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Title:
PROCESS FOR EDIBLE FILAMENTOUS FUNGAL CULTIVATION AND ITS INTEGRATION IN CONVENTIONAL SUGAR TO ETHANOL PRODUCTION
Document Type and Number:
WIPO Patent Application WO/2017/208255
Kind Code:
A1
Abstract:
The present invention relates to a process for edible fungal cultivation by employing vinasse or spent wash of first generation ethanol production as a substrate. More particularly, the present invention relates to a process for edible fungal cultivation in the form of pellets and filamentous (mycelial) biomass and its applications as a feed composition for animal, aquatic feed or as human consumption either as itself or in any other form.

Inventors:
NAIR RAMKUMAR BALACHANDRAN (SE)
TAHERZADEH MOHAMMAD JAFAR (SE)
Application Number:
PCT/IN2017/050206
Publication Date:
December 07, 2017
Filing Date:
May 29, 2017
Export Citation:
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Assignee:
NAIR RAMKUMAR BALACHANDRAN (SE)
International Classes:
C12N1/14; C12P1/02
Domestic Patent References:
WO2015086803A12015-06-18
Foreign References:
US20100196994A12010-08-05
Other References:
PIETRZAK ET AL.: "ETHANOL, FEED COMPONENTS AND FUNGAL BIOMASS PRODUCTION FROM FIELD BEAN (VICIA FABA VAR. EQUINA) SEEDS IN AN INTEGRATED PROCESS", BIORESOURCE TECHNOLOGY, vol. 216, September 2016 (2016-09-01), pages 69 - 76, XP029631925
NITAYAVARDHANA ET AL.: "INNOVATIVE BIOREFINERY CONCEPT FOR SUGAR-BASED ETHANOL INDUSTRIES: PRODUCTION OF PROTEIN-RICH FUNGAL BIOMASS ON VINASSE AS AN AQUACULTURE FEED INGREDIENT", BIORESOURCE TECHNOLOGY, BIORESOURCE TECHNOLOGY, vol. 101, December 2010 (2010-12-01), pages 9078 - 9085, XP028320918
NAIR ET AL.: "VALORIZATION OF SUGAR-TO-ETHANOL PROCESS WASTE VINASSE: A NOVEL BIOREFINERY APPROACH USING EDIBLE ASCOMYCETES FILAMENTOUS FUNGI", BIORESOURCE TECHNOLOGY, vol. 221, December 2016 (2016-12-01), pages 469 - 476, XP055444099
See also references of EP 3464555A4
Attorney, Agent or Firm:
P., Aruna Sree (IN)
Download PDF:
Claims:
We claim,

1. A process for cultivating edible fungal biomass selected from phylum Ascomycota, excluding the order, Saccharomycetales in high yield, the said process comprising;

a) preparing a culture medium comprising vinasse in a concentration ranging from 1 to 50% (v/v) and optionally atleast one nutrient supplement for fungal cultivation;

b) i noculati ng fungal culture i n the culture medi um of step and mai ntai ni ng a pH of upto 7 followed by incubation at a temperature range 20e to 40eC for 24 to 96 hours to obtain edible fungal biomass in high yield and additionally ethanol production;

wherein viscosity of the vinasse medium is reduced.

2. The process according to claim 1, the culture medium obtained from industrial streams comprising constituents selected from the group consisting of soluble sugars ranging from about 1 g/L to about 30g/L, and organic metabolites rangi ng from about 0 to about 120 g/L as that of vi nasse.

3. The process according to claim 1, wherein the fungal biomass cultivated is in the form of pellets.

4. The process according to claim 2 for cultivating fungal biomass in the form of pellets, the said process comprising

a) preparing a culture medium comprising vinasse in a concentration ranging from 5 to 10% (v/v) and optionally at least one nutrient supplement for fungal cultivation;

b) i noculati ng fungal culture i n the culture medi um of step and mai ntai ni ng a pH ranging from 6 to 6.5 followed by incubation at a temperature range 20 to 40eC for 24 to 96 hours to obtain edible fungal biomass in high yield additionally ethanol production; wherein the viscosity of the vinasse medium is reduced.

5. The process according to claim4, wherein the pellet size of fungal biomass is i n the range of 2 mm to 4 mm.

6. The process according to claim 1, wherein the fungi cultivated are selected from the group consisting of Aspergillus spp., and Neurospora spp.

7. The process according to claim 1, wherein yield of fungal biomass cultivated is rangi ng from about 50g/L to about 250g/L .

8. The process according to claim 1, wherein the nutrient supplement is selected from a carbon source, nitrogen source.

9. The process according claim 1, wherein the edible fungal biomass has a total protein content ranging from about 45% to about 55%, fat content from about 5%-20 %, lysine content from about 2% -5 % and methionine content from about 1 %-3 %.

10. The process according to claim 1, wherein ethanol is produced in the range of 0.1 g/L to 10 g/L .

11. The process according to claim 1, wherein fungal cultivation is carried out in reactors selected from the group comprising reactors with type as stirred tank, airlift, bubble column or bubble distributor or bubble riser.

12. The process according to claim 1, wherein the reactors comprise, steel, concrete, fabric, metal, plastic, carbon fiber or any chemically developed material in any form as of a container.

13. A fungal feed composition having high protein content comprising fungal biomass cultivated by the process as claimed in claim 1, wherein the protein content is ranging from about 45% to about 55% of the total biomass.

14. The fungal feed composition according to claim 13, further comprising fat content ranging from about 5% to about 20 %, lysine content ranging from about 2% to about 5% and methionine content ranging from about 1 % to about 3 % of the total biomass.

15. The fungal feed composition according to claims 13 and 14 is in the form of pellets.

Description:
PROCESS FOR EDIBLE FILAMENTOUS FUNGAL CULTIVATION AND ITS INTEGRATION IN CONVENTIONAL SUGAR TO ETHANOL PRODUCTION

T E C H NICA L FIE L D O F T H E INV E NTION:

The present invention relates to a process for edible fungal cultivation by employing vinasse or spent wash of first generation ethanol production as a substrate. More particularly, the present invention relates to a process for edible fungal cultivation in the form of pellets. Further, the present invention relates to the integration of fungal cultivation into the first generation sugar to ethanol production.

