WO2004108609A1

WO2004108609A1 - Fermentation media comprising wastewater and use hereof

Info

Publication number: WO2004108609A1
Application number: PCT/DK2004/000385
Authority: WO
Inventors: Anne Belinda Thomsen; Helene Bendstrup Klinke
Original assignee: Forskningscenter Risø
Priority date: 2003-06-06
Filing date: 2004-06-04
Publication date: 2004-12-16
Also published as: EP1641713A1

Abstract

The present invention relates to a fermentation medium comprising wastewater and a process for production of fermentation products such as lactic acid, combustible fuel product e.g. ethanol or cellbiomass by using wastewater derived from sewage such as municipal sewage, slaughterhouse waste, household waste or manure. The process comprises the steps of: (I) providing wastewater, (II) adding a biomass material to the wastewater of step (I) to obtain a biomass slurry, and (III) subjecting the slurry of step (II) to a fermentation process to obtain a fermentation product (IV) separating the fermentation product resulting from step (III).

Description

FERMENTATION MEDIA COMPRISING WASTEWATER AND USE HEREOF

FIELD OF INVENTION The present invention relates in general to the field of producing a fermentation product. In particular, there is provided a novel process for the production of a fermentation product such as lactic acid, combustible fuel product e.g. ethanol or cell biomass by using wastewater derived from e.g. municipal sewage, slaughterhouse waste, household waste or manure as a source of water and nutrient supply for a hydrolysis process and/or a fer- mentation process.

TECHNICAL BACKGROUND AND PRIOR ART

The problem of disposing sewage in particular municipal sewage has been increasing for many years along with the growth of population, and now the disposal starts being critical especially in and around larger cities. The usual manner of disposing of municipal sewage has been to use it or use part of it as landfill, to dump it into water bodies, or to incinerate it. Suitable landfill areas are becoming increasingly hard to find and together with dumping into water, sewage provides increasing pollution problems in the soil, seawater and groundwater. Incinerating sewage pollutes the air with harmful gases and increases its C0₂ content. A significant factor in air pollution is the increasing level of gaseous airborne pollutants, which combine with moisture in the air to produce acids, e.g., carbon dioxide, sulphur dioxide, nitrogen monoxide and dioxide, and compounds of chlorine and fluorine. The carbon dioxide content in some industrial districts is as high as ten times normal. Acid forming pollutants have been found in some instances to increase the acidity of rainwater from its normal pH of about 6.9 to values of 4.0. Rainwater having a pH of 5.5 or lower will destroy aquatic life and can do substantial harm to buildings, monuments, and other structures.

Numerous efforts have been made to solve the problems associated with sewage but none have been entirely satisfactory. One proposal is to subject the sewage to dry distillation or pyrolysis at temperatures between about 900°C and about 1000°C without the injection of steam or oxygen, thereby attempting to produce crude oil. Scrubbing the gas products of this process is required and the scrubbing treatment causes pollution of water and the atmosphere.

Another proposal is to include the step of applying heat externally to a retort in which sewage is heated without internal combustion. The sewage is distilled or pyrolysed at a tern- perature of about 400°C. The resulting gas is cracked, enriched, and scrubbed to make it suitable for heating and ligating purposes. However, scrubbing of the gas creates pollution problems and about 30% of the carbon remain unused and will be discarded with the ash.

Another proposal calls for sewage to be destructively distilled while on a travelling grate in an open system. Organic material is thermally decomposed at temperatures between about 450°C and about 1100°C in the absence of oxygen. Some 34-36% of the starting material remains at the completion of the process. Most of the gas produced during the process is consumed in the process and the gas which is not so consumed is heavily di- luted with carbon dioxide and nitrogen and is not suitable for use in the chemical industry or release to the atmosphere.

As the public concern about air pollution has increased sewage, stack heights have been increased to affect better dispersion of pollutants. Increasing stack heights add to the cost of constructing and maintaining stacks, yet provides no solution to the underlying problem, i.e., emission of harmful substances such as sulphur oxides, chlorine gases, phosphor oxides, etc.

A specific aspect of sewage disposal relates to separation and handling of the aqueous phase of sewage in the following referred to as wastewater. Not much attention has been given to the disposal problems associated with wastewater.

Wastewater derived from sewage comprises a range of compounds such as dissolved organic matter (proteins, sugars, fats etc), nutrient salts, minerals, and metals, thus, with- out an efficient cleaning technology, it becomes a hazard for the environmental stability.

The pollution effect of wastewater can be expressed as Chemical Oxygen Demand (COD) which relates to a standard test that measures the amount of the organic matter in waste- water that can be oxidised (burned up) by a chemical oxidant. Besides the contribution of N₂0 released during conventional aerobic wastewater treatment (denitrification) is of environmentally importance.

Other types of compounds present in wastewater are compounds such as sodium, potassium, magnesium, calcium, sulphur, phosphorus, nitrogen, iron, copper, iodine, fluorine, chlorine cobalt etc. Nutrients like nitrogen and phosphorous serve as nutrients for a range of microorganisms. These compounds have been linked to dangerous toxic microorganisms such as Pfisteria piscicida. Pfisteria is believed to be responsible for major fish kills and disease events in several Mid-Atlantic States and may pose a risk to human health. Nitrogen and/or phosphorus stimulate aquatic algae growth, thus depleting water bodies of oxygen and killing fish and other aquatic organisms. Nutrient pollution comes from runoff of excess fertilisers, industrial wastewater, municipal wastewater, animal sewage, and other diffuse sources, as well as from wastewater treatment plants and some industries.

Several disclosures of prior art teach that the organic matter present in sewage may be used as a carbon source in the microbial fermentation production of ethanol or other fermentation products.

US 6,267,309 disclose a method for producing ethanol or other chemicals from the organic portion of a waste stream of municipal solid sewage. The content of heavy metals present in the organic portion is reduced by treating the solid sewage with sulphuric acid or via ionic exchange. The treated organic portion is subsequently shredded followed by acidification and thermal treatment. The resulting composition can be utilised in a fermentation process.

DE 19946299 discloses a method for fermenting readily decomposable solid sewage products and difficult-to-decompose solid sewage products to produce methane, which method includes the following steps: 1) the readily decomposable sewage products and the diffi- cult-to-decompose sewage products are mixed with water in two separate vessels and heated for 1 hour at 70 °C; 2) the difficult-to-decompose sewage products are mixed with an enzyme suspension and subjected to aerobic hydrolysis for 3 days and subsequently mixed with the readily decomposable sewage products; 3) the mixture then undergoes an anaerobic hydrolysis and acidogenesis for 3 days followed by separation of the formed sediment from the remaining liquid of the anaerobic hydrolysis and acidogenesis; 4a) the formed sediment are mixed with enzyme, 10% paper sewage and 5% whey and subjected to aerobic hydrolysis, and 4b) the remaining liquid is mixed with sewage and used as a medium for the production of methane.

It has been shown in the prior art that methanogens are capable of producing fermentable products more or less independent on the medium if just any kind of energy source and nutrients are present. This characteristics makes the methanogens useful in cleaning procedures of sewage, furthermore they are naturally occurring in sewage and grow well under the anaerobic conditions present in sewage. On the contrary, specific fermentation products, such as ethanol, is provided by substrate specific organisms which require a more specific and/or well defined constituents of energy source and nutrients, therefore wastewater has not until now been suggested as a source for the fermentation of specific fermentation products. Thus, the disadvantage of the methods in the prior art is that the wastewater is discarded after a suitable cleaning procedure and as such, makes no use of the nutrients, minerals and the water present in the collected wastewater in a fermentation, but mainly the solid phase of the sewage is utilised. Furthermore, additional handling steps are required in or- der to provide a suitable solid phase, which is usable as carbon source and in which the amount of heavy metals is removed or substantially reduced.

As illustrated above, there is a need for further utilising the wastewater of sewage in order to overcome environmental problems as well as providing useful fermentation products in a cheap manner.

Accordingly, it has not until now been possible to provide a process wherein the wastewater derived from sewage is utilised in a medium for the production of various fermentation products. Thus, the present invention discloses a suitable process and medium utilising wastewater derived from sewage for the fermentation of useful products, such as ethanol. Additionally, it has been found possible to replace nutrients, minerals and/or water conventionally added to fermentation media as pure compounds by using wastewater derived from sewage. Thereby, it has become possible to provide a cheap and efficient process and medium for the production of fermentation products.

SUMMARY OF THE INVENTION

Thus, in the broadest aspect of the present invention, a process for production of fermentation products is provided. The process comprises the steps of:

(i) providing wastewater,

(ii) adding a biomass material to the wastewater of step (i) to obtain a biomass slurry,

(iii) subjecting the slurry of step (ii) to a fermentation process to obtain a fermentation product, and

(iv) separating the fermentation product resulting from step (iii).

In a further aspect of the present invention a fermentation medium comprising wastewater is provided. A still further aspect relates to the use of a fermentation medium according to the invention for the production of a fermentation product.

