. A process as claimed in claim 12 in which the biomass feedstock is ruminant dung.

14. A process as claimed in any one of the preceding claims in which the feedstock is at least partially liquefied before its introduction into the reactor vessel by the addition of a liquid.

15. A process as claimed in claim 14 in which the liquid is water.

16. A process as claimed in claim 14 or in claim 15 in which the liquid also contains nutrients which, in use, stimulate initial growth of the microbial consortia, to the raw feedstock.

17. A process as claimed in any one of the preceding claims in which the feedstock is partially comminuted.

18. A process as claimed in any one of claims 1 to 16 in which the feedstock is chopped.

19. A process as claimed in any one of claims 1 to 16 in which, where the feedstock is sufficiently liquefied such as, for example, in the case of ruminant dung sourced from a feed lot, the liquefaction step is omitted.

20. A process as claimed in any one of the preceding claims in which the feedstock is inoculated with at least one microbial consortium prior to its introduction into the reactor vessel.

21. A process as claimed in any one of claims 1 to 19 in which the feedstock is inoculated with at least one microbial consortium after its introduction into the reactor vessel.

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22. A process as claimed in any one of the preceding claims in which the hydrocarbon fuel is a gas which is drawn from the top of the reactor vessel for storage and later use or for immediate use.

23. A process as claimed in claim 22 in which the gas is methane.

24. A process as claimed in any one of claims 1 to 22 in which the hydrocarbon fuel is a liquid.

25. A process as claimed in claim 24 in which the liquid is an alcohol or a related compound, which is drawn from liquid in the reactor vessel.

26. A process as claimed in any one of claims 1 to 22 in which the hydrocarbon fuel is a a gas, preferably methane, and a liquid, preferably an alcohol or a related compound, which are drawn from the top and from liquid in the reactor vessel respectively.

27. A process as claimed in any one of the preceding claims in which the feedstock and microbial consortia are heated in the reactor vessel to a temperature of between 25°C and 35°C.

28. A process as claimed in claim 26 in which the feedstock and microbial consortia are heated in the reactor vessel to a temperature of about 30°C.

29. A process as claimed in claim 27 or in claim 28 in which energy required to heat the feedstock is obtained from the utilization of biofuel produced in the reactor vessel.

30. A novel process for producing a hydrocarbon fuel from a biomass feedstock substantially as herein described and exemplified.

31. A biofuel reactor for producing a hydrocarbon fuel from biomass in accordance with the process as claimed in any one of the preceding claims, said reactor comprising a reactor vessel having an inlet for receiving, in use, a biomass feedstock, and at least one outlet for biofuel produced, in use, in the reactor.

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32. A reactor as claimed in claim 31 in which the reactor vessel is generally circular cylindrical and has a feedstock inlet towards its base, a gaseous fuel outlet towards its top and a liquid fuel outlet between the feedstock inlet and the gaseous fuel outlet.

33. A reactor as claimed in claim 31 or in claim 32 in which the reactor vessel includes an agitator located towards the base which, in use, stirs the reactor vessel contents thus inhibiting collection of sediment and/or sludge at the base of the reactor vessel.

34. A reactor as claimed in claim 33 in which the agitator is a set of paddles.

35. A reactor as claimed in any one of claims 31 to 34 in which the reactor vessel has a microbial consortia inoculant inlet for fresh inoculants to be introduced into the reactor vessel.

36. A reactor as claimed in claim 35 in which and, preferably the inoculant inlet includes a feedback for material drawn from the reactor vessel as well as for fresh inoculant.

"37. A reactor as claimed in any one of claims 31 to 36 in which the reactor vessel includes a means for heating the contents of the vessel to a temperature of between 25°C and 35°C.

38. A reactor as claimed in claim 37 in which the reactor vessel includes a means for heating the contents of the vessel to a temperature of about 30°C.

39. A reactor as claimed in claim 37 or in claim 38 in which the means for heating the contents of the reactor vessel is an internal heating element which is located within the reactor vessel.

40. A reactor as claimed in claim 37 or in claim 38 in which the means for heating the contents of the reactor vessel is an external heating system.

41. A reactor as claimed in claim 39 or in claim 40 in which in which the heating means is an electric element.

42. Areactor as claimed in claim 39 or in claim 40 in which in which the heating means is a series of steam charged hollow tubes.

43. A novel biofuel reactor for producing a hydrocarbon fuel from biomass substantially as herein described with reference to and as illustrated in Figure 6. DATED THIS 6™ DAY OF OCTOBER 2010 BOWMAN GILFILLAN INC. FOR THE APPLICANT