METHOD AND APPARATUS FOR CONVERTING BIOWASTE INTO USEFUL COMMERCIAL PRODUCTS

Field of the Invention

The present invention relates to a method of and apparatus for reducing the volume and/or mass of waste material, specifically bio waste, and extracting useful by-products by active conversion thereof into commercially viable products. More particularly, the invention relates to scalable biofuel production from localised biomass waste sources. The invention further relates to the application of the method and apparatus to the reduction and/or recycling of municipal, domestic and business waste.

The method and apparatus as described hereinbelow are directed primarily with reference to the recovery of materials from localised biomass waste sources which can be converted locally. It will be appreciated by the skilled addressee that the invention may be applied to any waste source having a high biomass content.

By being fully scalable, the invention may be applied to small businesses, residential properties and specific mobile applications in addition to large-scale municipal and similar applications.

Background of the Invention

A significant amount of domestic, municipal and business waste is biodegradable, however, the degradation is known to take time, in some cases years.

It is known that once sorted the bulk of biodegradable waste matter available from domestic, municipal and business sources, hereinafter referred to simply as "biomass" comprises cellulose fibre. This originates from paper, cardboard, food waste, agricultural waste, wood products, natural fibres and the like.

Although there are a number of recycling processes in existence, the most common ways of dealing with domestic and commercial waste are by incineration or dumping in landfill sites.

There are well-established problems associated with the disposal of domestic, municipal and business waste, most significantly being the space it occupies in landfill sites. Although there are recycling systems available, these generally add to the cost of processing refuse and do

not generate an income stream. As a result, such recycling systems are seen as a financial burden. Consequently, most waste is buried without sorting, and in the conditions prevalent within a landfill site, degradation is often hampered by lack of aeration thereby increasing the period in which bio waste decomposes. Consequently, bio mass takes up valuable landfill site space, and a significant potential resource is buried rather than utilised.

The usual alternative to landfill is incineration; however, this has inherent problems also. Significant amongst these problems are the need for sorting of combustible and non- combustible waste, the high fuel requirements to achieve pyro lytic conversion of toxic fumes to more benign fumes, and the need for tall stack chimneys, which are both unsightly and hugely expensive to construct, as well as being significant, immediate contributors of greenhouse gases to the atmosphere.

Both of these methods have inherent problems associated with them. For most municipal collections, waste is collected and subsequently dumped without sorting and is not compacted prior to burial. When placed in a landfill site, waste material takes up excessive space and renders the land unusable for decades. Additionally, toxic material or effluent can leak into surrounding land and aquifers. Furthermore, there are fewer and fewer available sites suitable for landfills, and planning authorities are often very reluctant to give permission for new sites.

Where incineration is used, the waste must first be sorted into suitable combustible material, and the non-combustible waste must be diverted again to the landfill site. When burnt, the combustible material emits carbon dioxide and sulphurous emissions, leaving elevated carbon and energy footprints and contributing to acidification of precipitation. There is also a necessity for elevated exhaust of fumes usually via high chimneys which create an aesthetic nuisance and are difficult to build within planning restrictions. Additionally, incinerators are notoriously inefficient and require high-energy inputs.

Most significantly, all current existing methods of disposing of waste add to the total "carbon footprint" of residences, businesses generally, and local authority or government (adding by inefficient collection and processing of waste). For example, composting leads to a release of large quantities of carbon dioxide, nitrous oxide and methane over extended periods, each of the latter being up to 5 times as powerful a greenhouse gas as carbon dioxide. Similarly, the

sorting and transport demands of conventional recycling are major contributors to greenhouse gases and are very wasteful of energy. This situation is exacerbated where the waste has to be transported over a significant distance. The use of waste in anaerobic digestion leads to the production of an explosive gas requiring 500 times as much storage capacity for the resultant fuel (methane) which is itself between 3 and 5 times as potent a greenhouse gas as carbon dioxide and is explosive.

It is an object of the present invention to seek to alleviate the disadvantages associated with the above methods of disposing of municipal and commercial waste.

It is a further object of the present invention to provide a method of reducing the volume of waste and utilising matter extracted therefrom to produce commercially valuable by-products.

It is a further object of the present invention to produce a substantially inert waste material suitable for reduced volume landfill, which material is not prone to methane production or the leaching of toxic materials into surrounding land or aquifers.

It is a yet a further object of the present invention to provide a method that accelerates and controls the degradation of waste without the intervention of microorganisms or toxic or corrosive materials and has predictability in the constitution of the output by virtue of the specificity of the enzyme mix which is selected or adjusted so that a wide variety of waste can be accommodated.

It is further an object of the invention to provide a scalable apparatus and method for the conversion of biomass into fuel and associated by-products for the production of energy.

Summary of the Invention

Accordingly, the present invention provides a method of reducing the volume of municipal waste and extracting matter therefrom, the method comprising, adding water to and macerating or shredding the waste into a liquid biomass mix; and chemically hydro lysing the biomass substances therein using an enzyme mix chosen to maximise output from the waste material, wherein hydrolysis is carried out in the presence of less than about 5% alcohol or detergent.

Preferably, the biomass derived waste is separated first and has high concentrations of cellulose. Preferably, the liquid biomass mix is in a substantially liquid form.

In a preferred embodiment of the invention, hydrolysis is carried out in the presence of about between 1% and 4%, between 1% and 3%, between 1.5% and 2.5%, or 2.4% alcohol or detergent. In one aspect of the invention, the alcohol is ethanol, methanol, butanol, or propanol. In another aspect, the detergent is Tween® 20.

In a preferred embodiment of the invention, the enzyme mix comprises cellulase, amylase, pectinase, xylanase, lignase, hemicellulase, protease, kinase, nucleic-acid splitting and conjoining enzymes, or a combination thereof. Preferably, different enzymes are chosen to deal with specific substances within the liquid biomass mix and for converting those substances into extractable bi-products.

In another aspect, the present invention provides for a method for reducing the volume of waste comprising starch, the method comprising a pre-hydrolysis step in which acid amylase is added to the liquid biomass mix at a temperature of about 40° C or more.

In another embodiment, the method further comprises sterilising the liquid biomass mix prior to hydrolysis.

In another aspect, the present invention provides for fermenting the hydro lysed liquid biomass mix to produce a specific output from the waste material and extracting the specific output material from the fermented liquid. Preferably, the fermentation step comprises adding yeast or Clostridium to the hydrolysed liquid biomass mix. Preferably, the specific output material is extracted from the fermented liquid for subsequent purification and processing.

In a preferred embodiment, the method further comprises separating water and remaining solids from the remaining fermented liquid following extraction of the specific output material so that said water may be recycled or flushed and said solids may be composted, compressed for fuel use or put to landfill.

In a preferred embodiment, alcohol produced during fermentation is extracted from a fermentation vessel before reaching a level where alcohol concentration interferes with the extraction process.

Preferably, the specific output is selected from ethanol, butanol, acetone, long-chain hydrocarbon esters, peptides, glucose, xylose, mannose, agarose and lignine derived substrates, including terpenes and complex oligosaccharides, and a combination thereof.

In another aspect, the invention provides for extruding the alcohol produced during fermentation and distilling it to provide a source of fuel either for addition to petroleum products or to power a fuel cell.

In another aspect, the invention provides for reducing the volume of waste by about 50% or more, by about 60% or more, by about 70% or more, by about 80% or more, by about 90% or more, or by about 95% or more.

By adjusting the enzyme mix, a wide variety of waste can be accommodated, and similarly by adjusting the microbes used in the fermenting stage, different commercially valuable outputs can be generated.

The present invention further provides an apparatus for reducing waste and extracting materials therefrom, the apparatus comprising: a macerator for breaking down solid matter within the waste into smaller pieces for accessibility by enzymes; a hydrolysis vessel for containing a reaction mixture comprising the broken down waste and the enzymes; a heating element for heating the reaction mixture during hydrolysis; and a belt filter for extracting un- hydrolysed solids from the hydro lysed liquid.

Preferably, the apparatus further comprises a fermentation chamber. Preferably, the apparatus further comprises a motorised valve for transferring the hydrolysed liquid to the fermentation chamber. Preferably, the apparatus further comprises a distillation tank. In a preferred embodiment, the distillation tank comprises a water chamber and a heating source submerged in water within said water chamber. In a further preferred embodiment, the distillation tank comprises a water jacket for preventing volatile liquids produced during distillation from coming into contact with said heating source.