BAC K G ROU ND A ND PRIOR A RT OF T H E INV E NTION:

Sugarcane or sugar beet is one of the most abundantly used feedstocks for bioethanol production worldwide. Ethanol is produced by fermentation of plant crops containing starch and sugar such as wheat, sugarcane, sugar beet and corn. Ethanol production from sugar is either directly obtained from sugarcane juice which is extracted from sugarcane or from beet sugar leaving behind bagasse that is generally used for electricity or energy generation or from molasses, a low value co-product of raw sugar production. While cane juice is the dominant feedstock for ethanol production in most Brazilian factories, a large number of producers in Indonesia, India, the Caribbean, and a significant number in Brazil, mostly manufacture ethanol from molasses.

Molasses is used either as a cattle feed supplement in specialized yeast propagation, as a fertilizer, or as a flavoring agent in some foods. Although fermentable sugars in molasses cannot be further upgraded to raw sugar, they can be converted to ethanol in a distillery using conventional ethanol fermenting microorganisms such as yeast. Hence, integrated sugarcane factories having a sugar manufacturing industry co-located with an ethanol distillery can use molasses as a feedstock for ethanol production in addition to raw cane juice directly from the mill. A significant number of sugarcane factories in Brazil and India and several other such factories employ this strategy of integrating sugar industries with ethanol distilleries.

Several research attempts by fermentation technologists have suggested integration models for fungal biomass cultivation at ethanol production industries. An instance of an integrated ethanol production industry has been disclosed in PCT International Publication No. WO/2015/086803 by Patrik R. Lennartsson et al. This publication discloses an alternative to the direct implementation of an industrial scale second generation bioethanol process with integration of second generation into the existing first generation bioethanol processes, which aims to reduce the current barriers to process change/investments. However, the conditions employed to obtain fungal biomass production have not been illustrated in this document. Demonstration studies show fungal biomass production to be at a negligible yield of 5.3g/l. Furthermore, this document mostly focusses on obtaining ethanol production.

During the process of ethanol distillation from fermented sugar juice or molasses, a large amount of vinasse also called as still age or spent wash is being produced. V inasse is the final by-product of biomass distillation, mainly for production of ethanol, from sugar crops (beet and sugarcane), starch crops (corn, wheat, rice, and cassava), or cellulosic material (harvesting crop residues, sugarcane bagasse, and wood). V inasse is usually produced as an acidic compost having pH varying from about 3.5 to about 5, a dark brown slurry, with high organic content (chemical oxygen demand 50 " 150 g L B1 ), and characterized by an unpleasant odor, with high contents of potential hazardous substances (Christofoletti et al., 2013). It has been estimated that for every liter of ethanol, around 12 " 15 L of residue is produced.

In Brazil, around 28 million cubic meters of ethanol in 2013-14 was produced, according to a report issued by the Brazil's Agriculture Ministry Report (2015). Therefore, handling huge volumes of vinasse is done by its direct application on the fields to partially replace mineral fertilizers during the sugarcane cultivation (J anke et al., 2016). Due to enormous volumes of vinasse being produced, alternative treatments and uses have been developed, such as recycling of vinasse in fermentation, fertirrigation, concentration by evaporation, and yeast and energy production. The practice of field application, commonly referred to as fertirrigation, totally or partially facilitates replacing use of chemical fertilizers, mainly those containing phosphorus (Christofoletti et al., 2013).

However, vinasse applied to farming lands as fertilizer can cause serious water and soil pollution, such as leaching of metals to groundwater, changes in soil quality, increase of phototoxicity, unpleasant odor, as well as leading to considerable methane emissions during temporary storage or transportation and also nitrous oxide emissions (Carmo et al., 2013; Christofoletti et al., 2013; De Oliveira et al., 2013; Dias et al., 2016).

Recently, several researchers have used vinasse for the process of anaerobic digestion, which allows the recovery of part of its energy content owing to biogas production (J anke et al., 2016) (Dias et al., 2016) (Djalma Nunes F errazJ ηϊθΓ et al., 2016; Moraes et al., 2015). However, due to the sulfating process (calcium sulfate precipitation) used in raw sugar production or addition of sulfuric acid to lower pH of the yeast cream (contamination control) during alcoholic fermentation, usually high amounts of sulfate are found in vinasse (Moraes et al., 2015). Hence, while using vinasse as the substrate for biogas production, hydrogen sulphide(H2S), a toxic and malodorous gas is produced in significant amounts (Krayzelova et al., 2014), which can inhibit microbial activity and also lead to reactor corrosion. The difficulty in finding a suitable solution to this problem is what hinders from developing a promising approach to handling large amount of vinasse produced from sugar- to- ethanol industries. The economical sustai nability of the existing bioethanol facilities can greatly depend on its intrinsic capacity to upgrade the process, adapting the concept of the biorefinery. The sugar- based bioethanol production process results in a large volume of vinasse, the stillage from the distillation process. V inasse finds application as a soil fertilizer or as feed stocks for biogas production. Though these processes had merits, they are not deprived of short comings such as for example, causing environmental pollution with increasing phototoxicity or leaching of metals to ground water upon soil application of vinasse or production of high amounts of sulfide and less methane production etc. during its use in the biogas processes (J anke et al., 2016; Moraes et al., 2015). The low pH, electric conductivity, and chemical elements present in sugarcane vinasse may cause changes in chemical and physical " chemical properties of soils, rivers and lakes with frequent discharges over a long period of time, and also have adverse effects on agricultural soils and biota in general. Thus, new studies and green methods need to be developed aiming at sugarcane vinasse recycling and disposal (Christofoletti et al., 2013).