DETAILED DISCLOSURE OF THE INVENTION Accordingly, the inventors of the present invention surprisingly found that wastewater obtained from sewage scan be used directly, i.e. without any or at least a minimum of additional modifications, in the fermentation production of a range of products. The conventional supply of individual nutrients, minerals and/or water required for fermenting microorganisms can be replaced by wastewater obtained from sewage. This finding is also the basis for providing a fermentation medium which is cheap and relieves the environment of pollution problems due to the use of conventionally discarded wastewater components (e.g. nitrogen compounds, phosphor-containing compounds).

The process of the present invention, as illustrated in Figure 1, involves the use of waste- water derived from sewage, which, prior to use may optionally be subjected to any kind of degradation or digestion to release nutrients, minerals, organic material into the waste- water. Such a digestion comprises the steps of thermal treatment and/or a optionally fermentation by aerobic or anaerobic digestion. Subsequently, the wastewater is separated from the solid phase. The wastewater is then mixed with a carbohydrate source to provide a biomass slurry which is mixed with a microorganism capable of producing the product of interest. The product produced by the fermentation is then separated and optionally isolated and purified.

The present invention also discloses a fermentation medium comprising wastewater, which thereby limit the additional supplementation of nutrients, minerals and/or water. This fermentation medium is useful in the production of a fermentation product in the petrochemical industry, pharmaceutical industry, biotech industry, chemical industry, and food and feed industry.

Wastewater

In accordance with the invention the sewage may be separated into two phases (i) an aqueous phase (wastewater) and (ii) a solid phase. The separation of the two phases is as described later.

In the present context the term "wastewater" refers to any type of discarded aqueous phase derived from sewage. The wastewater may comprise a community's used water and water carried solids, including used water from industrial processes such as the pharmaceutical, food and feed industry, that flow to a sewage treatment plant. Whey, storm wa- ter, surface water and groundwater infiltration that enters a wastewater treatment plant may also be included in the term "wastewater".

In the present context the term "sewage" relates to the waste, i.e. liquid and solid phase, 5 discarded such as municipal waste, household waste, slaughterhouse waste, e.g. meat/bone flour and blood, garbage and/or industrial waste such as waste from the food, feed and pharmaceutical industry, and waste derived from animal farming, such as manure and solid manure, and from gardening. In a preferred embodiment of the present invention the sewage may be collected from a human or an animal waste sources. 10

In a preferred embodiment of the present invention the wastewater is derived from sewage by separating the aqueous phase (the wastewater) from the solid phase.

When performing fermentation processes the supply of nutrients, minerals and/or water 15 constitutes a significant part of the costs involved in the fermentation process. As mentioned above, the present inventors surprisingly found that naturally occurring water, nutrients and minerals originally present in the wastewater are preserved and used in a fermentation process, in the present context also referred to as "the mandatory fermentation process". In this way the process and the fermentation medium according to the present 20 invention becomes much cheaper than conventionally used processes and fermentation media and relieve the pressure on the environment.

In an useful embodiment, the sewage and/or the wastewater may be subjected to a treatment, as discussed below, for the release of nutrients and/or minerals as well as for

25 releasing any carbohydrate-containing material already present in the wastewater and making the carbohydrate-containing material more accessible for the microorganisms used in the mandatory fermentation. Thus, in an useful embodiment, the wastewater comprises an increased amount of nutrients and/or minerals compared to untreated wastewater, such as at least 5% more nutrients and/or minerals, e.g such as at least 10% such as at least

30 25%, including at least 50% or even at least 75% more nutrients and/or minerals compared to untreated wastewater.

In most countries there is a significant cost involved in water disposal where wastewater disposal plants are paid to purify and dispose wastewater. It is an object of the present 35 invention that the wastewater may be used with or without any further treatment for removing nutrients and minerals. In one preferred embodiment of the present invention the wastewater is obtained directly from the sewage discharge source or it can be obtained after the sewage has been subjected to at least one optionally treatment to remove solids, microorganisms, chemicals and/or enzymes. Treatments for removing nutrients and in^¬ hibitory substances are discussed later.

In an embodiment of the present invention the content of ammonium in wastewater is in the range of 500-10,000 mg/l, such as 1,000-8,000 mg/l, e.g. 1,500-6,000 mg/l, such as 2,000-2,500 mg/l, such as 2,000-4,000 mg/l.

In another embodiment of the present invention the content of phosphor in wastewater is in the range of 25-5,000 mg/l, such as 50-3,000 mg/l, e.g. 80-2,000 mg/l, such as 90- 1,000 mg/l, such as 150-200 mg/l, e.g. 100-500 mg/l.

In yet an embodiment of the present invention the content of chemical oxygen demand (COD) in wastewater is in the range of 100-250,000 mg/l, such as 150-150,000 mg/l, e.g. 200-100,000 mg/l, such as 300-75,000 mg/l, e.g. 350-50,000 mg/l, such as 400-25,000 mg/l, e.g. 500-10,000 mg/l, such as 1,000-10,000 mg/l, e.g. 10,000-100,000 mg/l, such as 15,000-90,000 mg/l, e.g. 25,000-80,000 mg/l, such as 50,000-75,000 mg/l.

According to the present invention, the content of ammonium, phosphor and chemical oxygen demand (COD), in wastewater resulting from thermal treatment, fermentation by anaerobe digestion and filtration is in the range of 2,000-4,000 mg/l, 100-500 mg/l and 1,000-10,000 mg/l, respectively.

In another embodiment of the present invention the amount of any of ammonium, phosphor and chemical oxygen demand (COD), respectively resulting from thermal treatment is in the range of 2,000-4,000 mg/l, 100-500 mg/l and 50,000-75,000 mg/l, respectively.

When separated from the solid phase, the wastewater is used, either alone or in combination with other solutions or substrates in microbial fermentation production of any kind of desired product. In one embodiment of the present invention the wastewater comprises at least one nutrient selected from the group consisting of nitrogen, phosphor, magnesium, zinc, manganese, cobalt, copper, calcium, iron, molybdenum, boron, urea, proteins, amino acids, a compound containing any of such elements and any combination hereof.

In the present context the term "solutions or substrates" relates to different types of me- dia or aqueous solutions with or without nutrients, minerals, carbohydrate-containing materials, vitamins, detergents, amino acids, lipids and salts. In order to avoid or reduce any interference from microorganisms, such as methanogens, originally present in the wastewater, the wastewater may be sterilised prior to being subjected to the mandatory fermentation process.

If needed, the wastewater is supplemented with at least one additional nutrient. The nutrient used to supplement the wastewater is selected from the group consisting of urea, nitrogen, phosphor, magnesium, zinc, manganese, cobalt, copper, calcium, iron, molybdenum, boron, any compound containing any of such elements and any combination hereof.

The amount of the supplementary nutrient, if added, calculated on the final medium, is typically at the most 10%, e.g. at the most 25%, such as at the most 50%, e.g. at the most 75%, such as at the most 90%, e.g. at the most 95%.

Separation of the solid phase When providing wastewater which is suitable for production of a fermentation product of interest the remaining solid phase of sewage may be at least partially removed, e.g. by a process selected from the group consisting of filtration, centrifugation, sedimentation and decanting.

To improve separation of the solid phase the sewage can be treated thermally, by ultra wave, enzymatically, by wet oxidation, steam explosion or combinations hereof. Such treatments are discussed below.

Additionally, solids, microorganisms, chemicals or enzymes may be removed or deacti- vated from the wastewater by any conventional process for such removal or deactivation, such as sterilisation by e.g. heating, boiling or cooking, simple filtration, chromatography, microfiltration, diafiltration, centrifugation or neutralisation.

In a further embodiment of the present invention the heavy metals present in the waste- water need not be removed or reduced before the wastewater are used in the fermentation of a product of interest. Thus, no additional treatment is needed in order to remove heavy metals from the wastewater.

Carbon source In another preferred embodiment of the present invention the biomass material is a carbohydrate-containing material (hexoses and pentoses) including a glucan and pentosan containing material such as a lignocellulosic material, starch containing material, cellulose, starch, an organic waste material, household wastes, paper materials, paper pulp, return paper, straw, maize stems, forestry waste (log slash, bark, small branches, twigs and the like), sawdust, wood-chips, simple monomeric sugars and molasse from sugar beet or sugar cane.

In yet another embodiment of the present invention the carbohydrate-containing material used in the mandatory fermentation process of the invention is not present in the waste- water, but an additional carbohydrate-containing material is added to the fermentation mixture.

Carbohydrate containing materials that can be used in the mandatory process of the present invention include materials of directly fermentable sugars (e.g. molasses) starch containing materials as well as lignocellulosic materials of plant origin, the lignocellulose, which is the principal component of such materials, in general being built up predominantly of cellulose, hemicellulose and lignin.

Cellulose, which is a β-glucan comprising of anhydro D-glucose units, is the main structural component of plant cell walls and normally constitutes about 35-60% by weight (% w/w) of lignocellulosic materials.