In another aspect, the invention provides an apparatus comprising a control mechanism for regulating process times and temperatures according to the composition of the waste and the type of enzyme chosen. Preferably, the apparatus comprises a variable temperature control, a variable mixing control, and/or a control for adjusting dwell time and rate of transfer between process steps carried out within the apparatus.

In another aspect, the invention provides an apparatus for reducing waste adapted specifically for use in vehicles, including submarines, aircraft, passenger ferries and cruise ships.

In another aspect, the invention provides an apparatus for reducing waste adapted specifically for use in the disposal or conversion of food waste from food service outlets, food processing sites, municipal disposal sites, apartment complexes, or individual homes.

Brief Description of the Drawings

Figure 1 shows the action of cellulase as a multi-enzyme mixture.

Figure 2 depicts Clostridium acetobutylicum metabolic pathways.

Figure 3 shows absorbance indicating activation/inhibition by ethanol of acid cellulase

(NBS-SIG), accelerase (ACC-SIG), cellulase 13L-CO13L (CO13L-SIG), and Depol 740L (740-SIG).

Figure 4 shows absorbance indicating activation/inhibition by butanol of the enzymes indicated in Figure 3.

Figure 5 shows absorbance indicating activation/inhibition by propanol of the enzymes indicated in Figure 3.

Figure 6 shows absorbance indicating activation/inhibition by detergent (T ween® 20) of the enzymes indicated in Figure 3.

Figure 7 shows absorbance indicating activation/inhibition of acid cellulase by ethanol, propanol, butanol, and detergent.

Figure 8 shows absorbance indicating activation/inhibition of accelerase by ethanol, propanol, butanol, and detergent.

Figure 9 shows absorbance indicating activation/inhibition of cellulase 13L - CO13L by ethanol, propanol, butanol, and detergent.

Figure 10 shows absorbance indicating activation/inhibition of Depol 740L by ethanol, propanol, butanol, and detergent.

Figure 11 shows methanol activation/inhibition of the enzymes indicated in Figure 3. Figure 12 shows ethanol activation/inhibition of the enzymes indicated in Figure 3. Figure 13 shows methanol activation/inhibition of Depol 740L. Figure 14 shows ethanol activation/inhibition of Depol 740L. Figure 15 shows propanol activation/inhibition of Depol 740L. Figure 16 shows butanol activation/inhibition of Depol 740L. Figure 17 shows detergent activation/inhibition of Depol 740L. Figure 18 shows ethanol activation/inhibition of Depol 740L.

Figure 19 shows a diagrammatic representation of a process apparatus according to one embodiment of the present invention.

Figure 20 shows a diagrammatic representation of a fermentation vessel according to one embodiment of the invention.

Detailed Description of the Preferred Embodiments

One embodiment of the present invention is directed to transforming bio waste into sugar, preferably in about 2 and 8 hours, to give a mass/volume reduction of solid waste derived from biological sources of about 50-95%. Preferably, the sugar is then processed into a fuel output, yielding approximately 200 to 400 litres of fuel per tonne of biowaste. For example, largely cellulosic waste may be hydrolysed to produce a glucose rich liquid which can be fermented to generate ethanol. The process has a negligible carbon footprint because of its waste origin and can be distilled to provide a source of fuel either for addition to petroleum products or to power a fuel cell, thereby providing a source of electrical power directly from the waste. The process times and temperatures vary according to the composition of the waste and the type of microbes chosen.

In one embodiment, the first step in processing biowaste is determining the nature of the waste and/or sorting the waste by category. Examples of categories of waste or feedstock to be used in the present invention include green waste, paper or card waste, food waste, agricultural waste, other biologically originated feedstock whether as a result of another process or in their native state, including aquatic plants. Preferably, hazardous and/or non- usable waste is removed from the biowaste to be processed. This removal may be accomplished through manual picking or metal detectors, in certain aspects of the invention. Preferably, plastic materials such as plastic bottles are also separated from the biowaste. In use, a biowaste material of predominantly one type is selected for processing, so that the

appropriate enzyme mix is used to maximize the recovery of appropriate sugars for subsequent conversion to ethanol.

Municipal solid waste, for example, typically contains a high proportion of usable cellulosic biomass contained within paper and cardboard, food waste, and garden waste. These are currently low value, or unrecyclable waste streams and are generally sent to landfill. The cellulosic biomass also contains quantities of both hemicelluloses and lignin bound to the cellulose.

Usable waste, for example as obtained above, is preferably macerated as a subsequent step. Preferably, the waste is cross-shredded or macerated to a size of approximately 50 mm or less, more preferably between about 5 mm and 40 mm, more preferably between about 5 mm and 20 mm. If the waste used is predominantly paper waste, the paper may be shredded into strips which may be longer than 50 mm; however, preferably its lateral dimension is not less than about 5 mm. Maintaining a lower limit such as approximately 5 mm on the dimension of the macerated waste prevents excessive homogenisation and reduction in enzyme activity.

In a further aspect of the invention, hot water is added to the macerated waste. Preferably, the temperature of the water is approximately 80° C or more, for example between about 80° C and 100° C. More preferably, the temperature of the water is between about 80° C and 98° C. The temperature and amount of water used depends on the type of waste and pump used, as the water facilitates an initial breakdown of the waste, and optionally the pumping of the macerated waste into a vessel in which further sterilisation may be carried out, and the mixture may be hydro lysed. Preferably, this first water mixed into the waste also begins to sterilise the waste, or accomplishes the sterilisation all together. The water used in this step is optionally recycled from a later distillation stage of the process.

In one embodiment, for every one tonne of waste (for example, paper waste), approximately 1200 litres of water are added. In another embodiment, for every one tonne of waste (for example, green waste), approximately 800 litres of water are added. In another embodiment, for every one tonne of mixed waste (for example, approximately half paper and half green waste), approximately 1000 litres of water are added. In another embodiment, for every one tonne of waste (for example, fruit waste), approximately 500 litres of water are added.

After the pumpable waste is placed into a vessel, sterilisation and/or mixing can be carried out, either in addition to or instead of previous sterilisation and maceration. This vessel is preferably the same vessel in which hydrolysis will be carried out; hereinafter, this vessel will be referred to as the hydrolysis vessel. In one embodiment, water is added to the waste within the hydrolysis vessel. Preferably, the waste is mixed/macerated and heated within the hydrolysis vessel, the heat preferably sterilising the waste.

The amount of water mixed with the waste and the temperature to which the waste mixture is heated depends on the type of waste present. The water preferably allows stirring of the waste mixture, helps to break it up, and sterilises it. Preferably, the temperature of the water added or water-waste mixture is approximately 25° C to 125° C, more preferably between 80° C and 120° C, most preferably above about 80 ° C or above about 90° C. When dry waste, such as paper, card or material with relatively higher proportion of lignin present than in other types of waste, is used, a lower temperature may be sufficient as an aid to the physical disaggregation of the material. For example, for such "dry waste" as herein described, the temperature of the water or waste-water mix may be between about 25° C and 90° C, more preferably between about 45° C and 80 ° C. For food or fruit waste, a temperature range of between about 65° C and 85° C serves to sterilise the waste by denaturation of DNA at the lower end (about 65° C) and denaturation of proteins and breaking up of other polymers at the upper end (about 85° C). The higher temperature treatment serves to cause disaggregation of cells. Additionally, structural breakdown of the gross material at higher temperatures aids the later enzymatic digestion of the material in the case of vegetable, fruit, and other food wastes.

In one embodiment, the ratio of water to waste is between approximately 1 : 1 and 4:1. For example, dry waste, such as paper, typically requires more water than food waste. Accordingly, the ratio of water to paper, card, and other water deficient material is preferably about 3:1; the ratio of water to other waste such as food waste or mixed waste is preferably about 2:1; the ratio of water to green waste such as vegetables and fruits is preferably about 1 :1.

Thus, the addition of hot water to the macerated waste within the hydrolysis vessel preferably causes a first stage waste breakdown and also sterilises the waste. During this first stage waste breakdown, the structure of the waste, for example cellulose, is made more accessible

to enzymes used in the latter stages of the procedure, allowing better break down during hydrolysis. Sterilisation is also an advantageous step before hydrolysis is to occur because of the typically high microbial contamination of waste. Allowing subsequent hydrolysis of waste that has not been previously sterilised would lead to a reduction of end product yield because sugar produced during hydrolysis would be taken up by those unwanted organisms.