A research study by S.B. Sartori et al. published in Mycology, 2015, Vol. 6, No. 1, 28 disclose the cultivation of fungi Pleurotus sajor-caju, P. ostreatus, P. albidus and P. flabellatus in vinasse and its use as a complementary diet for Zebra fish. The fungi mycelium is cultured in vinasse for 15 days. However, the vinasse constituents and concentration of the organic content have not been demonstrated by S.B. Sartori et al. Further, the incubation period of the culture media comprising vinasse inoculated with fungal mycelia was for an extended period therefore rendering the process to be time consuming.

Other studies, for instance an article by Saoharit Nitayavardhana et al, published in Bioresource Technology 133 (2013) 301 " 306 have employed a high concentration of vinasse of 75% v/v for protein rich fungal biomass production. This high concentration of the vinasse substrate increases the oxygen demand of the substrate. Further, the additional use of nutrient supplements for fungal biomass production is disclosed therein.

The use of the edible A scomycetes fungi, N. intermedia and A. oryzae for ethanol and fungal biomass production has been extensively studied with lignocellulose or starch based industrial waste streams, for example, disclosed in US8481295 and WO/2015/086803.

A foremost drawback is encountered by filamentous growth forms of the fungi causing several cultivation challenges, while scaling up an integration process at the existing sugar to ethanol facilities. The broth viscosity caused by the filamentous nature of the fungi negatively affects the mixing and aeration of the culture; mycelial clumps can wrap around impellers; all of which can lead to a decrease in production efficiency and bioreactor performance (Gibbs et al., 2000, Critical Reviews in Biotechnology, 20(1), 17-48).

Hence special focus was made to determine the pelletization potential of fungi in vinasse medium. From the basal screening experiments, it was observed that the vinasse dilution (% v v) and cultivation media pH were the most critical factors influencing pelletization in A. oryzae. In light of the drawbacks imposed by conventional fermentation practices of the art it becomes important to cultivate fungal strains of economic importance in large batches which are convenient to retrieve from fermenters. Therefore, the present inventors have provided an efficient approach of using vinasse obtained from sugar to ethanol fermentation process as a substrate for cultivation of protein rich fungal biomass for feed application and ethanol from fungal fermentation.

OBJ E CT OF T H E INV E NTION:

It is an object of the present invention to provide a process for edible fungi cultivation of fungi selected from members of the phylum A scomycota, excluding the order, Saccharomycetales by employing a substrate comprising stillage from conventional sugar to ethanol production processes so as to generate higher yield of fungal biomass and ethanol.

Another object of the present invention is to optimize fungal culture conditions to obtain high yield of the fungal biomass.

Y et another object of the present invention is to provide animal feed including cattle, or poultry, or fish feed; or human feed comprising the fungal biomass of the present process and an agricultural fertilizer.

SU M MA RY O F T H E INV E NTION:

In an aspect, the present invention provides a process for cultivating edible fungal biomass selected from members of the phylum Ascomycota, excluding the order, Saccharomycetales in high yield, the said process comprising;

(a) preparing a culture medium comprising vinasse in a concentration ranging from 1 to 50% (v/v), and optionally one or more nutrient supplements for fungal cultivation;

(b) inoculating fungal culture in the culture medium of step (a), and maintaining a pH of upto 7 followed by incubation at a temperature range 20 to 40eC for 24 to 120 hours to obtain edible fungal biomass in high yield.

High fungal yields were obtained at the aforesaid optimized conditions for fungal biomass culture, however the present invention provides the preferable concentrations wherein higher fungal yield was obtained. Accordingly, the culture medium comprising vinasse in a preferable concentration of 5% to 20% (v/v), at the most preferable incubation temperature at 35eC and incubation duration of 72 hours.

Accordingly, fungal biomass is produced employing the present process in the range from about 50g/L to about 250g/L . In another aspect, the present invention provides the cultivation of edible fungal biomass selected from Ascomycetes, the fungus including, but not limited to species of Aspergillus spp., and Neurospora spp are employed herein.

In a further aspect, the present invention provides the integration of the process for cultivating edible fungal biomass into a conventional process of ethanol production in sugar industries.

In yet another aspect, the present invention provides the reduction in the heavy metal content, soluble chemical oxygen demand (COD), and medium viscosity of diluted vinasse used as a substrate for edible fungal cultivation.

In one aspect the present invention provides a culture medium comprising vinasse obtained from conventional alcohol production in a concentration ranging from about 1 % to 50% (v/v) with the optional addition of nutritional supplements being optional.

In one more aspect, the present invention provides a fungal biomass product for use as an aquaculture or animal or human feed supplement, as a fertilizer or as a flavoring agent in certain foods.

DE TAIL E D DE SC RIPTION OF T H E DRAWINGS:

F igure 1 depicts integration of fungal cultivation into a conventional fermentation process, wherein fungal cultivation is carried out in a bioreactor with spent wash/ vinasse obtained from evaporation stage of conventional ethanol production process;

F igure 2 depicts the integrated fungal cultivation step is added after the di sti 11 ati on step, pri or to the evaporati on step;

F igure 3 depicts fermentation using fungal biomass as inoculum at optimum conditions: vinnase dilution 10% v/v, pH 5.5 (for N. intermedia) and 6.0 (for A. oryzae), temperature 35eC and time 72 h; F igure 4 depicts Sugars and organic acids profile: Fungal fermentation on vinasse at optimum conditions in bench scale airlift reactor (a) N. intermedia (pH 5.5) and (b) A. oryzae (pH 6.0) at vinnase dilution 10%, (v/v), temperature 35eC and time 72 h. The said figure represents- lactic acid (IV); glycerol ( 1 ; acetic acid (v¾ glucose (a); cellobiose ( ° ) and ethanol ( * ) concentration (g/L of dilute vinasse- 10%).

DE TAIL E D D E SC RIPTION O F T H E INV E NTION:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

V inasse refers to the unwanted residual liquid waste generated during alcohol production from sugarcane or sugar beet; therefore, references in the present invention to vinasse are to be construed as references to spent wash.