Hemicellulose is the term used to denote non-cellulosic polysaccharides associated with cellulose in plant tissues. Hemicellulose typically constitutes about 20-35% w/w of lignocellulosic materials, and the majority of hemicelluloses consists predominantly of polymers based on pentose (five-carbon) sugar units, such as D-xylose and D-arabinose units, although minor proportions of hexose (six-carbon) sugar units, such as D-glucose and D- mannose units, are generally also present.

Lignin, a complex, cross-linked polymer based on variously substituted p-hydroxyphenyl- propane units, generally constitutes about 10-30% w/w of lignocellulosic materials. It is believed that lignin functions as a physical barrier to the direct bioconversion (e.g. by fer- menting microorganisms) of cellulose and hemicellulose in lignocellulosic materials which have not been subjected to some kind of pre-treatment process (which may suitably be a wet-oxidative process as described in the following) to disrupt the structure of lignocellulose.

In addition to the use of wastewater to minimise the production costs of the fermentation products produced in the process of the present invention it is an important embodiment of the present invention to use biomass in the form of low-cost by-products from gardening such as garden refuse, waste materials from agriculture, forestry, the timber industry and the like. Thus, processes of the invention are applicable to any kind of hemicellulose-con- taining lignocellulosic materials. Relevant materials thus include wooden or non-wooden plant material in the form of stem, stalk, shrub, foliage, bark, root, sugar beet pulp, shell, pod, nut, husk, fibre, vine, straw, hay, grass, bamboo, sugar cane bagasse, or reed, singularly or in a mixture.

Further specific sources of carbohydrate-containing materials are described in WO 01/60752 which is incorporated herein by reference.

In a still further preferred embodiment of the present invention the ratio between the solid carbohydrate-containing material and the wastewater is in the range of 1:99-1: 1, preferably in the range of 1:49-1: 2, e.g. in the range of 1:9-1:4.

In another embodiment of the present invention the initial proportion of carbohydrate- containing material in wastewater will be in the range of 0.02-1 kg/litre of wastewater, often 0.05-0.35 kg/litre, such as 0.05-0.25 kg/litre, depending on the form, bulk and/or dimensions of the lignocellulosic material. On an industrial scale it will normally be economically most advantageous to perform the process of the invention at the highest practicable ratio between carbohydrate-containing material and wastewater i.e. at the highest ratio which permits adequate mixing of the carbohydrate-containing material in the wastewater comprising the oxidising agent and which leads to a satisfactorily high rate of degradation of carbohydrate-containing material.

In a preferred embodiment of the present invention additional water is added to the biomass slurry obtained in step (ii) of the present process. The source of additional water can be tap water, distilled water and wastewater.

It may be desirable to subject the carbohydrate-containing material in question, before releasing the sugar residues, to a grinding step (e.g. milling, abrading, grinding, crushing, chopping, chipping or the like) in order to enhance the overall reactivity of the carbohy- drate-containing material by enhancing, e.g., the physical mobility, mixability, ratio of surface area to mass and the like of the material.

In a preferred embodiment of the present invention the slurry obtained in step (ii) contains, calculated on the total carbohydrate content, at the most 90% microbially femnent- able sugars, e.g. at the most 80% microbially fermentable sugars, such as at the most 70% microbially fermentable sugars, e.g. at the most 60% microbially fermentable sugars, such as at the most 50% microbially fermentable sugars, e.g. at the most 40% microbially fermentable sugars, such as at the most 25% microbially fermentable sugars and e.g. at the most 5% microbially fermentable sugars. Microorganisms and mandatory fermentation

When performing the mandatory fermentation process according to the invention a suitable microorganism is selected depending on the product of interest. In a preferred em- bodiment of the present invention the microorganism is selected from the group consisting of bacteria, yeast and fungi.

It will be understood that the mandatory fermentation process may be either an anaerobic fermentation process or an aerobic fermentation process or a combination hereof.

As an alternative embodiment of the present invention, the mandatory fermentation process is substantially not a methane producing fermentation process. In the present context, the term "substantially" relates to that the mandatory fermentation at the most produces 1% methane, such as at the most 0.5%, including at the most 0.1%.

With regard to the production of ethanol, from e.g. hexoses (mainly glucose and man- nose), any microorganism capable of converting glucose and mannose to ethanol can be used in the process according to the invention. For example, a suitable microorganism for this purpose includes a mesophilic microorganism (i.e. one which grows optimally at a temperature in the range of 20-40°C), e.g. a yeast including Saccharomyces cerevisiae, also referred to as "baker's yeast".

With regard to fermentation of e.g. xylose to yield ethanol, any microorganism capable of converting xylose to ethanol can be used in the process according to the invention. Useful microorganisms include e.g. certain types of thermophiles (i.e. organisms which grow optimally at an elevated temperature, typically a temperature in excess of about 50°C) and genetically engineered microorganisms derived therefrom. In useful embodiments, a suitable organism for the ethanol fermentation is selected from the group consisting of Ther- moanaerobacter species including T. mathranii, Zymomonas species including Z. mobilis and yeast species such as Pichia species. An example of a useful strain of T. mathranii is described in Sonne-Hansen et al. (1993) or Ahring et al. (1996) where said strain is designated strain A3M4.

It will be appreciated that a useful ethanol-fermenting organism can be selected from a genetically modified organism of one of the above useful organisms having, relative to the organism from which it is derived, an increased or improved ethanol-fermenting activity. As used herein the expression "genetically modified bacterium" is used in the conventional meaning of that term i.e. it refers to strains obtained by subjecting an organism to any conventionally used mutagenization treatment including treatment with a chemical muta- gen such as ethanemethane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to spontaneously occurring mutants, including classical mutagenesis. Furthermore, as it is possible to provide the genetically modified bacterium by random mutagenesis or by selection of spontaneously occurring mutants, i.e. without the use of recombinant DNA-technology, it is envisaged that mutants of the above mentioned organism can be provided by such technology including site-directed mutagenesis and PCR techniques and other in vitro or in vivo modifications of specific DNA sequences once such sequences have been identified and isolated.

For the production of lactic acid or other desired products, such as vitamins, antibiotics, amino acids and colours, the industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species, Pediococcus species and Brevibacte um species. Also the strict anaerobes belonging to the genus Bifidobacterium is generally included in the group of lactic acid bacteria. A group of lactic acid bacterial species which are used as so-called probiotics or used in the fermentation of probiotics include e.g. Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactocoocus lactis subsp. cremoris, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus (Lactocidin), Bifidobacterium infantis, Bifidobacterium adolescent^'s, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Enterococcus faecium, Streptococcus saliva us and Streptococcus lactis (Nisin).

With regard to the fermentation of acetic acid, species from Aetobactor spp. are useful or Closteridium thermoaceticum. For the fermentation of propionate and butyrate Closte d- ium propionicum and Closteridium tyrobutyricum, respectively, can be used. Caldicellulosiruptor saccharolyticus may be used in the production of hydrogen and different kind of species of Penicillium are useful in the production of various kinds of antibiotics. With regard to the a fermentation process for producing vitamins organisms such as Propi- onibacteria shermanii (B12) and Corynebacterum sp. (vitamin C) are useful. An example of an amino acid producing species is Corynebacterium glutamicum which produces the amino acid L-lysine and colours may be produced by Curvularia lunata (Anthraquinone).

For the production of various kinds of enzymes, such as cellulose and hemicellulose- degrading enzymes e.g. endoglucanase (EC 3.2.1.4), β-glucosidase (EC 3.2.1.21) filamentous fungi may be used such as Aspergillus niger, Botrytis cinerea, Penicillium brasilianum, Schizophyllum commune or Trichoderna reesei (Thygesen et al. 2003). In one embodiment of the present invention the production of the fermentation product is a continuous process. In an embodiment of the present invention the wastewater obtained from the production of a fermentation product according to the present invention may be recycled or at least part of the wastewater may be recycled. This recycling is provided in order to reuse the wastewater as an aqueous liquid phase in the process of the invention thereby reducing the consumption of water and minimising the quantity of waste material emerging from the process. This recycling may be processed mostly without any substantial inhibition of the pre-treatment of the lignocellulosic biomass or of the subsequently hydrolysis or the fermentation of sugars.

In a preferred embodiment of the present invention the mandatory fermentation process is performed using a mixed culture of organisms. This mixed culture may comprise at least one type of microorganism capable of producing the product of interest and at least one type of microorganism capable of reducing the level of inhibitory substances. Examples of the latter type of organisms are disclosed below.

Fermentation products

The mandatory process and the medium according to the present invention can be used for the fermentation production of any kind of product.

In a preferred embodiment of the present invention the fermentation product produced is selected from the group consisting of ethanol, lactic acid, acetate, propionate, butyrate, formate, hydrogen, H₂, C0₂, vitamins, antibiotics, amino acids, colours, proteins and enzymes.

It will be understood that the fermentation process and the fermentation medium according to the invention also can be useful in the production of cell biomass of a selected microorganism such as a bacteria, a fungi such as Mucor indicus or yeast, such as S. cere- visiae.