A second stage waste breakdown is preferably further achieved by using a mechanical agitator (for example a mixer/macerator/shredder) within the vessel. The first stage breakdown, sterilisation, and second stage breakdown of the waste preferably takes between approximately 2 and 8 hours, more preferably about 6 hours or less, more preferably about 4 hours or less, more preferably about 3 hours or less, most preferably approximately 2 hours, during which time a sterilised slurry is produced.

Other treatments preceding hydrolysis may include treating the waste with a dilute acid, organosolvents, hot water, and combinations thereof, which may be used to break down these structures and make the crystalline cellulose, in the example of cellulosic biomass, more accessible to enzyme hydrolysis. Some papers and food waste may have less lignin than others, as they have already been processed in some way during manufacture.

Following sterilisation and breakdown, the waste mixture, or slurry, is cooled. Preferably, cooling occurs after the first and second stages of breakdown have substantially been carried out. In one embodiment, a constant temperature water jacket is used, cooling and maintaining the slurry at an optimal temperature in preparation for hydrolysis. The optimal temperature for hydrolysis depends on the selection of enzyme and is preferably between about 20° and 70° C, more preferably between about 30° and 60° C, more preferably between about 40° and 55° C, and most preferably above 40° C.

If starch is present in the waste (such as in high proportions of food waste, mostly vegetable waste, and in higher grade, office-type paper) a pre-digest with thermostable amylase is optionally carried out. Preferably, this step is carried out when starch is present in a quantity of about 5% or more, more preferably about 10% or more by weight of the total sugar polymer content. In one embodiment, acid amylase is first added to the sterilised slurry within the hydrolysis vessel, at a temperature of above about 40° C, before any other enzymes are added in order to remove a substantial amount of the starch. Optionally, the

supernatant liquid in the hydrolysis vessel is evacuated after the determined amylase digestion time (approximately 2 to 8 hours, more preferably about 6 hours or less, more preferably about 4 hours or less, more preferably about 3 hours or less, most preferably approximately 2 hours) to avoid the inhibition of the further hydrolysing enzymes, such as cellulase, by the glucose liberated during amylase breakdown. The vessel is then refilled with water, and hydrolysis allowed to continue as described below.

Thus starch, which is composed of a polymer of glucose molecules, may be broken down into the subunits of which it is composed, in the presence of water, through the intervention of the enzyme group described as the amylases. The resulting dimer of glucose - maltose - itself may be further hydro lysed to glucose in the presence of maltase. Also, glucose is released as a function of terminal glucose residues of the starch chain being released. The enzymes of the amylase group and the maltase specifically coordinate their active sites to the orientation of the glycosidic bonds holding the glucose molecules together and catalyse the addition of the elements of water to the exposed hydrogen and oxygen groups exposed by the separation of the individual glucose residues from its neighbour.

In one example of this embodiment, approximately 20,000 international units of thermostable amylase per kilogram of waste is mixed with the waste for between approximately 1 hour and 3 hours, at a temperature of approximately 50° C to 65° C, and a pH of approximately 4.5 to 7.0 depending on the pH optimum of the amylase used.

In another embodiment, amylase is added instead of, or in addition to the pre-treatment, to starch-containing waste simultaneously with other enzymes used during hydrolysis, preferably as part of an enzyme complex.

In another embodiment, sugars and other soluble products are removed from the hydrolysis vessel to prevent product inhibition of the enzymes or competitive/steric hindrance of enzyme activity, thereby improving the rate of reaction and yield of fermentable sugars and other desirable products.

It was surprisingly discovered that by controlling the amount of alcohol present during hydrolysis, or by adding or removing amounts of alcohol and/or detergent or a surfactant, acceleration of the bio waste processing can be achieved as compared to reactions in which

the amount of alcohol is not actively controlled. Example 1 discusses and Figures 3-18 show experimental data indicating activation and inhibition by alcohols and detergents at different percentages of various enzymes.

In a preferred embodiment, a small quantity of alcohol or detergent is added to the waste slurry at the hydrolysis stage. Preferably the amount of the alcohol or detergent added is about 5% or less, more preferably between about 2% and 5%, more preferably about 2.5% or less, and more preferably about 2.4% and less. This addition has an activation effect on the enzyme used during hydrolysis. Preferably, the alcohol used is ethanol or butanol. Preferably, the detergent is TWEEN® 20. In certain embodiments, approximately 2.4% ethanol may be employed as an activator in a butanol producing system, and 2.4% butanol may be employed as an activator in an ethanol producing system.

Control of alcohol production can be of particular importance in simultaneous saccharification and fermentation (SSF) processes. SSF is an advantageous process because it prevents end point inhibition of cellulase by glucose (when a cellulosic waste is used, for example), it reduces the chance of bacterial contamination, it reduces initial plant cost, and it improves efficiency. However, SSF produces alcohol while hydrolysis action is taking place, and this alcohol may adversely impact the enzymatic action. By controlling the alcohol concentrations throughout the process, the production of sugar during hydrolysis, and the alcohol yield from the optional fermentation/distillation steps, can be increased.

Using alcohol at the percentages disclosed herein as an activator for enzymes can allow less enzymes to be used, or can decrease the residence time. Using a detergent such as Tween® 20 can increase solubility, thus increasing accessibility of the enzyme to the substrate. Detergent used at the percentages disclosed herein can also have positive thermodynamic effects on the hydrolysis process.

In one embodiment directed to a separate hydrolysis step from fermentation, approximately 2.5% alcohol/detergent or less is added during the enzymatic reaction. Control of alcohol during separate saccharification processes can be accomplished with balanced enzyme mixes or sequential additions and washings.

In another embodiment, the alcohol content during SSF is controlled by removing alcohol (e.g. volatile ethanol) during fermentation to prevent the concentration rising to inhibitory levels as discussed herein (e.g. above about 2.5% in some cases). Preferably, volatile alcohol is removed by low pressure distillation from the SSF mixture.

In another embodiment, a sequence of separate fermentations and enzyme/chemical treatments is carried out each using the products of the preceding fermentation/treatment, or parallel processing of the output of the initial hydro lysate by feeding proportions of it to different next steps. Alcohol/detergent levels are preferably controlled as herein discussed.

Variations in organisms, enzyme mixes, chemical treatments, physical treatments and alcohol/detergent concentrations can be used in order to obtain a range of products from the same hydro lysate. Inhibitory effects of the substrate and experimental variables such as temperature and pH have a large impact on the process at this point.

During hydrolysis, enzymes are added to the hydrolysis vessel according to the type of waste. The enzyme mix may be selected, for example, from cellulases (including exogluconase, endogluconase and cellobiase), amylases, proteases, lipases, lactases, lignases and hemicellulases, pectinases, xylanases, or combinations thereof, adjusted accordingly to the waste used. Ideally, the enzyme mix is adjusted actionally in response to monitored analysis of relative sugar concentrations, particularly cellulose, starch, lactose and cellobiose. Additionally, the mix can be adjusted accordingly to the desired output of the process whether alcohols, ketones or aldehydes for optional subsequent conversion to fuels.

For example, a predominantly or solely paper waste is preferably treated with cellulose enzymes, since the sole component from which sugars could be released (or any other biologically usable substance) is cellulose - the polymer of glucose found in cell walls. Where the biowaste comprises green plant material, the components would include cellulose, but there would also typically be starch present. Therefore, this biowaste is preferably treated with amylase to release the sugars from that component. Similarly, plant materials are preferably broken down by a mixture of enzymes because they are made up predominantly of cellulose, starch, pectoses and xylan, which are various configurations of sugar monomers and other components. The enzymes best suited for breaking down plant materials are therefore able to effectively break these chain sugars down into monomers, e.g. cellulose and

starch into glucose; xylan into xylose. For waste comprising paper, garden and/or food waste, cellulases, amylases, pectinases, xylanases, or combinations thereof are employed. Waste comprising meat and/or other protein rich material would preferably be treated with a protease (protein hydrolysing enzyme) to break down the meat and like material into amino acids and short chain, soluble chains thereof. These are nitrogen rich and may be used as a starting point for the recovery of fertiliser and other products from which fuel or foodstuffs may be derived. Lipases cause the degradation of fats and oils and offer an energy efficient way of liberating fuel length carbon chains and starting points for these from waste fats.

In one exemplifying embodiment, where the waste is predominantly paper, card and green waste, a cellulase complex is used in a quantity of between about 80,000 international units and 1,000,000 international units per kilogram of waste. Predominantly endogluconase first, then exogluconase, and then cellobiase give a progressive opening of the cellulose (which in this case is the main component of the waste) to break down the waste to yield monomers and fermentable oligomers of glucose.