In a preferred embodiment, the present invention provides a process for cultivating edible fungal biomass selected from members of the phylum Ascomycota, excluding the order, Saccharomycetales in high yield, the said process comprising;

(a) preparing a culture medium comprising vinasse in a concentration ranging from 1 to 50% (v/v) and optionally one or more nutrient supplements for fungal cultivation;

(b) inoculating fungal culture in the culture medium of step and maintaining a pH of upto 7 followed by incubation at a temperature range 20e to 40eC for 24 to 96 hours to obtain edible fungal biomass in high yield and additionally ethanol production.

In accordance with the preferred embodiment, the present invention provides a process for cultivation of edible fungal biomass selected from Ascomycetes, the fungus including, but not limited to species of Aspergillus spp. and Neurospora spp are employed herein.

In an embodiment, the concentration of vinasse employed is ranging from about 1 % to 50% (v/v), more preferably the concentration of vinasse in culture medium is ranging from about 5% to 20% (v/v). The most preferable concentration of vinasse used in the present invention is 10% (v/v).

The present invention also provides applications of spent wash from fermentation processes including industrial waste streams having constituents similar to that of vinasse used in the present invention. The constituent make-up of vinasse as described in Tables 1(a) to 1(d) is conducive for the growth of edible fungi by the process of the present invention.

O her culture conditions such as the pH of the culture medium is in the range from about 5 to 6.5, the temperature of cultivation is in the range from about 25eC to 45eC, and the duration for cultivation ranges from about 24hrs to 96hrs.

Accordingly, the present process for cultivation of fungi is most preferably performed in a culture medium having vinasse concentration ranging from about 5% to 20% (v/v), at a temperature of 35eC for duration of 72 hours. With respect to the pH, cultivation is carried out at pH of 5.5 for Neurospora intermedia and 6.0 for Aspergillus oryzae. Table 2 describes the optimization of the fungal conditions to achieve a high yield of various edible fungi.

In another embodiment, the present invention provides introduction of the fungal inoculum, which is the edible ascomycete Aspergillus oryzae var. oryzae C BS 819.72 and Neurospora intermedia CBS 131.92 (Centraal bureau voor Schimmelcultures, Netherlands), maintained on potato dextrose agar (PDA) slants containing (in g/L) potato extract 4; dextrose 20; agar 15 (renewed every six months), i n the cultivati on medi um rangi ng from 1 B10 1 to 1 B10 8 spores /mL . The fungal inoculum added to the vinasse media as the seed culture is either in the form of spores or as mycelial filamentous biomass.

In yet another embodiment, the present invention provides the yield of fungal biomass to be ranging from about 50g/L to about 250g/L of vinasse.

Accordingly, batch cultivations employing vinasse as a substrate without additional nutrient supplementation, presented an efficient fungal growth ranging from about 202 g and 223 g dry fungal biomass (per liter of vinasse) for N. intermedia and A.oryzae respectively.

In an optional embodiment, the present invention provides addition of nutrient supplements not limited to but selected from the group consisting of carbon, nitrogen and other nutrients or salts.

In yet another preferred embodiment, the present invention provides integration of the process for cultivating edible fungal biomass into a conventional process of sugar to ethanol production by using any forms of fungal bioreactor.

The integration of the present fungal cultivation process into the existing conventional -process stream of the sugar-to- ethanol facility is provided herein. Accordingly, the present invention discloses the use of vinasse from the existing or new ethanol plants that uses either sugar-juice or molasses for ethanol production process. The sugar juice or molasses in this context is either from sugar cane or from beet sugar. The two scenarios of the integration processes are described in Figures 1 and 2.

Figure 1 depicts fungal cultivation carried out in a bioreactor with vinasse obtained from the evaporation stage of conventional ethanol production process in existing or new sugar industries. In Figure 2, an integrated fungal cultivation step is added after the distillation step, prior to the evaporation step. The dilute spent wash/vinasse from distillation is sent directly to the fungal cultivation step. Accordingly, spent wash and vinasse may be interchangeably used for the purposes of the present i nvention.

In both the processes the vinasse/spent wash dilution is made ranging between 1 to 50% (v/v) resulting in the diluted vinasse with reduced content of total carbon and other constituents such as heavy metals and other ions selected from K, S, Na. The fungal inoculum either in the form of spore or mycelial filamentous biomass is added to the vinasse media as the seed culture. The fermentation media optionally is supplemented with additional nutrient sources. The fungal cultures include the members of phylum Ascomycota, excluding the order Saccharomycetales.

In one more preferred embodiment, the present invention provides the cultivation of fungal biomass in the form of pellets having size in the range of 2 mm to 4mm.

Fungal pellet form was observed at a pH range of 6 to 6.5 and vinasse concentration 5 to 10% v/v, with the pellet size (diameter) ranging from 2.29 to 4.1 mm.

The fungal cultivation results in ethanol production in the range of but not limited to 0.1 to 12 g/L, which in the present invention may also be referred to as additional ethanol.

The : additional ethanol " as described in the present invention is ethanol produced from vinasse substrate using the filamentous fungi under the process conditions. The additional ethanol is directed to the fermentation step or directly to the distillation step of the current existing ethanol process line without additional recovery step ( F igure 2). The additional ethanol production improves the overall ethanol production process of any existing or new fermentation facility, typically as between 1-20%, such as the least between 1-2.5% or between 2.5-5% or between 5-10%.

Further, the fungal biomass is harvested by conventional processes such as drying or simple sieving, filtration or any other process of solid-liquid separation method.

In one preferred embodiment, the present invention comprises fungal biomass having protein content ranging between 10 to 70% such as in between the range of 50% to 55 % or 60%.

Accordingly, a representative non-purified fungal biomass according to the present invention comprises 45-55% (% of DS) protein, 5-20 % (% of DS) fat, 2-5 % (% of protein) lysine and 1-3 % (% of protein) methionine.

In one embodiment, the present invention provides the process of fungal cultivation is carried out in reactors selected from the group comprising reactors with type as stirred tank, airlift, bubble column or bubble distributor or bubble riser.