Additional features

As mentioned above, prior to addition of biomass material, the sewage, biomass or waste- water itself may by subjected to a treatment for the release of nutrients, minerals and/or water as well as for releasing any carbohydrate-containing material already present in the sewage, biomass or wastewater and making the carbohydrate-containing material more accessible for the microorganisms used in the mandatory fermentation. Such a treatment of the sewage, biomass and wastewater can be a thermal treatment, an optionally fermentation/digestion, enzymatic treatment, acid hydrolysis, wet oxidation, steam explosion, filtration or any combination thereof. In a preferred embodiment of the present invention, prior to adding the biomass material, the sewage or the wastewater obtained from sewage is subjected to any one of the following steps in any given order: (a) thermal treatment, (b) fermentation (anaerobic diges- tion), or (c) filtration.

According to the present invention a wet oxidation or elevated temperature treatment, e.g. thermal treatment or steam explosion of carbohydrate-containing material may be used. Such treatments are water consuming so it is an embodiment of the present invention to use the provided wastewater during such treatments. When using wastewater during thermal treatment the wastewater can function directly as process water, due to this high temperature treatment the wastewater will also be sterilised. In steam explosion as described below, water is mainly needed before a high temperature steam pre-treatment for presoaking and after a treatment as a fermentation medium; in this case, as well as other similar cases, the provided wastewater need to be sterilised before added to the treated biomass (as described later).

In an embodiment of the present invention the biomass material is pretreated by thermal treatment, wet oxidation, steam explosion, dilute sulphuric acidic or other relevant acids, alkaline solutions, organic solvents or any combination hereof before being subjected to fermentation.

In a further embodiment of the present invention the pretreated, untreated biomass and/or the biomass slurry is hydrolysed by enzymatic treatment and/or chemical hydroly- sis, this chemical hydrolysis may either be by acidic treatment or by alkaline treatment.

The terms "wet oxidation" and "wet-oxidative" as used herein refers to a process which takes place in an aqueous medium, i.e. sewage, wastewater, liquid water or a liquid medium containing at least a substantial proportion of water, in the presence of an oxidising agent which reacts oxidatively with one or more components or species present (as a solid or solids, and/or in dissolved form) in the medium. The process normally takes place at an elevated temperature, i.e. at a temperature substantially above room temperature or normal ambient temperature (usually at a temperature of at least 100°C), and at a pressure at least equal to the vapour pressure of water above the liquid aqueous medium at the temperature in question plus the partial pressure(s) of any other gas or gasses, e.g. oxygen, or (when using air) oxygen plus - primarily - nitrogen, present. The conditions (temperature, pressure) employed are such that the aqueous medium does not boil. As an alternative to wet oxidation the more well-known steam explosion (Puls, 1993) or steaming that may be successfully used in the process according to the invention. Steam explosion or steaming operate at the same temperature range of 170-220°C, e.g. a range of 180 to 210°C and reaction time of 2-20 minutes, but the chemicals used differ and addi- tion of water, prior to the treatment by soaking the biomass in weak acidic, alkaline solutions or in water e.g. waste water is only optional. Steaming operates with saturated steam with or without prior addition of oxygen, ammonia, carbon dioxide, sulphur dioxide or sulphuric acid as catalyst.

Oxidising agent as mentioned above relates mainly, but is not limited, to oxygen or hydrogen peroxide that under suitable concentrations and under suitable conditions of temperature and reaction time are appropriate for use in a wet-oxidative process in the manner of the invention.

Hydrogen peroxide is highly soluble in water, is readily available commercially as aqueous solutions of concentration ranging from relatively dilute (e.g. hydrogen peroxide concentrations of around 3% or 5% w/w) to relatively concentrated (e.g. hydrogen peroxide concentrations of about 30-35% or 30-49 w/w) and is, like oxygen, a very acceptable oxidising agent from an environmental point of view. The initial concentration of hydrogen per- oxide in the liquid, aqueous medium is typically in the range of 0.5-10% w/w.

When oxygen is used as oxidising agent, it is preferred that the process is performed in the presence of oxygen introduced at an initial partial pressure of oxygen equal to or exceeding the ambient partial pressure of oxygen (i.e. the partial pressure of oxygen in the surrounding air, which at sea level is normally around 0.2 bar, typically about 0.21 bar), and initial oxygen partial pressures which lie in the range from about 0.2 to about 35 bar will normally be of interest. It is, however, generally preferable to employ initial oxygen partial pressures of at least 0.5 bar, normally in the range of 0.5-35 bar. Typical initial partial pressures of oxygen will be in the range of 1-15 bar, such as 3-12 bar, e.g. 5-12 bar. The solubility of oxygen in water at temperatures of relevance for the process of the invention increases with oxygen partial pressure, and the use of such elevated partial pressures of oxygen can thus be advantageous in ensuring the availability of sufficient oxygen in dissolved form.

The oxygen used may be added in the form of substantially pure oxygen or in the form of an oxygen-containing gas mixture (such as atmospheric air) which in addition to oxygen is constituted by one or more other gases (e.g. nitrogen and/or an inert gas, such as argon) that are not detrimental to the performance of the process of the invention; it will, how- ever, often be advantageous to use substantially pure oxygen (such as oxygen of >99% purity, which is commercially available in conventional gas cylinders under pressure).

Preferred conditions for wet oxidation and other thermal processes including dilute acid treatment include the use of temperatures in the vicinity of, or in excess of, •100°C. In general, temperatures in the range of 120-300°C, e.g. 120-240, such as 180-210°C, e.g. 180-240°C, more typically in the range of 180-220°C, will be appropriate for the vast majority of such embodiments of the process according to the invention, and when using lignocellulosic materials of preferred types it will be usual to employ temperatures in the range of 160-210°C, such as 180-210°C. Good results appear to be obtainable with temperatures around 185-195°C or 170-190°C. As already indicated, the temperature applied should be a temperature at which boiling of the liquid, aqueous medium does not occur under the pressure conditions in question. However, in preferred embodiments, the temperature is less than 220°C, such as less than 200°C, e.g. less than 195°C including less than 190°C, e.g. less than 185°C, such as less than 180°C including less than 175°C, such as less than 125°C, e.g. less than 100°C including less than 80°C, e.g. less than 70°C. In a useful embodiment, the temperature is 70°C, 80°C, 90°C, 100°, 110°C or 120°C.

The wet oxidation and the steam explosion convert a large portion of the biomass material to C0₂, H₂0 and simpler, more oxidised organic compounds, mainly low-molecular weight carboxylic acids.

As mentioned above, under some circumstances it is desirable to reduce the level of inhibitory substances in the fermentation medium or wastewater. To provide this reduction a treatment or digestion, in the present context also referred to as the "optionally fermentation/digestion", is performed using methane-producing microorganisms (also known as methanogens) which constitute a unique group of prokaryotes which are capable of forming methane from certain classes of organic substrates, methyl substrates (methanol, methylamine, dimethylamine, trimethylamine, methylmercaptan and dimethylsulfide) or acetate (sometimes termed acetoclastic substrate) under anaerobic conditions.

In the present context the term "inhibitory substances" relates to substances produced during thermal treatment. Such substances include carboxylic acids such as acetic acid, formic acid and lactic acid, and furans including 5-hydroxymethylfurfural, 2-furfural and 2- furoic acid and phenols including guaiacol, syringoi, 4-hydroxy benzalde-hyde, vanillin, sy- ringaldehyde, 3,4,5-trimethoxybenzaldehyde, 4-hydroxy aceto-phenone, acetovanillone, acetosyringone, 3,4,5-trimethoxyacetophenone, 4-hydroxy benzoic acid, vanillic acid, sy- ringic acid, p-coumaric acid and ferulic acid. Methanogens are found within various genera of bacteria, and methanogenic bacteria of relevance in the context of the present invention include species of Methanobacterium, Methanobrevibacter, Methanothermus, Methanococcus, Methanomicrobium, Methano- genium, Methanospirillum, Methanoplanus, Methanosphaera, Methanosarcina, Metha- nolobus, Methanoculleus, Methanothrix, Methanosaeta, Methanopyrus or Methanocor- pusculum; some of these, notably species of Methanopyrus, are highly thermophilic and can grow at temperatures in excess of 100°C. Only three genera of methanogenic bacteria, viz. Methanosarcina, Methanosaeta and Methanothrix, appear to contain species capable of carrying out the acetoclastic reaction, i.e. conversion of acetate to methane (and carbon dioxide). It will be appreciated that useful methanogenic bacteria can be selected from a genetically modified bacterium of one of the above useful organism having, relative to the organism from which it is derived, an increased or improved methane producing activity. Such a genetically modified organism can be obtained by the methods discussed below.

In the context of the present invention it will generally be most appropriate to apply, in addition to one or more methanogens, other types of microorganisms which, alone or in combination, are capable of degrading organic substances present in the material to be treated in the optionally anaerobic fermentation/digestion step of the process of the invention, but which are not directly suited as substrates for the methanogen(s) employed in the anaerobic fermentation step. Such other types of microorganisms include certain fermentative anaerobic bacteria capable of converting, for example, glucose to products such as acetate, propionate, butyrate, hydrogen and C0₂, and so-called acetogenic bacteria, which convert organic substances such as propionate, butyrate and ethanol to acetate, formate, hydrogen and C0₂.