As discussed above, where starch is present in the waste, amylase is optionally used. The presence of green waste indicates that pectinases be preferably employed to accelerate the disaggregation of the cells and cause the release of the sugars comprising the pectin/pectose polymer for fermentation. The presence of food waste indicates that preferably amylases, sucrose, maltase, or a combination thereof be used to accelerate the release of the glucose and fructose monomers for fermentation. The presence of meat and oils/fats indicates that preferably proteases, in particular pepsin and trypsin, be employed because of their effectiveness in acid situations.

The addition of acid optimised lipase to deal with the disaggregation of the fats to release carboxylic acids and glycerol offers a potent additional feedstock to the fermentation as do the released amino acids and oligopeptides. For wastes comprising meats, oils, fats, sewage, or combinations thereof, lipase are preferably employed. The pH of the mixture is preferably monitored when lipases are added and carboxylic acids are released; preferably, a pH of approximately 5 is maintained by using an acetate, citrate, phosphate buffer solution.

The addition of the proteases, lipases, deaminases, pancreatine, pancre lipases, or combinations thereof, in such instances, is optimally sequential with the addition of the amylases and cellulases since the temperature of the mixture is preferably reduced to between 30° C and 40° C to ensure the activity of the enzymes is retained and optimised. The complete mix, in the sequence described above, under higher sterilisation temperatures, is also preferably used for the treatment of sewage, to release the feedstocks described for fermentation. The outputs from the digestion of sewage or meat rich feedstocks are rich in phosphate and nitrogen and may be used as fertiliser. The addition of an amphoteric detergent such as Tween® 20 or a low molecular weight primary alcohol such as ethanol or n-propanol (e.g, propan-1-ol, propan-2-ol) at a concentration of between about 1% and 2.5% by volume may be added to the reaction mixture with the cellulase addition, to enhance the activity of the cellulase. The detergent/ alcohol concentration optimally should not be allowed to exceed 2.5% at any time.

The relative density of the liquid digestate post-hydrolysis preferably does not exceed 1.2 kg/L, and the volume is preferably equal to or slightly greater than the starting volume of liquid added in the case of dry waste to up to twice the volume of liquid added in the case of fruit waste.

For alcohol fuel production, municipal solid waste with high cellulose content is preferably used, so the hydrolysis focus is on cellulose complexes. The main enzyme used for breakdown of cellulose-containing waste is cellulase. However, hydrolysis in these instances may also be carried out in the presence of assisting enzymes, for the increase of accessibility to cellulose polymers and for improving the efficiency of the process and increasing yield. For example, high food stuff waste may also benefit from the addition of amylase, due to its high starch content, while agricultural waste may benefit from the addition of xylanase.

In one embodiment, the hydrolysis vessel is continually agitated during enzyme digestion, preferably at a speed of 10-30 rpm, depending on mix viscosity. Waste is preferably agitated at about 10-15 rpm, as a starting rotation; this preferably self adjusts as a consequence of the changing mechanical resistance of the mixture as the digestion proceeds. In one embodiment, cardboard and paper waste starts at about 10 rpm and rises whilst maintaining a consistent torque to a maximum of about 30 rpm. In another embodiment, food waste starts at about 15-20 rpm increasing to a maximum of about 30 rpm adjusted through the

acceleration so as to maintain the same torque. The agitation may be accomplished by a motor-run impeller.

The time in which digestion by enzymes is carried out is preferably about 2 to 8 hours, depending on the waste composition. For example, paper waste is normally digested at around 2.5 hours or less, biologically recognisable materials such as fruit with a waxy cuticle or hard fruits (e.g. oranges and apples) or garden waste, may take up to about 8 hours. The density of the final mixture is preferably about 1.2 to 1.5 kg/L or less.

In one embodiment, a substantially cellulosic starting waste (such as one substantially comprising paper), is broken down by cellulase, which in itself is a multi-enzyme mixture. Figure 1 shows the enzyme action in this embodiment. Preferably, endocellulase first breaks the crystal structure of the cellulose down into more accessible strands. These strands are then broken down further into oligomers and dimers of glucose such as cello bio se by exocellulase (aka cellobiohydrolase). Finally these di-mers and oligomers are broken down into glucose by the action of beta-glucosidase (aka cellobiase).

In another embodiment, exoglucohydrolase is added, followed by endoglucohydrolase, followed by cellobiohydrolase. This can be simultaneously achieved by adding a cellulase preparation with a high concentration of the exoglucohydrolase. The reaction mixture is preferably monitored for changes in rate of enzyme activity in response to glucose concentration in the mixture to identify the point of optimisation, that is, where the product inhibition due to the glucose reduces the rate of release of new glucose beyond an optimum. At this point, the continued reduction may be accepted until the rate becomes asymptotic with the base line of reaction or the liquor may be washed to fermentation and the mixture recharged with water and enzyme.

In one embodiment of the invention, the amount of cellulase enzyme does not exceed 1 kg/tonne. Weight values of enzyme are determined using the KU value of the specific enzyme, e.g. about 1 KU activity to release 1 mmol of glucose in one minute. The maximum glucose output can be determined by using the weight of the waste matter. Based on the maximum glucose output value and the desired time for the hydrolysis to occur (for example, approximately 2 hours), the required KU value to produce this maximum glucose output in the desired time can be calculated. This KU value is then used to calculate the weight or

volume of enzyme required, as enzyme is supplied in a KU/g or KU/ml form. Each enzyme produced by each supplier has a different KU per gram value, so weight/volume values cannot be used as a measure of enzymatic activity. The reaction time however is theoretical as it assumes complete accessibility by the enzyme to the substrate, which does not occur in a viscous solution. Therefore, the hydrolysis time must be tested experimentally to establish this. For example the dissolving properties of paper allow easy accessibility; however some steric hindrance occurs in the case of fruit with a waxy cuticle, or particularly hard vegetables and other items which cannot be solubilised or allow water to flow freely between the substrate. Reaction time for 40 KU/g cellulase is approximately 2-3 hours, however the KU values of other enzymes, combined with the accessibly of the substrate, determines the reaction time. Preferably, the reaction time does not exceed about 8 hours.

The action of the following enzymes is compared and discussed in greater detail below:

α- Amylase: By acting at random locations along the starch chain, α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase.

β- Amylase: From the non-reducing end, β-amylase catalyzes the hydrolysis of the second α- 1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time.

γ- Amylase: In addition to cleaving the last α(l-4)glycosidic linkages at the non-reducing end of amylose and amylopectin, yielding glucose, γ-amylase cleaves α(l-6) glycosidic linkages.

Xylanase: Xylanase degrades the linear polysaccharide beta-l,4-xylan into xylose, thus breaking down hemicellulose, which is a major component of the cell wall of plants.

Pectinase: Pectin, a polysaccharide substrate that is found in the cell walls of plants is broken down with the use of pectinase into the natural sugars mainly D-galactose, L-arabinose and D-xylose, the types and proportions of neutral sugars varying with the origin of pectin.

Once the sugar solution yield has been optimised, the sugar solution and residual solids mix may be pumped into a low level storage tank. The mix can also be returned to the hydrolysis

tank for additional hydrolysis if desirable, for instance, because of a less-than-optimal sugar solution yield.

From the low level storage tank, the sugar solution and residual solids mix is preferably transferred to a belt filter, for example by pumping, where residual solids are separated from the sugar solution. In one embodiment, the belt filter comprises a feed hopper which positions the mix onto a slow-moving perforated biologically and chemically inert conveyer belt. The sugar solution drains through the perforations and is collected in a biologically and chemically inert holding tank, located below the belt filter. As the mix is conveyed, rollers which are preferably biologically and chemically inert apply pressure to compress the solid waste thus releasing further sugar solution, the solids being retained on the belt. The complete belt filter is preferably sealed from the atmosphere, the air within the sealed area being cleaned and filtered to minimise recontamination.

The residual solids comprise organic polymers not susceptible to hydrolysis through the enzymes introduced. These can be collected for further processing, or can be air dried and collected as a powder/granule to exploit their calorific value. Applications of this material include: soil bulker; composition filler; insulation (preferably mixed with a flame retardant); incineration; fuel for heating, where the calorific value warrants this; pyro lysis, where the organic content merits this; feed stock for gasification. In one embodiment, when powdered residual solids are used as a soil bulker, they have a long break-down period, thus enhancing the water retention and aeration of soils, their progressive breakdown by natural pedo logical processes resulting in the organic enhancement of the soil and an absence of undesirable residues from the material. The residual waste may also be compressed with or without the addition of a resin and preferably with the addition of a fire retardant material, to be used as structural materials, whether primary or for insulation in building. Alternatively, they may be dried and used as an insulating material for buildings in place of foams, hemp, paper, fleece, hair or other insulating materials. A further use is to burn the solids either in simply dried form or as briquettes for any of the above applications, or as a material for building or insulation. The residual waste may also be extruded to form logs for burning in suitable processes, such as pyro lysis, steam generators for electrical or mechanical power generation, or heating for domestic or industrial use. These options for processing and applications are not exhaustive.