Further, the reactors are constructed of material selected from the group comprising steel, concrete, fabric, metal, plastic, carbon fiber or any chemically developed material in any form as of a container.

The fungal biomass can be used either directly or in any other forms as a nutritional substitute for human or domestic food or feed applications. Domestic feed in the present invention includes, animal, poultry, fish feed products, either as direct or as constituent of any existing or new food or feed products. The spent liquid resulting from the fungal biomass separation is used as the fertilizer, either directly or with the inclusion of the evaporation or drying step, as indicated in Figure 1. Alternatively, the spent liquid can be used post the evaporation of ethanol and water mixture as in Figure 2. In Figure 2, after the fungal biomass separation or harvest, the spent liquid generated is evaporated with additional ethanol produced during the fungal cultivation and is sent back (together with the water) to the existing fermentation step of the current process line.

In one embodiment, the present invention provides a fungal feed composition having high protein content comprising fungal biomass cultivated by the process of the present invention, wherein the protein content is in the range of 45% to 55% of the total biomass and further comprising fat content from about 5%-20 %, lysine content from about 2% - 5% and methionine content from about 1 %-3 % of the total biomass.

The spent liquid or the spent wash can be used as a fertilizer in the agricultural field either as directly or supplemented with any other forms of solid or liquid residues such as press- mud.

The present invention reduces the organic content of the spent wash / vinasse, with the total chemical oxygen demand (TCOD) or soluble chemical oxygen demand (SCOD) reduction in the range 5 to 100%. The reduction in organic content is described as the difference between the vinasse composition feeding into the fungal cultivation bioreactor and those going out of the bioreactor as the spent liquid.

The present invention also reduces the viscosity of the spent wash or vinasse by about 1 to 50% reduction. The viscosity of the spent wash before and after fungal cultivation is considered for this purpose. The present invention also describes the glycerol utilization by the filamentous fungi with the maximum of 1 to 100 % reduction in the total glycerol content of the vinasse substrate either in the diluted or in the crude state. In one more preferred embodiment, the present invention provides edible fungal biomass produced by the present process for use as cattle or poultry or fish feed supplement, as a fertilizer or as a flavoring agent in certain foods. The edible fungi can be used in appropriate concentrations and in formulations for its intended use.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

E xamples:

E xample 1 : Inoculum preparation

Food grade- edible ascomycetes fungi, Neurospora intermedia CBS 131.92 and Aspergillus oryzaevar. oryzae C BS 819.72 (Centraal bureau voor Schimmelcultures, Netherlands), were obtained from the microbial Culture Collection at the University of GQeborg (Sweden). The cultures were maintained on potato dextrose agar (PDA) plates containing (in g/L) potato extract 4; dextrose 20; agar 15. For preparing spore suspension, fungal plates were flooded with 20 mL sterile distilled water and the spores were released by gently agitating the mycelium with a disposable cell spreader. An inoculum of 3 mL spore suspension (with a spore concentration of 3-4 B 10 6 spores/mL) per liter of the medium was used for the cultivations, unless otherwise specified. For preparing fungal biomass inoculum, the spores were inoculated into 100 mL Y PD broth containing (in g/L) dextrose 20; peptone 20; yeast extract 10. The culture was incubated for 48 h at 35eC and 125 rpm. The fungal biomass was harvested at the end of the cultivation and used as the inoculum, with its dry weight calculated by drying the biomass at 105eC overnight. E xample 2: Optimization of fungal growth conditions

Optimal growth factors such as vinasse concentration (% v/v dilution), media pH, culture temperature, and cultivation time were screened (Table 2). Batch cultivations employing vinasse as a substrate without additional nutrient supplementation, presented efficient fungal growth of upto 202 g and upto 222 g dry fungal biomass (per L of vinasse media) for N.intermedia and A.oryzae respectively. As the nutrient constituents in vinasse mostly favored the fungal biomass production, ethanol production remained limited upto 12 g (per L of vinasse media) for N. intermedia and upto 10.5 g (per L of vinasse media) for A. oryzae, at 10% (v/v) vinasse dilution (Table 2). Nevertheless, while considering the overall integration model, the production of around 1.0 to 12 g/L ethanol being produced from the process could potentially contribute to about 100 to 100,000 m 3 ethanol together with 100 to 10,000 tons of dry fungal biomass per year at the existing sugar- to- ethanol facilities.

E xample 3: V inasse dilution

The concentration of vinasse post the ethanol distillation at the industrial production stream (i.e. 20% v/v) was kept as the basal screening media, with levels of dilution 5 to 15% v/v, tested for the fungal growth. Prolific fungal growth was observed in the diluted vinasse samples, 10% (v/v dilution) with the maximum biomass production, 196 and 209 g/L dry fungal biomass for N. intermedia and A. oryzae respectively (Table 2). The significantly higher amount of chemical constituents, mainly organic acids and cations such as K, Ca and Mg (Christofoletti et al., 2013) together with low fermentable sugar levels resulted in reduced fungal growth at 20% v/v vinasse media as compared to the diluted sample. Maximum ethanol production of 9.1 g and 6.5 g per liter of vinasse (N. intermedia and A. oryzae respectively) was also observed at vinasse dilution of 10% (v/v). E xample 4: Screening of fungal factors

As the vinasse in the present concentration supports no microbial activity, the initial fungal growth screening was made on the vinasse diluted to 5, 10, 20, 30, 40, 50 (% v/v). Further screening was made on the factors such as cultivation time (24, 48, 72 and 96 h); media pH (4.5, 5.0, 5.5, 6.0, 6.5 (e 0.1)); temperature (25, 35 and 45eC) and media sterilization (with and without). These factors were optimized in a sequential order, where the optimum values obtained for one specific factor was kept constant to obtain the optimum values of the following factors. Ethanol concentration and fungal biomass yield were used as the variables to determine the optimum conditions. Effect of nitrogen and phosphorus on fungal growth in vinasse medium was studied with the presence of either nitrogen (N) or with phosphorus (P) or both (NP). A vinasse based semi -synthetic media was custom made with the addition of ammonium sulfate, (NhU SC ) 7.5 g/L and (or) potassium dihydrogen phosphate (K H2PO4) 3.5 g/L, as sources of nitrogen and phosphorus respectively, as modified from (Sues et al., 2005). A culture without any additional nutrient supplements served as the control.