In an interesting embodiment of the present invention, the level of the above inhibitory substances, including nitrogen, phosphate or toxins, in the fermentation medium and/or wastewater is reduced by employing various kinds of plants in a plant clarifying unit capable of degrading or uptaking such substances. Any kinds of known plants capable of de- grading such inhibitory substances can be used, including water hyacinth (Eichornia crassipes) and St. Augustine Gras (Stenotaphrum secundatum).

The derived wastewater from the above treatments may be sterilised alone or together with the biomass to be pretreated and fermented. In a preferred embodiment of the pres- ent invention paper pulp used as biomass material can be added without further modification to the sterilised wastewater together with enzymes and yeast. In case of using ligno- cellulose (e.g. straw or wood) as biomass the sterilisation can be performed by a wet oxidation process, steam explosion or dilute acid treatment resulting in subsequently at least partial hydrolysis of the cellulose and hemicellulose to obtain a slurry of wastewater and biomass containing an amount of microbially fermentable sugars as well as nutrients that permits the slurry or wastewater to be used as an ethanol fermentation medium.

The purpose of such a hydrolysis treatment is to hydrolyse oligosaccharide and polysac- charide species as well as nutrient salts and substances during the pretreatment (e.g. steam explosion, wet oxidation and acidic hydrolysis) in step (ii) of cellulose and/or hemicellulose origin to form fermentable sugars (e.g. glucose, xylose and possibly other mono- saccharides). Such treatments may be physical, chemical or enzymatic.

Chemical hydrolysis of e.g. lignocellulosic material can normally be achieved in a manner known per se by treatment with an acid, such as treatment with dilute (e.g. 2-10% w/w, typically 4-7% w/w) aqueous sulphuric acid, at a temperature in the range of about 100- 150°C, e.g. around 120°C, for a period of 5-15 minutes, such as 5-10 minutes. Treatment with ca. 4% w/w sulphuric acid for 5-10 minutes at ca. 120°C is often very suitable.

Additionally, the release of the carbohydrate-containing material as well as the provision of accessible sugars from the carbohydrate-containing material can be obtained by enzymatic treatment. In case of cellulosic materials cellulases and hemicellulases may be used. Suitable microorganisms may be used to produce such enzymes.

In case of using starchy materials such as corn and grains for ethanol fermentation, the sterilised wastewater can serve as water phase during the process. Prior to enzymatic hydrolysis and fermentation the grain must be opened by physical methods to release the starch. The two best-known and most widely used processes in starchy materials are wet milling and dry milling. In wet milling the corn is first steeped in a solution of water and sulphur dioxide for 24-48 hours, at a temperature of around 52°C, and then passed through mills to loosen the germ and the hull fibres. In the dry milling process the grain is first broken up as small particle size as possible in order to facilitate subsequent penetration of water in the following cooking process described below in which waste water can be used.

Before fermentation, the milled starchy material must be processed ("saccharified"), to convert the starch into fermentable sugars by distillery yeast (Baker's Yeast). This can be done by use of starch hydrolysing enzymes - amylases. In its natural state, starch exists as compact crystalline granules, which are resistant to enzymatic attack. To enable the enzymes to operate more efficiently, the starch molecules are brought into solution e.g. by using wastewater by a heat treatment.

The raw material is first prepared as a slurry of found meal in wastewater. A small quantity of α- amylase is added to reduce the viscosity of the slurry during the following cooking procedure. The slurry is then cooked at 130-160°C. Once the starch has gelatinised the mash is cooled to 80-90°C at which temperature the -amylase can be added to produce rapid liquefaction. At 32°C the amyloglycosidase can be added together with yeast to carry out the hydrolysis and fermentation. In traditional fermentation strategies, enzymatic pre-treatment and glucose fermentation is performed separately at different conditions, known as SHF (separate hydrolysis and fermentation). However, by performing the enzymatic glucose liberation and the microbial fermentation in one process step - referred to as SSF (simultaneous saccharification and fermentation) - product and substrate inhibition will be minimized, yielding higher overall ethanol productivity.

In further aspects the present invention relates to a fermentation medium comprising wastewater and to the use of such a fermentation medium for the production of a fermentation product selected from the group consisting of ethanol, lactic acid, acetate, propionate, butyrate, formate, hydrogen, H₂, C0₂, vitamins, antibiotics, amino acids, colours, proteins, enzymes and cell biomass.

An alternative embodiment of the present invention, the fermentation medium and/or the use of such a fermentation medium is not for the production of methane.

Furthermore, the fermentation medium may be useful in the production of biomass of a selected microorganism such as bacteria, fungi or yeast, including S. cerevisiae. It will be understood that the above-described embodiments of the fermentation process of the invention also relate to the fermentation medium of the present invention and its use in fermentation processes.

Accordingly, in a preferred embodiment the fermentation medium comprises wastewater which is obtained from sewage selected from the group consisting of municipal sewage, household waste, slaughterhouse waste, human waste, animal waste and/or industrial waste such as waste from the food, feed and pharmaceutical industry such as cell biomass including genmodified cell biomass.

In an useful embodiment of the present invention the fermentation medium comprises a biomass material which may be a carbohydrate-containing material as described above. The biomass material may be, as discussed above, pretreated by thermal treatment, wet oxidation, steam explosion, dilute sulphuric acidic or other relevant acids, alkaline solutions, organic solvents or any combination hereof before being subjected to fermentation. In further useful embodiments, the pretreated or untreated biomass is hydrolysed by enzymatic treatment or chemical hydrolysis, as discussed above. In preferred embodiments, the fermentation medium is one wherein the ratio between the above discussed carbohydrate-containing material and the wastewater is in the range of 1:99-1 : 1, preferably in the range of 1:49-1:2, including the range of 1:9-1:4.

As described above, the wastewater may be treated prior to adding the biomass material, such a treatment may be a thermal treatment, fermentation, enzymatic treatment, acid hydrolysis, wet oxidation, filtration or any combination thereof. The wastewater may be supplemented with at least one nutrient compound selected from the group consisting of nitrogen, phosphor, magnesium, zinc, manganese, cobalt, copper, calcium, iron, molybde- num, boron, a compound containing any of such elements and any mixture thereof.

The fermentation medium according to the present invention may be used in the production of a fermentation product selected from the group consisting of ethanol, lactic acid, acetate, propionate, butyrate, formate, hydrogen, H₂, C0₂, vitamins, antibiotics, amino acids, colours, proteins, enzymes and cell biomass.

The following non-limiting examples and drawings will illustrate the invention further, wherein

Fig. 1 shows a schematic view of the process according to the present invention;

Fig. 2 shows the ethanol production at two enzyme loadings during fermentation of paper pulp in wastewater derived from thermally treated and anaerobic digested sewage. In the experiments there was used a concentration of cellulose of 7 FPU/g dry matter (triangles) and 10 FPU/g of dry matter (crosses);

Fig. 3 shows the cellulose conversion at two enzyme loadings during fermentation of paper pulp to ethanol in wastewater derived from thermally treated and anaerobic digested sewage. In the experiments there was used a concentration of cellulose of 7 FPU/g dry matter (triangles) and 10 FPU/g of dry matter (crosses);

Fig. 4 shows the production of ethanol during fermentation of wet oxidised wheat straw and paper pulp, respectively, in wastewater (derived from thermally treatment and anaerobic digestion of sewage) with and without addition of urea. In the experiments there was used a paper pulp (circles), paper pulp and urea (star), wheat straw (crosses and dashed line) and wheat straw and urea (squares);

Fig. 5 shows the conversion of cellulose during fermentation of paper pulp and wet oxidised wheat straw in wastewater (derived from thermally treatment and anaerobic diges- tion of sewage) with and without urea addition. In the experiments there was used a paper pulp (circles), paper pulp and urea (star), wheat straw (crosses and dashed line) and wheat straw and urea (squares);

Fig. 6 shows the conversion of cellulose during ethanol fermentation using wastewater derived from slaughterhouse waste (MBF). In the experiment there were used MBF treated by wet oxidation (WO-MBF, triangles), MBF treated by wet oxidation followed by anaerobic digestion (WO-AD-MBF, diamonds) and MBF treated wet oxidation followed by anaerobic digestion and addition of urea (WO-AD-MBF+urea, squares). Wet oxidised wheat straw was used as biomass;

Fig.7 shows the production of ethanol during fermentations using wastewater from slaughterhouse waste, i.e. meat and bone flour (MBF). In the experiment there were used MBF treated by wet oxidation (WO-MBF, triangles), MBF treated by wet oxidation followed by anaerobic digestion (WO-AD-MBF, diamonds) and MBF treated wet oxidation followed by anaerobic digestion and addition of urea (WO-AD-MBF+urea, squares). Wet oxidised wheat straw was used as biomass;

Fig 8 shows the conversion of cellulose during ethanol fermentation using wastewater from manure. In the experiment there were used manure treated by wet oxidation (WO- manure, triangles), manure treated by wet oxidation followed by anaerobic digestion (WO- AD-manure, diamonds) and manure treated wet oxidation followed by anaerobic digestion and addition of urea (WO-AD-manure+urea, squares). Wet oxidised wheat straw was used as biomass;