Post-hydrolysis, there may be additional chemical co-products from the process which may have useful applications. The products present in the enzyme hydrolysate or fermentations (whether performed simultaneously with the hydrolysis or whether performed separately) depends on the composition of the initial feedstock and on the specific enzyme mixture used. Thus, in addition to the specified output, the liquid containing this output may also contain a variety of other useful products which can be separated and sold or further processed according to an identified demand. Examples of these secondary products include: nitrogen rich and phosphorus rich compounds suitable for use as fertiliser; vitamins; anti-microbials; single cell protein sources for fertiliser and animal feed; sugars other than those passed on for an optional fermentation step.

After the biowaste has been shredded or macerated and subsequently processed through the enzyme digestion stage where it is agitated physically and broken down enzymatically using free enzyme solutions or immobilised enzymes capable of breaking down some or all of the macro molecular residues in the waste, the hydro lysed slurry is preferably passed to a fermentation stage. In one embodiment, glucose can be anaerobically digested by yeasts or bacteria. In the case of yeasts, ethanol and carbon dioxide are produced as they respire. Other bacterial systems are able to produce alcohols such as butanol and methanol as they respire.

In this further embodiment of the invention directed to fermentation, the sugar solution separated out from any residual solids post-hydrolysis is pumped to a sugar solution storage tank or to a fermentation vessel/tank, where fermentation may be carried out. The sugar solution storage tank is preferably kept at a slight positive pressure (i.e. just above ambient pressure). Preferably, all air entering the storage tank is filtered to minimise microbial contamination. In the storage tank, the sugar solution is preferably constantly mixed. The sugar solution may be passed over a UV light(s) to minimise microbial contamination. The resulting sugar solution can be used in a stable feedstock for anaerobic digestion or for non- seasonal feedstock for conventional bioethanol and biobutanol production.

During the fermentation stage, which may be carried out in the sugar solution storage tank or within a separate fermentation vessel, specific microorganisms are added to the hydro lysed, filtered sugar solution. A mixture of fermentable sugars produced for effective utilisation by

the selected microorganism may also be added to the hydrolysed, filtered sugar solution, to create a new fermentation mixture.

Fermentation can be carried out at any scale, large or small. Preferably, all fermentation equipment is modular. In one embodiment, an onsite culturing facility exists. This provides the flexibility to culture any microorganism depending on the desired output.

Preferably, the fermentation mixture undergoes agitation. In one embodiment, the fermentation mixture is pumped in and out of the vessel in constantly changing directions. Agitation may be maintained with the unique use of two modulating 3-port valves, controlled via a timer unit. This promotes complete and random mixing in the fermentation vessel. Optimally, fermentation is carried out at about 37° C.

The microorganisms utilised during fermentation are identified and selected for their ability to digest the micromolecular residues, released by the enzymatic action described herein, and converting these to a desired end product. For example, for butanol, preferably Clostridia is used with acid addition. For ethanol, yeast is preferably used. The microorganisms are selected from yeasts and bacteria, including Saccharomyces spp. and Clostridia spp. Further species known by those skilled in the art are hereby included by way of reference and without limitation.

Fermentation may also be aided by nitrogen and/or recycled carbon dioxide addition, preferably via injection into the bottom of the vessel, just above the fresh media addition point. This upwards gas movement aids dispersal of the media and also spurges and provides anaerobic conditions for the microorganism. Injection of gas and gas levels can be altered to suit the microorganism selected. Yeast has a high tolerance to oxygen levels. It can utilise the oxygen for the production of more yeast biomass. Once the oxygen has been used up and waste CO ₂ fills the vessel, the conditions are anaerobic and solvent production can occur. Therefore, the use of sugar substrate is for the formation of more yeast cells which can in turn produce ethanol. However the presence of oxygen with Clostridium causes the cells to form spores, which are not useful for the production of butanol. Therefore it is more efficient to keep the process of butanol production anaerobic from the start and spurge the sugar medium. Also Clostridium is grown in anaerobic conditions and sudden inoculation into an aerobic environment could case cell death and decrease efficiency in that manner.

Clostridia are commonly found to inhabit soil, sewage, and marine sediments, and also the animal and human intestines. There are several species of Clostridium. Clostridium acetobutylicum is the most known due to its first use in 1916 by Chaim Weizmann to produce acetone and biobutanol from starch for the production of gunpowder and TNT. The process by which this is achieved is known as the ABE process (Acetone Butanol Ethanol process) for industrial purposes such as gunpowder and Cordite (using acetone) production. This has led to the C. acetobutylicum organism being referred to as the Weizmann organism. In the 1940s, low oil costs drove more efficient processes based on hydrocarbon cracking, and petroleum distillation techniques were utilized, resulting in the ABE process being sidelined. C. acetobutylicum also extrudes acetic acid, butyric acid, carbon dioxide, and hydrogen, during the process of butanol production. When produced from a bio mass source, there is preferably no net carbon dioxide production.

Species of bacteria from the Clostridium genus are used for the production of primarily butanol with the coproduction of acetone and ethanol, including and not exclusively acetobutylicum, beijerinckii, ljungdahlii, butyricum and sporogenes. This process is carried out through the use of the metabolism of the Clostridium to utilise monosaccharide and polysaccharide-based carbon sources including pentoses derived from hemicellulose, as well as glycerol, hexoses, pentoses, and oligosaccharides like cellobiose, lactose, raffinose, mannose, xylose, and arabinose. Initially a mixture of acidic products (such as butyric acid, acetic acid and ethanoic acid) is produced, followed by reuptake and solvent production. Figure 2 depicts Clostridium acetobutylicum metabolic pathways.

This process is aided by the use of high solvent yielding bacteria through an initial selection process and improved upon by inducing of required function and/or genetic manipulation. The induction process of one embodiment is described in specific Example 2.

In a further embodiment, sugar media from enzymatic breakdown can be supplemented with the addition of salts: sodium chloride, magnesium sulphate, calcium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and sodium bicarbonate in varying required dosages. Other supplements to the media include acid and alkali for the alteration of pH, namely hydrogen chloride and sodium hydroxide or other alternatives of similar

properties, tryptone (peptides from casein digestion by trypsin), meat extract containing nitrogen and yeast extract containing amino nitrogen and vitamins (B-complex vitamins).

Metabolism of the sugar substrate by Clostridium is preferably continuous with continual addition of fresh media and nutrients and displacement of spent medium containing products into the holding and distillation part of the process. A percentage of this displaced media is used to re-inoculate the fresh media and maintain the cell count of the fermentation stage for effective substrate usage.

The fermentation vessel preferably has the facility to recapture released carbon dioxide expelled by the cells as a co-product. This allows for the release of pressure build-up within the fermentation vessel, storage of the carbon dioxide for selling or for use as an additional spurging gas in the maintenance of anaerobic conditions instead of, or in addition to, nitrogen gas.

An additional feature is the availability of utilising the recirculation system for further dosing of the microorganism, injection of media additives and nutrients, to take test readings, probes for biomass and pH measurements, as well as sampling for assessment of solvent production, sugar utilisation through various techniques including spectrophotometery assays (e.g. for biomass determination at 585 nm (OD585)), Gas Chromatography assays, and High Pressure Liquid Chromatography assays, for example.

Species of ethanol producing yeasts are also able to be cultured within the fermentation facility in much of the same way. Less concentration is focused on spurging of the media than during Clostridium fermentation, and nutrient and salt addition is modified as well as flow rate, to maximise the productivity and yield of the specific yeast strain. Selection of this strain is similar to that of Clostridium stated above, and carbon dioxide capture is also possible in this case.

By changing the growth conditions of the microorganisms, different products may be derived from the fermentation of the sugars and other products of fermentation. Thus Clostridia may produce butyric acid at higher pH and move to butanol production when the pH is lower. Similarly minor alterations in the fermentation can lead to the production of acetone and ethanol. Accordingly, the product of the process may be altered, giving different solvents

and organic acids in response to the market demand. This opportunity may be further enhanced by using different organisms or strains thereof to give other solvents, such as propanol, ethyl acetate and other oxygenated hydrocarbons.