E xample 5: C ulture conditions

The fungal cultivations were carried out aerobically in the liquid vinasse medium (with specific dilution), in 100 mL volume (in 250 ml E rlenmeyer flasks), unless otherwise specified. Cultivations were carried out for 96 h in an orbital shaking water bath (Grant OLS-Aqua pro, U K) at 35eC and 125 rpm, with samples taken every 24 h. The initial pH adjustment was made with either 2M NaOH or 1 M HCI addition. All experiments and analyses were carried out in duplicate and results reported with error bars and intervals representing two standard deviations.

During the culture pH optimization tests, A. oryzae, exhibited its adaptability to a wide pH range of the vinasse media, i.e. 5.0 to 6.5, with the maximum of 215 g/L dry fungal biomass production at pH 6.5. However, N. intermedia, showed constraints with the range of culture pH, with its maximum production, 184 g/L dry biomass produced at the growth optimum at pH 5.5. The cultures at the range 5.0, 6.0 and 6.5 showed about 65, 61 and 75% reduction (respectively) in the fungal biomass production compared to the optimum growth at pH 5.5. pH of the vinasse generally ranges from 3 to 5, depending on the process and the dilution rate used during the ethanol distillation. Hence, the addition of alkali/acid to maintain the pH optimum would not be a constraint while using both the fungal strains tested in the study, thereby reducing the additional maintenance cost of the industrial process. Similar observations were made while screening for optimum cultivation temperature, were both the fungi showed extended growth adaptability over the range 25e to 35eC. The maximum fungal biomass production recorded at 35eC, with a dry fungal biomass production of 202.4 g and 217.5 g per liter of vinasse for N. intermedia and A. oryzae respectively. Table 2 shows the fungal ethanol production rate at various culture conditions.

With an industrial perspective for the process, effect of media sterilization was also tested for the fungal biomass production. However, the optimum fungal growth requires media pH in the range 5.5 to 6.5, which could possibly maximize the growth of undesirable bacterial cultures in non-sterile conditions. A reduction in dry fungal biomass production by 42% (for N. intermedia) and 37% (for A. oryzae) was observed when non-sterilized vinasse was used. Ethanol production at the nonsterile conditions was however negligible. Nevertheless, the non-sterile process conditions can still be enhanced with higher fungal biomass production with the addition of fungal biomass as inoculum (as opposed to spores) at a larger scale. Hence, the optimum growth conditions for the maximum fungal biomass production were determined as: 10% (v/v) vinnase concentration, culture pH 5.5 (for N. intermedia) and 6.0 (for A. oryzae), culture temperature 35eC and time 72 h. The sugar and metabolite profile of the fungal growth at the optimum conditions is depicted in Figure 4a and 4b.

E xample 6: C ultivation with additional nutrient supplementation

Fungal biomass showed no significant increase in cultures where vinasse media was supplemented with extra nitrogen and phosphorus sources. With a p- value 1i 0.05, the difference between the fungal growth at cultures with supplementation of nitrogen or phosphorus or both (considering the replicates), as compared to the control (without supplementation) remained statistically insignificant. An improved ethanol production, as opposed to the biomass production, was observed in the cultures supplemented with additional nutrients (Table 1). This implied that the indigenous organic and inorganic constituents present in vinasse avoid the need for additional nutrient sources for an efficient fungal growth; hence would considerably reduce the process maintenance cost at the industrial scale.

T able 1 : C haracterization of vinasse substrate a) General characteristics b) Soluble sugar c) Organic acids/ metabolites and d) Ions and H eavy metals T able 1(a):

T able 1(b):

Soluble Sugar C oncentration (g/L )

A rabi nose 1.74

G I ucose 12.9 (before autoclaving)

16.8 (after autoclavi ng)

Fructose 10.64 Sucrose 4.53

Cellobiose 21.49

Galactose 3.11

Xylose 0.86

Mannose 0.99

T able 1(c):

Organic acid/ metabolites C oncentration (g/L )

Acetic acid 44.76

Lactic acid 132.28

Ethanol 0.87

Glycerol 107.9

T able 1(d):

H eavy metals C oncentration (ppm)

Selenium (Se) 2.5

Zinc (Zn) 0.7

Cadmium (Cd) 0

Strontium (Sr) 3.4

Barium (Ba) 0.1

Copper (Cu) 0.4

Nickel (Ni) 0.6

Arsenic (As) 2.7

Cobalt (Co) 0

Lead (Pb) 0

Molybdenum (Mo) 0

Manganese (Mn) 8.5

Chromium (Cr) 0.6

Aluminium (A I) 5.5

Potassium (K) 87220 E xample 7: Scale-up in bench scale airlift reactors and glycerol utilization The optimum culture conditions were validated in a bench scale airlift bioreactor (4.5 L) (Belach Bioteknik, Sweden), with a working volume of 3.5 L . An internal loop with cylindrical geometry with a diameter 58 mm, height 400 mm and thickness 3.2 mm was used to achieve the airlift-liquid circulation. The bioreactor and the draft tube were made of transparent borosilicate glass. Fungal fermentation on vinasse media (10% νΛ/) in the bench scale airlift reactor showed similar results as observed with the shake flask cultures, at the optimum conditions. A maximum fungal biomass production of 154 g and 199 g per liter of vinasse was observed with N. intermedia and A. oryzae respectively. The aeration rate maintained at 0. 71 vvm (volume a ir Λ olumemedia /min) made an effective mixing for the media possible. Filtration of inlet air was achieved by passing it through a polytetrafluoroethylene (PTFE) membrane filter (0.1 =m pore size, Whatman, Florham Park, NJ , USA). The cultivation was carried out at pH 5.5 e 0.2 (for N. intermedia) and 6.5 e 0.1 (for A. oryzae), initially adjusted with 1 M NaOH. The fermentation was carried out at temperature 35 e 2 eC for 72 h. High amount of foaming was observed, which was controlled by the addition of 5ml (50% νΛ/ dilution) sterile antifoam. The requirement of the antifoam could hence be considered as an add-on to the maintenance cost during the integration process. Biomass growth in the form of entangled filamentous mycelial clump was observed in the case of both the fungi. However, in the case of A. oryzae, this observation deviates from the shake flask experiments, where pellet form of growth was observed at the similar conditions. The biomass separation was carried out with much ease using a metallic sieve with pore size 0.11 m to 5 mm. A considerable degree of glycerol utilization was observed with A. oryzae fermentation, with about 74% reduction. The reduction in the amount of certain heavy metals such as zinc (57% reduction), manganese (91 %), and aluminium (60%) were observed while the fermentation was carried out using N. intermedia for 72 h in 10% (νΛ/) vinasse (Table 2). Though no significant ( p 1i 0.05) heavy metal reduction was observed with A.oryzae, the total COD content was reduced by 34% compared to the much lower COD reduction, by about 9%, with N.