Fig. 9 shows the production of ethanol during fermentation using wastewater from manure. In the experiment there were used manure treated by wet oxidation (WO-manure, triangles), manure treated by wet oxidation followed by anaerobic digestion (WO-AD-ma- nure, diamonds) and manure treated wet oxidation followed by anaerobic digestion and addition of urea (WO-AD-manure+urea, squares). Wet oxidised wheat straw was used as biomass; and

Fig. 10 shows the production of ethanol during fermentation using household waste (HW). In the experiment there were used household waste treated by wet oxidation (WO-HW, diamonds) and household waste treated by wet oxidation and addition of urea (WO- HW+urea, squares). EXAMPLES

Example 1

Use of wastewater in the fermentation of Saccharomyces cerevisiae (Baker's yeast)

This example illustrates the effect of wastewater in the fermentation of Saccharomyces cerevisiae (Baker's yeast) in order to produce ethanol. Ethanol has in recent years received attention as a potential replacement for or supplement to petroleum-derived liquid hydro- carbon products. To minimise the production cost of ethanol for use as fuel it is important to use low-cost by-products. Normally, ethanol for use in the food industry (liquor) is produced from molasses and starch, but now where ethanol may be used as a fuel (not to consume), sewage and/or wastewater have now surprisingly shown to be potential candidates for the supply of nutrients and water in the ethanol fermentation.

1.1. Materials and Methods

1.1.1. Wastewater

The wastewater derived from sewage was subjected to thermal hydrolysis at 180°C for one hour followed by anaerobic microbial digestion and filtration as indicated in Figure 1 (flow- diagram). The chemical data for the wastewater is shown in Table 1.1.

Table 1.1. Data for wastewater derived from sewage after thermal hydrolysis (I) and after thermal hydrolysis following anaerobic digestion and filtration (II)

1.1.2 Biomass material

Paper pulp derived from processing wastepaper for reuse. The cellulose content is estimate to 93% (w/w) and 7% inorganic material (mainly kaolin). 1.1.3 Methods

In 250 ml blue cap flasks 103 g wet paper pulp (corresponding to 30 g dry matter and 73 g H₂0) was mixed with 177 ml sterilised (15 minutes at 121°C) wastewater. The thick suspension was liquefied by enzymatic treatment at 50°C for 24 hours. The enzyme loading 5 during liquefaction was 1,5 ml Celluclast and 300 μl Novozym 188. As the activity of the Celluclast enzymes was 68 FPU/ml this corresponded to 3.4 FPU/g DM. After liquefaction the suspension was cooled on ice (to room temperature) and 0.8 g freeze dried Baker's yeast and 1 ml 24% urea (corresponding to 1000 mg/l) was added together with another portion of enzymes making the total enzyme loadings in the two experiments at 7 and 10 10 FPU/g DM, respectively. At this time the flasks were weighed (corresponding to fermentation time = 0 hours in the figures), following the flasks were weighed at regular intervals to follow the C0₂ production as the weight loss.

1.1.4 Ethanol production

15 The ethanol yield during fermentation was calculated from the C0₂ loss by multiplication of the conversion factor (i.e. the molar ratio of EtOH/C0₂) according to reaction scheme below: EtOH (g) = C0₂, loss(g)/1.045. The final ethanol concentration was also measured by HPLC.

20 H(C₆H₁₀O₅)_nOH -> C₅H₁₂0₆ -> 2 C₂H₅OH + 2 C0₂

Mw 162(monomer) Mw 180 2 x Mw 46 2 x Mw 44

Cellulose Glucose Ethanol Carbon dioxide

The theoretical yield of ethanol based on the glucose content is calculated by: 25

EtOH (g) = 0,51 x glucose

The yield of ethanol calculated from the weight loss of CO_z is:

30 C0₂, lost (g) x 1,045 = EtOH (g)

1.2 Results

The ethanol production is seen in Figure 2. As it can be seen the higher enzyme loading yields a higher ethanol yield after a short lag phase. Using the highest enzyme loading of 35 10 FPU/g DM the fermentation was completed after 120 hours reaching a level of 30 g/l. This corresponded to 62% conversion of the cellulose (Figure 3). This example demonstrates that paper pulp and wastewater derived from thermally treated and anaerobically digested sewage can serve as substrates in an ethanol process without specific removal of heavy metals or other components. Enzymes were added for hydrolysis of cellulose (to glucose) and urea was added as the only nutrient supply. Paper pulp was mixed with wastewater to a dry matter density of 10%. The fermentation was carried out in two step 1) a liquefaction step by enzyme treatments at 50°C enabling good mixing conditions 2) fermentation at 32°C using Saccharomyces cerevisiae (Baker's yeast) and extra enzymes added.

Example 2

Use of wastewater in the fermentation of Saccharomyces cerevisiae (Baker's yeast) without addition of additional nutrients to the wastewater

This example illustrates that the nutrients, such as urea, present in wastewater are usable in the production of ethanol and that no additional nutrient (urea) need to be added.

2.1. Methods and materials

2.1.1 Wastewater The wastewater derived from sewage was subjected to thermal hydrolysis at 180°C for one hour followed by anaerobic microbial digestion and filtration as indicated in Figure 1 (flow- diagram). The chemical data for the wastewater is shown in Table 1.1.

2.1.2 Biomass materials Paper pulp (as used in Example 1) derived from processing wastepaper for reuse. The cellulose content is estimate to 93% (w/w) and 7% inorganic material (mainly kaolin).

Pre-treated wheat straw: Wheat straw was treated by wet oxidation using 60 g in one litre of wastewater heated to 195°C for 10 minutes at initial 12 bars of oxygen pressure (giving a total pressure of 20 bars); after the treatment the slurry was cooled and filtered and washed with 200 ml of water. The composition of the wet oxidised wheat straw is seen in Table 2.1: Table 2.1: Chemical composition of pre-treated wheat straw and untreated wheat straw.

2.1.3 Ethanol production

The ethanol yield during fermentation was calculated as described in Example 1.

2.2 Results

This example illustrates that two different cellulosic materials, wheat straw and paper pulp respectively, each being mixed with wastewater derived from thermally treated and an- aerobically digested sewage can serve as the only substrates without addition of urea.

The experiments were carried out as outlined in Example 1, and compared with parallel experiments without addition of urea. The enzyme loading was in all experiments 7 FPU/g DM. The ethanol production (Figure 4) was highest using paper pulp as a substrate compared to wet oxidised wheat straw due to the higher cellulose content however the conver- sion of the cellulose in percentage was similar for the two substrates (Figure 5). The fermentation and ethanol productivity showed to be independent of urea addition. The ethanol yield measured after fermentation was in accordance with the calculated ethanol yields based on the C0₂ production with a small difference (5%) due to a minor yeast production in the initial fermentation phase facilitated by the amount of oxygen present in the flask head space.

Table 2.2 shows the ethanol produced during fermentation of wet oxidised wheat straw and paper pulp respectively in wastewater (derived from thermally treatment and anaerobic digestion) with and without addition of urea also as indicated in Figure 4. Table 2.2: Ethanol yield measured by HPLC.

Example 3

Use of wastewater from slaughterhouse waste in the fermentation of Saccharomyces cerevisiae (Baker's yeast) with and without addition of additional nutrients to the waste- water

This example illustrates that wastewater derived from wet oxidised slaughterhouse waste, i.e. meat and bone flour, can be used in ethanol fermentation.

3.1. Material and methods

3.1.1 Wastewater The wastewater used in this example was obtained from wet oxidised slaughterhouse waste, i.e. meat and bone flour (MBF). The content of MBF is shown in Table 3.1. Wet oxidation (WO) of meat and bone flour was carried out in a 2 L loop autoclave treating 1 litre of water containing 100 g MBF at 200°C for 15 minutes with 12 bars of 0₂ in the headspace of the reactor of one litre. After the treatment the suspension was filtered into a liquid and a solid fraction. The ash content of MBF before wet oxidation was 41.6% and after wet oxidation 83.4% (corresponding to 46.2 g mainly inorganic particles) showing that a significant amount of the organic components (protein) were dissolved during the wet oxidation process. The liquid fraction was analysed for Na⁺, K⁺ and NH₄ ⁺ (Table 3.2).

In order to study the effect of an anaerobic digestion, the optionally fermentation/digestion of the invention, of the liquid fraction, to experiments, i.e. wet oxidised wastewater (WO- MBF) and wet oxidised wastewater followed by anaerobic digestion (WO-AD-MBF), were performed parallel with each other. 500 mL of the liquid fraction was anaerobic digested (AD) to covert organic carbon to biogas (methane and C0₂) and further release inorganic nutrient components. The anaerobic digestion was performed in bluecap-flasks with yeast locks by adding methanogens to the liquid and leaving it on a lab shaker at anaerobic conditions for 6 days after which the organic carbon fraction was reduced by 2.6 g/L measure by weight loss. The anaerobic digested sample was then sterilised to stop biological activity.