Cells, including microorganisms may be stimulated to produce more of a given enzyme when in the presence of the substrate for that enzyme or a specific other molecule, described as an inducer. Interpreted strictly, this term refers to the production of multiple copies of the genes coding for the given enzyme through contact with the promoter, the consequence of which is the increased amount of the enzyme being produced in the cell and the level of activity associated with that enzyme consequently increasing. A further embodiment of the invention is therefore directed to the genetic modification of the organism to insert additional copies of the enzyme gene or genes for enzymes instrumental in the production of the enzyme or in production of co factors that enhance the activity of the enzyme. Thus during production monitoring, organisms from the fermentation vessel may be screened for enhanced productivity of the enzymes or for enzyme forms with greater activity necessary for the process or for additional activity, and these organisms may be further cultured and developed to pure strains.

In a further embodiment of the invention, following fermentation whereby the sugars released by the enzymatic action have been converted into the required end product (particularly ethanol), distillation may be carried out. For example, ethanol liquor obtained as described above may be evaporated and condensed in a distillation process to obtain higher, usable concentrations. Lower pressure and multi-plate systems can be used to increase the efficiency and purity of the product. This purified ethanol can then be mixed with petrol and sold at the pump or used as an alternative fuel source or to power a fuel cell.

The fermentation mix is preferably pumped into a vacuum distillation tank where the energy rich end product is released from the fermentation mix under reduced pressure and elevated temperature, the temperature being closely controlled and regulated to extract the specific volatile end product by vaporisation and collecting the end product as a condensate. The collected condensate is then fed to a storage vessel which may include a transporter tank or an underground storage tank. In one arrangement, the volatile end product is fed to a fuel cell capable of generating electrical power or to any other system capable of utilising the volatile

end product (or products) for the generation of usable energy, whether as engine fuel or for water heating systems or the like.

The separation of the alcohol component may use a reduced pressure distillation allowing the evaporation of the fuel at a lower temperature (for ethanol, this is preferably between about 46° C and 52° C at an overpressure of -1 bar). Butanol is encouraged to separate by this means, and is preferably driven to the surface of the fermentation suspension by the bubbling of carbon dioxide through the fermentation (whereby this carbon dioxide is preferably derived from the fermentation process and recycled). Where there is a shortage of carbon dioxide, nitrogen is preferably bubbled through as the separating agent.

A conventional plate distillation apparatus may be held as part of the plant as a backup system and to enable the separation of other desired products of reaction.

Where appropriate, a catalytic condensation reaction process may be added as a post distillation step to concatenate the derived oxygenated hydrocarbons (e.g. alcohols, ketones, ethers, carboxylic acids) to produce longer carbon chain residues for different applications, for example for fuels, lubricants, feedstocks, for generation of new compounds, or to enable separation of bioactive or chemically active substances arising from the process at any stage thereof.

The water from the distillation phase may be recycled for various uses. For example, this water may be recycled as a safe grey water addition for the initial suspension, in breaking down and/or sterilising the waste; as a source of heated water to warm the enzyme reaction and the fermentation without the need for additional heating; or as a condensate that may be re-fed to fermentation as a fermentable sugar containing supplement. Residual liquid in the distillation chamber may also be returned to the fermentation chamber for further processing.

The processing of biowaste according to the present invention is preferably designed as a series of modular steps; consequently, any stage can be carried out independently from the others. The plant or system employed for processing biowaste is also preferably of a modular design, allowing any apparatus to be modified in capacity quickly without altering the basic construction of the plant or disturbing the operation of the existing systems. This can be done either as an entirety or by the addition or removal of a module(s) or individual element(s)

thereof, including tanks for reaction or fermentation, connections, storage, etc.. Parts of the system can be isolated for cleaning, repair or trial processing under changed conditions. Examples of situations in which the need for such modification may arise include the following: in the event of chemical, physical, or microbial/bio logical contamination; repair and maintenance without impairing the overall process; replacement of units and linking elements; adjustment of plant capacity; upgrading of the system. This list is not exhaustive. The operating machinery may be constructed to meet any size of demand, from a few millilitres/grams to kilo tonne ranges of operation. This allows the process to be applied at a municipal level business level (e.g. supermarkets, factories, distributions centres), to a household level, as well as having application in ships and submarines or for emergency and temporary camps, such as military or refugee camps.

The process may, in certain embodiments, be operated as a batch process. For example, a series of digestions may be operated at different stages, and the hydro lysate to continuously be fed to the fermentation, with fermentation products drawn off as they reach a commercially and biochemically optimum concentration, while the volume drawn off is replaced by the new hydrolysate. The concentrations of the released sugars in the digestion and in the fermentation are either manually or automatically monitored, as are alcohol or other desired product levels, the flow rate between the vessels being accordingly adjusted to maintain a steady state. The flow rate may be manually or automatically controlled.

The system may also be connected to a combined heat and power system or to fuel cells, enabling the products of the biochemical processes to be used in the generation of electricity at a local level.

The invention will now be described more particularly with reference to the accompanying Figure 19 which shows, by way of example only, one embodiment of a biomass processing apparatus in accordance with the invention.

In this exemplifying construction, the apparatus comprises a first tank, the hydrolysis vessel (101). In one embodiment, the hydrolysis vessel is sized at 125 litres capacity and can preferably be filled to about 100 litres of material. The biological waste, for example unprocessed feedstock, is fed in the top (102) of the hydrolysis vessel (101). The waste is mixed with water which is regulated via a level switch (103) comprising a sensor, and

associated valve at a water inlet pipe (104). This is staged at intervals and may or may not reuse the hot water left from the distillation tank when the distillation phase is complete.

A heating element (105) raises the temperature of the water in the vessel to approximately 80° C, preferably for about 20 minutes, to sterilise the waste of unwanted bacteria and to at least partially break down the waste. However, heat may be retained by the waste for a longer period of time before it cools.

Mixing is facilitated by the rotation of a motorized mixer/shredder (106), disposed near the bottom of the vessel and powered via a belt drive from a motor (107). Blades are disposed on the mixer/shredder (106) both to assist in the mixing and circulation of materials in the vessel, but also to break down the waste to produce a slurry. The motor includes a reciprocating action to further break down the waste.

The slurry is then allowed to cool to an operational temperature (that of the optimal enzyme activity temperature e.g. between about 35° and 65° C) which is monitored by regulation of the heating element (105).

An enzyme or enzyme mix is added into the hydrolysis vessel (101) to the slurry to break down the waste and to prepare it for the later fermentation stage, preferably through the top of the vessel, when the temperature is at the optimum temperature for enzyme activity, determined by the enzyme mix used. The slurry is circulated by outwardly directing impellers of the mixer/shredder (106) which forces the slurry into the corners of the hydrolysis vessel (101) where corner baffle plates are preferably provided. These plates not only encourage circulation of the material towards the upper port of the vessel, but also act to protect less robust elements within the tank, including for example the water inlet pipe (104) and associated level switch (103) and the heating element (105).

Once hydrolysis has been carried out, a motorised pinch valve (108), preferably disposed in one corner at the base of the hydrolysis vessel (101), opens and drains the slurry into the fermentation tank or vessel (109). The mixer/shredder (106) may be used to assist this operation by running at a throughput commensurate with the incoming waste rate of addition to the process required to ensure no backlog. The maceration is used to increase the surface area of the waste, which increases collision between substrate and enzyme for improved

enzyme action upon the substrate. The mixer/shredder (106) also pressurises the area above the drain pipe thereby forcing the more solid particles through the valve. The motorised pinch valve (108) is closed when the drain cycle is complete allowing the hydrolysis vessel (101) above to be filled with the next batch.

The fermentation vessel (109) is maintained at the optimum temperature (about 37°C for Clostridium and about 25-30 ⁰ C for yeast) by a surface heater (110) clamped to the outside. Agitation is achieved in the fermentation vessel (109) by recirculating the contents with the fermentation pump (111); material is drawn from an outlet port A and reintroduced to the fermentation tank at a second inlet port B.

A selected fermentation agent, for example yeast, is added to the material in the tank to enable fermentation and the conversion of sugars within the fermentation vessel to alcohol, for example ethanol. An inspection door (112) is provided within one wall of the fermentation vessel to facilitate visual assessment of the mixture.