intermedia. The separated fungal biomass with the total protein content of 43% and 46%; ash content of 4.3% and 4.6% (for N. intermedia and A. oryzae respectively) could be considered as an efficient animal of fish feed supplement

(Table 3). A reduction in vinasse media viscosity by about 2.5% and 7.6% (for N.

intermedia and A. oryzae respectively) was observed while analyzing the viscosity of the biomass free fermentation slurry at 72h.

T able 2. F ilamentous fungal cultivation in vinasse: Optimization of the

growth conditions

g dry fungal biomass /L

C ulture C ultivation factors g ethanol /L vinasse) vinasse

conditions (variables)

N. intermedia A. oryzae N. intermedia A. oryzae

C oncentration (%

νΛ/)

pH 5.5 5 177.4 e 3.6 192.1 e 4.1 4.22 e 0.81 2.28 e 0.38

Cultivation time: 10 196.5 e 1.7 209.4 e 5.7 9.14 e 0.48 6.58 e 0.23

72h 15 102.7 e 3.8 137.3 e 2.6 5.26 e 0.32 5.07 e 0.75

Temp : 35eC 20

30 - - - 50

C ultivation time

Vinasse cone: 10%

24 31.7 e 1.3 37.9 e 0.9

temp: 35eC

48 57.5 e 1.7 118.8 e 2.7 8.51 e 0.28 5.27 e 0.47 pH: 5.5

72 159.7 e 5.2 199.4 e 3.6 5.72 e 0.62 2.62 e 0.19 96 63.5 e 1.9 171.9 e 4.2

C ulture pH

Vinasse cone: 10%

4.5 - - - - Time- 72h

5.0 64.3 e 1.3 214.8 e 7.5 3.52 e 0.22 2.37 e 0.51

Temp : 35eC

5.5 184.7 e 5.8 205.2 e 4.9 11.81 e 0.96 5.53 e 0.22

6.0 73.6 e 3.7 193.1 e 1.8 - 10.25 e 0.97 6.5 45.8 e 1.9 215.6 e 2.7 - 2.81 e 0.23

Vinasse cone: 10% T emperature (eC )

Time: 72h, temp 25 142.9 e 5.1 144.5 e 5.3 5.18 e 0.19 3.37 e 0.01

35eC 30 198.1 e 2.8 212.7 e 2.9 8.87 e 0.25 7.92 e 0.03 pH: 5.5 (NI) and 35 202.4 e 1.3 217.5 e 1.8 10.18 e 0.71 9.08 e 0.07

6.5 (AO) 45 - - - -

Vinasse cone: 10% Sterilization

Time: 72h, temp Non-S 111.5 e 3.9 138.6 e 7.8 - -

35eC

pH: 5.5 (NI) and Sterile 195.2 e 6.4 222.8 e 3.5 10.72 e 0.91 8.75 e 0.40

6.5 (AO)

Additional

Vinasse cone: 10% nutrient source

142.6 e 4.1 203.5 e 1.71 10.08 e 0.54 9.53 e 0.57 Time: 72h, temp Nitrogen

35eC Phosphorus 201.4 e.8.3 176.8 e 2.01 11.18 e 0.97 10.57 e 1.09 pH: 5.5 (NI) and Nitrogen &

6.5 (AO) 156.8 e 2.5 196.5 e 1.80 12.14 e 0.11 9.62 e 0.93 phosphorus

* Values represent mean e SD

E xample 8A: Pellet formation by A. oryzae in vinasse media

A statistically developed experimental design was used to determine the optimum culture conditions for pellet formation in A. oryzae. A full -factorial experimental model was designed using MINITA B÷ 17 (Minitab Inc, State College, PA, USA) software. The vinasse medium dilutions (vAY) at three levels (5, 10 and 15%) and media pH at three levels (5.5, 6.0 and 6.5) were studied to determine their combinational effect on the pelletization process. The concentration of the fungal biomass and the pellet size (diameter) were selected as the response variables. A composite desirability (D) value was calculated to validate the results (Nair et al.,