Table 3.1 Content of meat and bone flour

Table 3.2. Content of Na⁺, K* and NH₄ ⁺ in wastewater samples prepared by wet oxidation (WO) and combined wet oxidation and anaerobic digestion (WO-AD-MBF).

3.1.2. Biomass material

Wet oxidised wheat straw (WO wheat straw) was used as biomass material in this example. WO wheat straw was prepared by wet oxidation of 60 g of wheat straw in one litre of water supplied with 2 g Na₂C0₃ at 195°C, for 12 minutes with 12 bars of oxygen added to the headspace of the reactor volume of one litre. After the treatment the cooled suspen- sion was filtered and the filter cake was analysed (Table 3.3) for its content of cellulose, hemicellulose, lignin and ash. The filter cake was stored in a climate chamber before used as substrate in the fermentations.

Tabei 3.3 Chemical composition of raw and treated wheat straw

3.1.3 Ethanol fermentation

Simultanous saccharification and fermentation, also referred to the mandatory fermentation, was carried out in blue cap flasks with yeast locks. The wastewater obtained by the wet oxidation of MBF (WO-MBF) and wastewater obtained by wet oxidation of MBF followed by anaerobic digestion (WO-AD-MBF) with and without urea addition, were mixed with the wet oxidised wheat straw, respectively. The dry matter content of wet oxidised wheat straw was 8%. Liquefaction was obtained using 5 FPU/g DM (celluclast and Novozym 188, 5: 1) at 50°C for 24 hours after which the suspensions were cooled and supplied with more enzymes, 20 FPU/g DM (celluclast and Novozym 188, 5: 1) together with 0.3% w/v dry Bakers yeast. The fermentations were carried out at 32°C and the ethanol production measure by weight loss due to C0₂ and ethanol production (g/L), as described in Example 1.

3.2 Result and discussion The conversion of cellulose during ethanol fermentation and the production of ethanol are shown in Figures 6 and 7, respectively.

This example illustrates that wastewater derived from meat and bone flour (MBF), i.e. slaughterhouse waste, can be used as a source for water and nutrients in an ethanol fer- mentation using wet oxidised wheat straw as a carbon source. The highest ethanol yield was produced using wastewater derived from slaughterhouse waste which only had been treated by wet oxidation, suggesting that an anaerobic digestion of the wastewater is not needed. The fermentation was completed after 170 hours reaching a level of 23 g/l. This corresponded to about 93% conversion of the cellulose.

Furthermore, from the results shown in Figures 6 and 7 it can be concluded that wet oxidised MBF and/or wet oxidised and anaerobically digested MBF can serve as the only substrate in an ethanol fermentation without addition of additional nutrients such as urea.

Finally, the results demonstrate that wastewater derived from MBF can be an essential component in a fermentation medium the production of a fermentation product selected from the group consisting of ethanol, lactic acid, acetate, propionate, butyrate, formate, hydrogen, H₂, C0₂, vitamins, antibiotics, amino acids, colours, proteins, enzymes and cell biomass. Example 4

Use of wastewater from manure in the fermentation of Saccharomyces cerevisiae (Baker's yeast) with and without addition of additional nutrients to the wastewater

This example illustrates that wastewater derived from wet oxidised manure can be used in ethanol fermentation.

4.1. Material and methods

4.1.1. Wastewater

The wastewater used in this example was obtained from digested manure obtained from a biogas plant containing 3% solid matter followed by a wet oxidiation. Wet oxidation of manure (see composition in Table 4.2) was carried out in a 2 L loop autoclave treating 1 litre of manure at 200°C, 15 minutes with 12 bars of 0₂ supplied to the headspace of the re- actor of one litre. After the treatment no more solid was present and also the odour was vanished. The liquid fraction was analysed for Na⁺, K⁺ and NH ⁺ (Table 4.1).

In order to study the effect of an anaerobic digestion, i.e. the optionally fermentation according to the invention, of the liquid fraction, to experiments, i.e. wet oxidised wastewa- ter (WO-manure) and wet oxidised wastewater followed by anaerobic digestion (WO-AD- manure), were performed parallel with each other. 500 mL of the liquid was anaerobic digested (AD) to covert organic carbon to biogas (methane and C0₂) and further release inorganic nutrient components. The anaerobic digestion was performed in bluecap-flasks with yeast locks by adding methanogens to the liquid and leaving it on a lab shaker at an- aerobic conditions for 6 days after which the organic carbon fraction was reduced by 0.6 g/L measure by weight loss. The anaerobic digested sample was then sterilised to stop biological activity.

Table 4.1. Content of Na⁺, K* and NH₄ ⁺ in wastewater samples prepared by wet oxidation (WO-manure) and combined wet oxidation and anaerobic digestion (WO-AD-manure)

30

4.1.2 Biomass material

Wet oxidised wheat straw (WO wheat straw) was used as biomass material in this example. WO wheat straw was prepared by wet oxidation of 60 g of wheat straw in one litre of water supplied with 2 g Na₂C0₃ at 195°C, for 12 minutes with 12 bars of oxygen added to the headspace of the reactor volume of one litre. After the treatment the cooled suspension was filtered and the filter cake was analysed (Table 4.2) for its content of cellulose, hemicellulose, lignin and ash. The filter cake was stored in a climate chamber before used as substrate in the fermentations.

Table 4.2 Chemical composition of raw and treated materials

4.1.3 Ethanol fermentation Simultanous saccharification and fermentation, i.e. the mandatory fermentation according to the invention, was carried out in blue cap flasks with yeast locks/ The wastewater obtained by the wet oxidation of manure (WO-manure) and wastewater obtained by wet oxidation of manure followed by anaerobic digestion (WO-AD-manure) with and without urea addition, were mixed with the wet oxidised wheat straw, respectively. The dry matter content of wet oxidised wheat straw was 8%. Liquefaction was obtained using 5 FPU/g DM (celluclast and Novozym 188, 5: 1) at 50°C for 24 hours after which the suspensions were cooled and supplied with more enzymes, 20 FPU/g DM (celluclast and Novozym 188, 5:1) together with 0.3% w/v dry Bakers yeast. The fermentations were carried out at 32°C and the ethanol production measure by weight loss due to C0₂ and ethanol production (g/L), as described in Example 1.

4.2 Results and discussion

The cellulose conversion during ethanol fermentation and the production of ethanol are shown in Figures 8 and 9, respectively. This example illustrates that wastewater derived from manure can be used as a source for water and nutrients in an ethanol fermentation using wet oxidised wheat straw as a carbon source. The highest ethanol yield was produced using wastewater derived from manure which only had been treated by wet oxidation, suggesting that an anaerobic digestion of the wastewater is not needed. The fermentation was completed after 170 hours reaching a level of 23 g/l. This corresponded to about 98% conversion of the cellulose.

Furthermore, from the results shown in Figures 8 and 9 it can be concluded that wet oxidised manure and/or wet oxidised and anaerobically digested manure can serve as the only substrate in an ethanol fermentation without addition of nutrients such as urea.

Finally, the results demonstrate that wastewater derived from manure can be an essential component in a fermentation medium the production of a fermentation product selected from the group consisting of ethanol, lactic acid, acetate, propionate, butyrate, formate, hydrogen, H₂, C0₂, vitamins, antibiotics, amino acids, colours, proteins and enzymes.

Example 5

Use of wastewater from household waste in the fermentation of Saccharomyces cerevisiae (Baker's yeast) with and without addition of additional nutrients to the wastewater

This example illustrates that wastewater as well as the glucan containing solids derived from wet oxidised household waste can be used in ethanol fermentation.

5.1 Material and methods

5.1.1. Wastewater and biomass material

Household waste (HW) was collected from a municipal waste treatment plant in Frederikssund (Denmark). It consisted of source-sorted kitchen waste (Table 5.1) shred- ded to < 1 mm and enriched with wheat straw (8%) for stabilization of the waste. 60 g MSW in one liter of water was treated by wet oxidation for 10 minutes at 195°C, supplemented with 2 g of Na₂C0₃ and 12 bars of oxygen added to the headspace of the reactor volume of one liter. After the treatment the reactor was cooled and the suspension filtered. Table 5.1 Chemical composition of raw and treated household waste

Both the filter cake and filtrate were used in the fermentation as biomass material and wastewater, respectively. Fermentations were carried out with and without addition of urea. Liquefaction was obtained using 5 FPU/g DM (celluclast and Novozym 188, 5: 1) at 50°C for 24 hours after which the suspensions were cooled and supplied with more enzymes, 20 FPU/g DM (celluclast and Novozym 188, 5: 1) together with 0.3% w/v dry Bak- ers yeast. The fermentations were carried out at 32°C and the ethanol production measured by weight loss due to C0₂ and ethanol production (g/L), as described in Example 1.