When fermentation is complete, a valve redirects liquid from outlet port A to inlet port C, and the liquid is pumped into the distillation tank, or still (113), where heating of the liquid producing vapour in the cavity above the liquid. The distillation tank is preferably an indirectly heated arrangement and preferably includes a water jacket (114) so the volatile liquids within the jacket are never in contact with the primary distillation tank heating source (115) which is optimally kept submerged in water by being positioned in a water chamber (116), preferably positioned at the base of the distillation tank.

The distillation tank (113) is preferably a closed loop system to prevent any loss of vapours to the atmosphere and may optionally provide an expansion vessel (117) to accommodate thermal expansion and subsequent contraction of the enclosed chamber. Preventing vapour loss is important, both for preventing loss of the hydrolysis yield, and for preventing risks associated with volatile vapours coming into contact with electrical equipment, as well as environmental risks.

Careful temperature control allows the alcohol to be vaporised and the water to remain. A thermostat preferably maintains a temperature of about 80 ⁰ C, depending on the pressure of the distillation equipment e.g. low pressure equipment can allow a reduced temperature. The

primary heating element (115) optimally has an automatic topping up valve (not pictured) to maintain the water level and to cope with expansion.

Preferably, a hot gas fan (118) circulates the atmosphere inside the distillation tank. Vapour thereby flows upward (119) through the condenser (120) over an evaporator, preferably in the form of an evaporation coil (121) to condense the alcohol vapours. The coil may be cooled either by water or by ambient air.

The collection area, or condensate collection chamber (122) under the evaporation coil (119) gathers the liquid which can then be discharged via an output (123) into a suitable container.

A distillation tank inspection door (124) is optionally provided within one wall of the distillation tank (113) to facilitate visual assessment of the distillation process.

The heated water left in the distillation tank (113) may be pumped back to the hydrolysis vessel (101) to reduce energy consumption at the start of the process through a still pump (125), or it may be discarded all together. Recycling of this water reduces the requirement for heating and watering of the waste being processed in the hydrolysis vessel (101).

Figure 20 shows one example of a fermentation system which may be used in certain embodiments of the invention. In this embodiment, the fermentation vessel is 200 m ³ . The sugar solution enters the vessel through an input pipe (201). Random mixing is accomplished through modulating 3-port control valves (202). The system also comprises spray bars (203), a vent (204) and an external heating jacket (205) for maintaining a temperature of preferably about 25°C. The alcohol to air separator (206) outputs fermented liquid to storage. The complete cycle is preferably processor controlled, allowing changes in programming of the device to be made with minimal effort.

The apparatus additionally may comprise a pre-processing stage (not illustrated) where bio waste is shredded or macerated to reduce the size of the individual waste components and to expose a greater surface area for watering and for subsequent enzymatic action. The shredded or macerated biowaste is heated by a heating element, by microwave energy or another appropriate mechanism to enhance the disruption of the gross structure and macro molecular fabric of the material being processed.

As indicated above, the invention is intended to include modifications to deal with different biowaste types, for example, specific enzyme mixtures to deal with food waste which includes meat and fats or oils. Such modifications include the selection of defined cultures of bacteria chosen for their survival and multiple metabolising capabilities. Careful selection and use of specific types of waste can yield additional end product options including ammoniacal-nitrogen and phosphate rich fertiliser liquids (which can be dried and powdered where necessary); longer chain fuel compounds; aldehydes; and ketones. The conversion and purification of vegetable oils and used cooking oils to fuel components is also considered.

The apparatus of the invention is presented in modular form so that the method in accordance to the invention may be conducted at separate sites and intervals. Thus, the separation and selection of suitable feedstock may occur at a municipal waste depot for conveying to a second site where more selective sorting of materials occurs and is combined with materials from other municipal sources. Selected feedstock is then shredded and/or macerated for batch-wise processing in an enzyme mix as described hereinabove.

The slurry thus formed after the enzymatic action is completed to release the targeted components is transferred to the fermentation stage which may not necessarily be adjacent the enzyme tank (although commonly it is). Similarly, preferably conveniently disposed adjacent the fermentation stage, the distillation stage may take place remotely of fermentation. Indeed, considering the handling of volatile components, there are advantages in minimising the potential handling risk factors to be weighed against the advantages of processing and condensing the volatile components separately. In addition to the processing and recovery of volatile components suitable for fuel use, biowaste processing also produces nutrient rich materials suitable for fertiliser and other by-products which can be either dried and sent to landfill or in some carefully controlled instances can be flushed to a foul outlet or sewer.

Soiled by-products (undigested residues) may also be used as a burnable fuel when compressed or otherwise further processed or if not of fuel grade be used for productive purposes including insulation and building materials.

While the system of the invention may be sited at a point of bio waste productions, such as a paper mill, sugar beet processing factory, furniture manufacturing/MDF processing plant, food product plant or at a recyclable materials deposit site, such as those specified for domestically sourced garden waste or for paper and cardboard recycling, one of its primary advantages is its scalability. Although advantageous to produce fuels, electrical energy and fertilisers on site at such a point of production of biowaste, and thereby reducing demand on outside energy, total carbon footprint, etc., it is the flexibility of the system that gives it its great environmental impact.

By modifying the apparatus for specific applications, the system may be applied locally to solve immediately the problem of large volumes of waste which otherwise would require collection and municipal disposal. In addition to the system's ability to address the needs of municipal authorities to reduce landfill and in doing so generate fuel and income, the apparatus and method of the invention are fully scalable to meet any demand.

Mobile processor

A chassis mounted apparatus may be constructed so as to meet specific and localised needs arising from gluts of available biomass material infrequently or as part of timed events, for example, seaweed incursions and harvest time.

Househo Id/residential

The average household in the UK generates 500kg of waste per year; of this, approximately 250kg comprise biowaste from which glucose and other sugars can be extracted, primarily from cooked food and glucose polymers, including cellulosic, such as paper, cardboard, and green waste or starch from vegetable waste and food stuffs such as potato and baked goods. Using the process described, upwards of 300 g/kg of digestable material may be released. The sugar solution produced may either be washed away, sold for ethanol manufacture, fermented and the alcohol-rich liquid sold for distillation, distilled for sale, or the distilled alcohol may be fed to a fuel cell and electricity generated from it for supplying household/residents or for sale back to the grid. The system of the invention is capable of processing all paper and food waste, preferably eliminating the need for large volume collections of recyclable material and is able to deal with "contaminated" materials and food stuffs (including meat), unsuitable for conventional recycling. The process reduces the original waste by between about 50 and 95% dry weight and/or volume, depending on the type of original waste. It also renders the

material hygienic, and the residue may be compressed for further use (for example, as an insulator) or may be used as a burnable fuel or passed on to a generator to power a complex. The invention can also improve the odour, hygiene and pest attraction of the accumulated waste. The system may be integrated directly into a new build or may be added subsequently to a house, complex factory or municipal type installation.

The solubised fraction of the output from the process is controlled in its content by using specific enzyme mixtures to determine the exact outputs from the digestive step, providing a stable and reliable substrate for fermentation to derive fuel and other products. The reliability of the consistency of the output from the enzyme controlled step provides a feedstock that that is consistent and non-disruptive to microbial cultures, whether single species or associations of organisms from a diverse and inherently unstable raw material source.

Business/small industrial

The apparatus and process may be used in the conversion of municipal waste, the outputs of food service and food processing sites (restaurants and factories). A significant proportion of business related waste involves paper (which may already be shredded) and cardboard packaging materials. Most other waste either relates to foodstuffs or materials already recyclable by conventional means (such as those specifically for glass and metals). A system according to the invention of similar or increased size to that described above in respect of household/residential facilitates a significant reduction in collected waste, decreasing the financial burden in collection charges and allowing for generation of fuel materials for sale or for use in meeting the energy requirements of the business.

A cruise liner will accrue up to 9 tonnes of waste per day, of which normally about 80% is convertible to a fermentable material by the process of the invention. Where regulated, cruise liners have to store the waste and can only offload this at certain ports, where they are subject to very high costs. This requires room for holding the waste and presents a hygiene hazard on board. By processing the material on board, a fuel mixture may be derived which can either be used in conjunction with a fuel cell to generate electrical power for adding to the ships electrical system or for mixing with fuels used in other systems on board. The residual mass/volume of the waste is also reduced, and the material is rendered more hygienic.