2016). The optimum combinations of vinasse concentration and culture pH resulting in maximum desirable pellet size and fungal biomass yield was scaled up in a bench scale airlift bioreactor (4.5 L) (Belach Bioteknik, Stockholm, Sweden), with a working volume of 3.5 L and the aeration maintained at 1vvm. Table 4 shows the fungal morphology and biomass production at various levels of factorial combinations. Fungal pellet form was observed at the pH range 6 to 6.5 and vinasse concentration 5 to 10% v/v, with the pellet size (diameter) ranging from 2.29 to 4.1 mm. Fungal growth in the form of filamentous mycelial clump was observed at higher vinasse concentration (15% v/v) and at pH 5.5 indicating that the ionic concentrations of the media are the critical factors facilitating pellet formation. Hence, a strong combinational effect of the media pH and the vinasse concentration was observed to have an influence on the pellet formation (Table 3). For some strains of A. oryzae, pellet formation occurs by coagulation of spores at pH values higher than 5, whereas low pH (<3.5) results in growth as freely dispersed hyphal elements (Carlsen et al., 1996). Fungal pellet size also tends to possess an inverse correlation with the vinasse concentration, with uniform pellets growing at lower dilutions (5% v/v vinasse), at pH 6 and 6.5, with an average size of 3.96 e 0.25 mm and 2.29 e 0.19 mm respectively. However, higher biomass production with clump morphology was observed at 15% vinasse dilution, irrespective of the culture pH. Considering all the factorial combinations the A NOVA response optimization model showed a composite desirability value of 0.91 with the maximum (average) of 163.3 e 2.6 g dry fungal biomass per liter of vinasse and the pellet size maximum of 2.76 e 0.49 mm at the optimum conditions, pH 6.5 and vinasse concentration 10% v/v. It was observed that the glycerol utilization was also a combinational effect of higher pH conditions and pellet morphological state. Improved glycerol utilization (80 to 100% reduction) was observed by fungal pellet forms at all pH and vinasse concentrations (Table 3). However, mycelial clumps showed similar results only at pH 6.5 (97% reduction), indicating the significance of media pH on glycerol utilization. T able 3. Pelletization of A. oryzae : Statistical optimization of culture conditions

* Values represent mean e SD

E xample 8B: Pellet formation by N. intermedia in vi nasse media

For the pellet inoculum preparation, the fungal cultures were carried out aerobically in a liquid semi -synthetic potato dextrose medium (containing 20 g/L glucose and 4 g/L potato extract) or a defined synthetic medium with (g/L) glucose (or another carbon source such as sucrose, fructose, arabinose, maltose, mannose etc) 20, NH4C I 7.5, MgS04X7H20 0.5, NaCI 1.0, and K H2P04 3.5. T he fungus was grown aerobically at different acidic pH conditions such as 3, 3.5, 4 and 4.5, in the liquid medium. Cultivations were made in 100 mL volume (in 250 ml E rlenmeyer flasks), for 72 h in orbital shaking water bath (Grant OLS- Aqua pro, U K) at 35 eC and 125 rpm (with an orbital shaking radius of 9 mm and a flask diameter of 85 mm). The pH was adjusted with either 2 M HCI or 2 M NaOH. For cultivation on vinasse medium, the fungal pellets inoculum of size (10 mg dry fungal biomass /L media) was added to the vinasse substrate (10% v/v). Cultivations were made at pH 5.5. Fungal inoculum as filamentous form (10-15 mg dry fungal biomass/ L media) serves as the comparison experiment. The average size of the pellets ranges from 2.1 e 0.2 mm and 4.5 e 0.9 mm respectively. Results wherein, g of ethanol, total sugars and organic acids produced per liter of vinasse (10% dilution) are shown in table 4(a) and 4(b).

T able 4(a). F ermentation using N. intermedia pellets inoculum

Table 4(b). Fermentation using N. intermedia filamentous inoculum

T ime Ethanol G lucose T otal sugar L actic G lycerol Xylitol Acetic acid (h) (g/L) (g/L) (g/L) acid(g/L) (g/L) (g/L) (g/L)

0 0.2e 0.1 2.2e 0.4 3.0e 0.1 11.2e 1.7 10.7e 1.0 4.4e 0.7 3.9e 0.2

24 1.7e 0.2 3.2e 0.7 2.1e 0.0 11.4e 1.2 11.1e 0.9 1.8e 0.1 2.5e 0.8

48 1.1e 0.9 2.9e 0.1 2.3e 0.4 11.1e 0.9 10.8e 0.5 3.1e 0.8 3.5e 0.4

72 0.6e 0.0 4.4e 1.2 4.7e 0.9 10.6e 0.7 10.6e 1.1 2.7e 0.0 O.Oe 0.0

96 0.2e 0.0 3.1e 0.4 3.3e 0.7 10.5e 1.1 9.5e 0.9 5.2e 0.1 O.Oe 0.0 Advantages of the invention:

I The appropriate use of vinasse as a fermentation substrate for cultivating edible filamentous fungi enables recovery of part of the energy content or nutrients in the vinasse, in addition owing to the production of value added products such as enzymes, organic acids, ethanol or the protein- rich fungal bi omass for feed appl i cati ons.

I V inasse with Chemical Oxygen Demand (COD) -650 g/L is waste stream of sugar-to-ethanol industries, cultivation of fungi reduce the total COD by

34% and vinasse media viscosity by 21 %;

I T he culti vati on yi el ded a maxi mum of 223 g of fungi per I iter of vi nasse. I Protein-rich fungal biomass is potentially used for aquaculture feed applications

I The integration of fungal cultivation process brings the valorization of vinasse, also with the concomitant waste remediation potential; targeting a sustainable existence of the sugar-to-ethanol based industrial facilities.

I Production of additional ethanol could improve the overall ethanol production process of any existing or new facilities.

I The present process provides cultivation of fungal cells in the form of pellets.

I Batch cultivations at various culture conditions and vinasse dilutions, presented a maximum of 202 g and 223 g dry fungal biomass together with 11.8 g and 10.2 g ethanol per liter of vinasse, with N. intermedia and A. oryzae respectively.

I The present process could potentially make a new revenue stream for existing sugar-to-ethanol facilities with minimum investment, since the produced ethanol can follow the normal stream towards existing distillation columns and evaporators at the ethanol facility.

I Integration of the current process at an existing ethanol facility producing about 100,000 m 3 of ethanol per year could potentially produce around 200,000 to 250,000 tons of dry fungal biomass (40 to 45% protein) together with about 8800 to 12,600 m 3 extra ethanol at an existing ethanol facility. Fungal biomass produced from the vinasse could potentially contribute to the increasing global demand for the fish feed of about 51 million tons in 2015 (FAO, 2012) with about 8.8 to 12.6% improvement in the current rate of industrial ethanol production of the existing sugar- to- ethanol facilities.