5.2 Results and discussion

The production of ethanol is shown in Figure 10. This example illustrates that wastewater as well as the solid fraction derived from household waste (HW) can be used as a source for water and nutrients and substrate in an ethanol fermentation and that the solid phase of the waste contains sufficient carbon for a fermentation. There were not a significant difference in the production of ethanol using wastewater derived from wet oxidised household waste compared to wastewater derived from wet oxidised household waste supple- mented with urea. This demonstrates that household waste contains sufficient nutrients needed for an ethanol fermentation. The fermentation was completed after 250 hours reaching a level of 23 g/l. The final ethanol yield corresponded to a glucan conversion of 81-82%.

Finally, the results demonstrate that wastewater derived from household waste can be an essential component in a fermentation medium the production of a fermentation product selected from the group consisting of ethanol, lactic acid, acetate, propionate, butyrate, formate, hydrogen, H₂, C0₂, vitamins, antibiotics, amino acids, colours, proteins, enzymes and cell biomass. LIST OF REFERENCES

Ahring, B.K., Jensen, K., Nielsen, P., Bjerre, A.B. & Schmidt, A.S. 1996. Pretreatment of wheat straw and conversion of xylose and xylan to ethanol by thermophilic anaerobic bac- teria. Bioresource Technology 58: 107-113.

Sonne-Hansen, J., Mathrani, I.M. & Ahring, B.K. 1993. Xylanolytic anaerobic thermophiles from Icelandic hot-springs. Applied Microbiology and Biotechnology, 38:537-541.

Thygesen, A., Thomsen, A.B., Schmidt, A.S., Jørgensen, H., Ahring, B.K. & Olsson, L. 2003. Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet oxidised wheat straw. Enzyme and Microbial Technology 32:606-615.

Puls, J. 1993. Substrate analysis of forest and agricultural wastes, pp.13-32. In: J.N. Sad- dler (ed.), Bioconversion of forest and agricultural plant residues. CAB International, Wallingford, UK.

Claims

1. A process for production of a fermentation product comprising the steps of:

(i) providing wastewater,

(ii) adding a biomass material to the wastewater of step (i) to obtain a biomass slurry, and

(iii) subjecting the slurry of step (ii) to a fermentation process to obtain a fermentation product.

(iv) separating the fermentation product resulting from step (iii).

2. A process according to claim 1 wherein the wastewater in step (i) is obtained from sewage.

3. A process according to claim 2 wherein the sewage is selected from the group consisting of municipal sewage, household waste, slaughterhouse waste, human waste, animal waste and/or industrial waste.

4. A process according to any of claims 1-3 wherein the biomass material is a carbohydrate-containing material.

5. A process according to claim 4 wherein the carbohydrate-containing material is selected from the group consisting of a glucan containing material such as a lignocellulosic material, starch containing material, cellulose, starch, an organic waste material, a household waste, a paper material, paper pulp, return paper, straw, maize stems, forestry waste (log slash, bark, small branches, twigs and the like), sawdust, wood-chips, simple monomeric sugars and molasse from sugar beet or sugar cane.

6. A process according to claims 4 and 5 wherein the biomass material is pretreated by thermal treatment, wet oxidation, steam explosion, dilute sulphuric acidic or other relevant acids, alkaline solutions, organic solvents or any combination hereof before being subjected to fermentation.

7. A process according to claims 4-6 wherein the pretreated or untreated biomass is hydrolysed by enzymatic treatment and/or chemical hydrolysis.

8. A process according to any one of the preceding claims wherein the ratio between the carbohydrate-containing material and the wastewater is in the range of 1:99-1:1, preferably in the range of 1:49-1 : 2, including the range of 1 :9-1:4.

5 9. A process according to any of the preceding claims wherein, prior to adding the biomass material, the wastewater in step (i) is treated by a process selected from the group consisting of a thermal treatment, a fermentation, an enzymatic treatment, an acid hydrolysis, a wet oxidation, a filtration and any combination thereof.

10 10. A process according to any of the preceding claims wherein, prior to adding the biomass material, the wastewater in step (i) is subjected to any one of the following steps in any given order:

(a) thermal treatment,

15

(b) fermentation (anaerobic digestion), or

(c) filtration.

20 11. A process according to claim 10 wherein the amount of ammonium, phosphor and COD, respectively resulting from step (a) is in the range of 2000-2500 mg/l, 150-200 mg/i and 50,000-75,000 mg/l respectively.

12. A process according to claim 10 or 11 wherein the amount of ammonium, phosphor 25 and COD, respectively resulting from step (a), (b) and (c) is in the range of 2,500-3,000 mg/l, 200-250 mg/l and 2,000-5,000 mg/l respectively.

13. A process according to any of the preceding claims wherein the wastewater is sterilised prior to being subjected to a fermentation process.

30

14. A process according to any of the preceding claims wherein the wastewater is supplemented with at least one nutrient compound.

15. A process according to claim 14 wherein the nutrient compound is selected from the 35 group consisting of nitrogen, phosphor, magnesium, zinc, manganese, cobalt, copper, calcium, iron, molybdenum, boron, a compound containing any of such elements and any mixture thereof.

16. A process according to any of the preceding claims wherein water is added to the biomass slurry obtained in step (ii) of claim 1.

17. A process according to any of the preceding claims wherein the biomass material

5 and/or the biomass slurry is subjected to either chemical hydrolysis and/or to enzymatic hydrolysis.

18. A process according to any of the preceding claims wherein the fermentation product produced is selected from the group consisting of ethanol, lactic acid, acetate, propionate,

10 butyrate, formate, hydrogen, H₂, C0₂, vitamins, amino acids, antibiotics, colours, proteins, enzymes and cell biomass.

19. A process according to any of the preceding claims wherein the fermentation process is performed using a microorganism selected from the group consisting of bacteria, yeast and

15 fungi.

20. A process according to any of the preceding claims wherein the fermentation is performed using a mixed culture of organisms.

20 21. A process according to claim 19 wherein the fermentation process is performed using an ethanol fermenting microorganism such as Saccharomyces cerevisiae.

22. A process according to any of the preceding claims wherein the fermentation process is an anaerobic fermentation process.

25

23. A process according to any of the preceding claims wherein the fermentation process is an aerobic fermentation process.

24. A process according to any of the preceding claims wherein the production of the 30 fermentation product is a continuous process.

25. A process according to any of the preceding claims wherein the wastewater obtained from step (i) to (iv) of claim 1 is recycled in the production of a fermentation product.

35 26. A fermentation medium comprising wastewater.

27. A medium according to claim 26 wherein the wastewater is obtained from sewage.

28. A medium according to claim 27 wherein the sewage is selected from the group consisting of municipal sewage, household waste, slaughterhouse waste, human waste, animal waste and/or industrial waste.

5 29. A medium according to any of claims 26-28 further comprising a biomass material.

30. A medium according to claim 29 wherein the biomass material is a carbohydrate- containing material.

10 31. A medium according to claim 30 wherein the carbohydrate-containing material is selected from the group consisting of a glucan containing material such as a lignocellulosic matierial, starch containing material, cellulose, starch, an organic waste material, a household waste, a paper materials, paper pulp, return paper, straw, maize stems, forestry waste (log slash, bark, small branches, twigs and the like), sawdust, wood-chips,

15 simple monomeric sugars and molasse from sugar beet or sugar cane.

32. A medium according to claims 30-31 wherein the biomass material is pretreated by thermal treatment, wet oxidation, steam explosion, dilute sulphuric acidic or other relevant acids, alkaline solutions, organic solvents or any combination hereof before being

20 subjected to fermentation.

33. A medium according to claims 30-32 wherein the pretreated or untreated biomass is hydrolysed by enzymatic treatment or chemical hydrolysis.

25 34. A medium according to any one of claims 26-31 wherein the ratio between the carbohydrate-containing material and the wastewater is in the range of 1:99-1: 1, preferably in the range of 1:49-1:2, including the range of 1:9-1 :4.

35. A medium according to any of claims 26-32 wherein, prior to adding the biomass

30 material, a treatment of the wastewater is selected from the group consisting of thermal treatment, fermentation, enzymatic treatment, acid hydrolysis, wet oxidation, filtration and any combination thereof.

36. A medium according to any of claims 26-35 wherein, prior to adding the biomass

35 material, the wastewater is subjected to any one of the following steps in any given order:

(a) thermal treatment,

(b) fermentation (anaerob digestion), or (c) filtration,

37. A medium according to any of claims 26-36 wherein the wastewater is supplemented 5 with at least one nutrient compound.

38. A medium according to claim 37 wherein the nutrient compound is selected from the group consisting of nitrogen, phosphor, magnesium, zinc, manganese, cobalt, copper, calcium, iron, molybdenum, boron, a compound containing any of such elements and any

10 mixture thereof.

39. A medium according to any of claims 26-38 for use in a fermentation process utilising at least one microorganism selected from the group consisting of bacteria, yeast and fungi.

15 40. Use of a fermentation medium according to any of claims 26-39 for the production of a fermentation product.

41. Use of a fermentation medium according to claim 40 wherein the fermentation product is selected from the group consisting of ethanol, lactic acid, acetate, propionate, butyrate, 20 formate, hydrogen, H₂, C0₂, vitamins, antibiotics, amino acids, colours, proteins, enzymes and cell biomass.