Submarines

In the case of submarines, these vessels may be at sea for up to six months and cannot leave any indication of their presence in addition to the issues raised in regard to cruise liners. The waste generated by personnel on submarines will be of a similar composition to that on cruise liners and similar sea-going vessels, although in small volumes but requiring storage over a substantially longer period. The storage problem is exacerbated by the highly restricted space on board and the requirement to retain all materials on board so as not to portray position information to an enemy or reveal any information concerning origin or route. A submarine version of the invention allows waste volume to be reduced and the resultant alcohol to be used as a fuel source, either as combustible material, for feeding a fuel cell for electrical generation on board or as a processed material that can be disposed of without significant risk of identification or tracing or origin.

The waste material once processed yields solid residues which have been reduced to fine particulates, these may be compressed and either burned to fuel this or another process or may be used in further constructions such as insulation material in buildings. Thus, the process yields a sugar rich liquor that may be passed for processing by fermentation to fuel or used as a fertiliser and solids that may be utilised as fuel for pyro lysis in one form or another or to be incorporated in further manufactures.

Aircraft

Aircraft too generate waste and the turnaround time and hygiene is improved by the ability to reduce waste and the fuel potential from the generated alcohol may be used to contribute to the aircraft's fuel demand.

It will of course be understood that the invention is not limited to the specific details described herein, which are given by way of example only, and that various modifications and alterations are possible within the scope of the appended claims.

The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.

EXAMPLES Example 1

The aim of this experiment was to elucidate the nature and extent of cellulase inhibition by increasing alcohol concentrations typically found within simultaneous saccharifϊcation and fermentation (SSF) processes.

Sigmacell® Cellulose, type 20, and 6mm diameter circles of Whatman no.l filter paper were used as substrates for the saccharifϊcation. The mixture was agitated in a rotating incubator (an Eppendorf centrifuge of 250 μL) and incubated for 30 minutes at 50° C. 0.1M sodium acetate buffer of pH 5.0 was used, and the substrate concentration was kept constant of 2mg/sample.

20μl of a number of cellulase mixtures were assayed against differing concentrations of alcohols (ethanol, methanol, butanol and propanol) and detergent. Ethanol, butanol and propanol were chosen since they are widely accepted petro-diesel substitutes. Assays were performed over thirty minute incubation periods; the production of reducing sugars was assayed using the 2,4-dinitrosalicyclic acid (DNS) assay. See Figures 3-10 for the specphotometer results indicating activation/inhibition of the enzyme as a function of absorbance. See figures 11-18 for the results expressed as percentages of activation/inhibition.

The enzymes used were: Acid Cellulase (Trichoderma Reesei), 1 KU/g (NBS Biologicals); Accelerase 1000 (Trichoderma Reesei), 2.5 KU/g (Genencor); Cellulase 13L - CO13L (Trichoderma sp.), 1.5 KU/g (Biocatalysts); Depol 740L (Humicola sp.), 36 U/g (Biocatalysts).

The results show that in general, increasing the concentration of alcohol progressively inhibited cellulase activity regardless of the type of alcohol used. However it was also noted that in the case of some enzymes (but not all), there was a repeatable stimulation of activity in concentrations of alcohol between 0% - 2%. This was interpreted as being possibly due to an increase in the solubility of the enzymes and/or their substrates. Therefore, a further study was conducted to test this hypothesis using the detergent Tween® 20 which would also be expected to increase solubility and reduce surface tension. The results are shown in Figures 6-10 and 17, and show that Tween® 20 detergent also stimulates activity (i.e. negative inhibition) at 0% - 2% in some cellulases.

The addition of alcohol appears to affect enzyme-substrate interactions thereby amplifying the hydrolysis of cellulose at low concentrations. This might be considered to result from 1) a change in the solubility of the enzymes, 2), a change in the solubility of the substrate(s) or 3) both of these. In keeping with a solubilisation role by the alcohol, reactions in the presence of the detergent Tween® 20 generally increase enzyme activity at low concentration but not at higher concentrations. Nevertheless, there is a saturation point for the alcohols beyond which the alcohol has an increasing inhibitory effect. The observation that not all enzymes are stimulated by the same alcohols suggests that the effect of the alcohols is manifest specifically through the enzymes rather than through changes in the nature of the substrate. If the alcohol acted on the substrates, a general stimulatory effect across all enzymes might be expected. Furthermore, different substrates have little effect on the shapes of the inhibition curves.

The presence of alcohols in SSF reaction systems can thus be important in optimising cellulo lysis.

Figure 11 shows methanol inhibition of the enzymes as a function of percentage inhibition/activation. Figure 12 shows ethanol inhibition of the enzymes. Figures 13-18 focus on inhibition/activation of Depol 740L. Figure 13 shows methanol activation of Depol 740L at a methanol percentage of about 0% to about 0.8%, inhibition starting at about 0.9 or 1% and above. Figure 14 shows ethanol activation of Depol 740L rising to about 180%, activation in general occurring at an ethanol percentages of between about 0% and 5%, more significant activation occurring at about 1% to about 3.5%, most significantly at about 2-3%. Figure 15 shows propanol inhibition hampered at about 3% or less, more significantly at about 2% or less, most significantly at about 1% or less. Figure 16 shows butanol inhibition of Depol 740L. Activation occurred at between about 0% and 2.5%, more significantly at about 0.8% to 1.8%, most significantly at approximately 1% to about 1.5%. Figure 17 shows the effect of Tween® 20 detergent on Depol 740L, activation occurring at between about 1% and 4.5%, more significantly at about 1% and 3.5%, more significantly at between about 1.7% and 3%, and most significantly at approximately 2%. Figure 18 shows optimum activation of Depol 740L with ethanol occurring at less than about 3.7% ethanol, more significantly at between about 1% and 3%, more significantly at between about 2% and 3%, most significantly at about 2.4%-2.5%.

From these experiments, it appears that at over approximately 5% alcohol, there is a significant inhibitory effect on enzymes. At lower percentages of alcohol (approximately 1- 3%), activation of enzymes is observed. Ethanol appears to be the best activating alcohol out of the alcohols and detergent tested.

Example 2

The induction process entails aliquots of various cultures parent strain inoculated into cuvettes containing basic medium at 1.25 times the normal concentration in a ration of Clostridium to media 1 :8. After 30 min of incubation, cultures were challenged with a volume of n-butanol solution equal to that of the Clostridium inoculation and suitably diluted with sterile distilled water to a final concentration medium of 5 g/litre. OD585 of 0.8 or the highest OD585 attainable were transferred sequentially to fresh media containing increasing concentrations of n-butanol. After 12 transfers, a strain capable of growth in the presence of high concentrations of n-butanol was obtained.

Example 3

5 kg of waste comprising garden waste, food waste (including fruit and vegetables) and paper waste was macerated into pieces of between 10 cm and 4 cm. To this, 15 litres of water at 50 ⁰ C were added to the waste. The mixture was pumped into a hydrolysis vessel. A heating element maintained the temperature at between 40 ⁰ C and 50 ⁰ C. The mixture was further mixed within the hydrolysis vessel to make a slurry. The temperature was then dropped to 40 ⁰ C by adjusting the heating element.

1000 kU cellulase and lOOkU amylase together with a mixed amount of amylase, pectinase, and xylanase were added to the slurry along with 45 ml propan-1-ol. The temperature was maintained at 50 ⁰ C. Hydrolysis was carried out for a period of 120 minutes.

The hydrolysed slurry was pumped onto a belt filter adjacent to the hydrolysis vessel. The belt filter, comprising a perforated biologically and chemically inert conveyer belt revolving at 1 rpm allowed the sugar solution to drain through the perforations to be collected in a biologically and chemically inert holding tank, located below the belt filter. Biologically and chemically inert rollers compressed the solid waste thus releasing further sugar solution into the holding tank, the solids being retained on the belt and then discarded.

12 litres of sugar solution were collected in the tank, representing a volume reduction of solids from the initial starting waste of 90%.

Example 4

10 litres of sugar solution obtained as in Example 3 above were pumped into a fermentation vessel. 1 litre of a standardised culture amount of Clostridium was added at 35°C, and the mixture was agitated and recirculated within the vessel for 16 hours. A heater was used to maintain the temperature at 35°C. The fermented mixture was then pumped into a distillation tank comprising a water jacket to be indirectly heated by a heating element submerged in a water chamber outside of the water jacket to a temperature of 82°C. The vapour produced was allowed to pass over an evaporation coil to be collected in a condensate collection chamber positioned immediately beneath the coil. 750 mis of distilled butanol were collected.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications and patent applications, are specifically and entirely incorporated by reference. The term "comprising" as used throughout this application includes the more limiting terms and phrases "consisting essentially of and "consisting of." It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following