Integrated process for the production of food, feed and fuel from biomass

A feedstock containing a biomass such as lignocellulosic materials, e.g. forest biomass; agricultural residues; or manures, is pretreated and thereafter is fractionated into cellulose, lignin and hemicelluloses. New mutants are disclosed which include Chaetomium cellulolyticum IAF-101 (NRRL 18756), Aspergillus sp. IAF-201 (NRRL 18758), Penicillum sp. IAF-603 (NRRL 18759), and Trichoderma reesei QMY-1. With these new mutants and also known fungi including Pleurotus sajor-caju and other Pleurotus spp. unfractionated predetermined biomass is converted into feed. The same treatment can also be applied to hemicelluloses, and cellullose. Cellulose can also be hydrolyzed by means of a cellulase-system prepared from cellulose and Tricoderma reesei to prepare glucose which can be converted to alcohol with Saccharomyces cerevisiae, Kluyveromyces spp. and Zymomonas mobilis. The residual microbial biomass of these microorganisms from alcohol fermentation broth is also used as feed. The process is economical and non polluting.

BACKGROUND OF INVENTION 
1. Field of the Invention 
This invention relates to an integrated process for the production of food, 
feed and fuel from biomass. More particularly, the present invention is 
directed to an improved process wherein the carbohydrate fractions of 
biomass are converted into protein-rich food for humans, protein-rich feed 
for animals and liquid fuel such as ethanol. On the other hand, the lignin 
fraction can be converted into adhesives, various chemicals or can be used 
as a burning fuel. 
In the following description, concentrations of substances are expressed as 
wt/vol (weight per unit volume of total mixture), as vol/vol (volume per 
unit volume of total mixture), as wt/wt (weight per unit weight of total 
mixture on a dry basis). 
The term biomass used herein includes lignocelluloses, which are mostly 
derived from crop residues, wood and wood residues. Generally speaking, 
biomass can be fractionated into cellulose, hemicelluloses and lignin. It 
is the most important source of organic carbon that can be used for 
bioconversion into food, feed and fuel. The term biomass also includes 
solid and liquid manures. 
2. Description of Prior Art 
In view of the world supply and demand of calories and proteins, the 
development of unconventional sources of food and feed is considered to be 
most important for mankind. Heavy demands of grain for the production of 
ethanol fuel would further aggravate present shortages. Moreover, 
escalating food/feed prices throughout the world have established the need 
for a high energy feed (70-75% digestability). It has already been 
proposed to produce high energy feed from poplar, and recently renewable 
biomass (especially forest biomass) has been found to be very attractive 
feedstock for the production of food, feed and also ethanol fuel. 
The forest is a very important renewable wealth and forest biomass is 
produced in large quantities every year throughout the world. Similarly, 
large quantities of mill and logging residues are also available. 
The biomass of plant origin, generally called lignocelluloses, contains 
45-56% cellulose, 10-29% hemicelluloses, and 14-30% lignin. It 
would,therefore, appear that bioconversion of carbohydrates (75-50%) into 
animal feed rich in protein (40-47%) would save millions of dollars which 
are presently spent to import soybean as a source of protein to supplement 
animal feed. On the other hand, the composition of biomass of manures of 
animal origin varies considerably depending on the feed given to the 
animal. Usually, this source is rich in starch lipids, protein and 
microbial biomass, and also contains little cellulose, hemicelluloses and 
lignin. Similarly, the affluents from food factories are rich in starch, 
protein and minerals but contain very little cellulose, hemicelluloses and 
lignin. 
In an article entitled "In Search of Microbial Food and Feed: Chaetomium 
cellulolvticum--the Obvious Answer", which I presented at the Symposium 
"New Sources of Food" of the National Meeting of AICHE at Denver, Col., 
Aug. 28-31, 1983, I indicated that the production of single cell protein 
SCP from biomass with Chaetomium cellulolyticum seems to be more promising 
than all the other microorganisms being tried in the world. C. 
cellulolyticum is a new fungus which I discovered in 1972 and is deposited 
at the ATCC under accession number of 32319. The taxonomy, morphology and 
physiology of this new fungus were reported in Mycologia, 68:600-610, 
1976. 
A few processes have been proposed or used to convert "lignocellulosic 
biomass" into protein-rich feed for humans and animals. However, they 
suffer with one or more of the following drawbacks. 
1. In most cases the "lignocellulosic biomass" is pretreated with various 
processes to make it suitable for bioconversion into protein-rich feed by 
fermenting with various microorganisms. During these pretreatments lignin 
is to be removed; however, by doing so, almost all the hemicelluloses, 
which are a potential source of carbohydrates, are also removed. These 
hemicelluloses end up in a waste stream which creates pollution problems 
because of its very high Biological Oxygen Demand(BOD). 
2. Most of the microorganisms used in such processes have poor conversion 
efficiency and a considerable amount of lignocelluloses are left utilized 
which cannot be separated from the final product thus lowering its value 
as protein supplement because of low protein content of the final product. 
3. Some of the organims such as Chaetomium cellulolyticum ATCC 32319, being 
used in some processes for production of protein-rich feed for animals, 
has recently been found to produce toxic compounds. 
The following Patents deal with a simple treatment of biomass with the 
known microorganism, Chaetomium cellulolyticum ATCC 32319. 
U.S. Pat. No. 4,379,544 
U.S. Pat. No. 4,401,060 
U.S. Pat. No. 4,447,530 
U.S. Pat. No. 4,526,791 
Canadian Patent No. 1,124,131 
Canadian Patent No. 1,129,709 
The disadvantages associated with fuel ethanol production from 
"lignocellulosic biomass.revreaction. through enzymatic hydrolysis are the 
following: 
1. Cost of enzyme production is very high. 
2. Substrate conversion into sugars is low (about 50%). 
3. Final concentration of sugars in the hydrolysate is 3-5% which may give 
1.5-2.5% ethanol in the fermentation broth. This small quantity of ethanol 
in the broth is not economical for distillation into 95% ethanol. 
4. Hemicelluloses are not properly utilized which end up in pollution 
stream. Although there are many methods to convert pure xylose (a major 
component of hemicelluloses into ethanol, none is capable to give more 
than 40% of theoretical yield from hemicelluloses. 
SUMMARY OF INVENTION 
It is an object of the present invention to provide new mutants which 
enable to obviate the problems associated with the prior art. The new 
mutants are the following: 
Chaetomium cellulolyticum IAF-101 (NRRL 18756) 
Aspergillus sp. IAF-201 (NRRL 18758) 
Penicillum sp. IAF-603 (NRRL 18759) 
Trichoderma reesei QMY-1 (NRRL 18760) 
It is another object of the present invention to provide an integrated 
process for the production of food, feed and fuel from biomass involving 
the use of the above microorganisms and also Pleurotus sajor-caju and 
other Pleurotus spp., as well as other micro-organisms such as 
Saccaromyces cerevisiae, Kluyveromyces sp. and Zymomonas mobilis and the 
like. 
It is another object of the present invention to provide a cellulase-system 
with high cellulase per unit volume, to give glucose and optionally 
ethanol. 
It is another object of the present invention to utilize native cellulose 
derived from wood and/or crop residues as such or pretreated by 
physicochemical methods, such as with alkali, acid, steam at 100.degree. 
C. or higher, and/or high pressure. 
It is another object of the present invention to provide a hydrolysis of 
high concentrations of cellulose with the cellulase-system according to 
the invention, thereby enabling to obtain high concentrations of sugars 
for the economical fermentation and distillation of ethanol. 
It is another object of the present invention to provide an integral 
process to produce inocula of the above microorganisms on hemicelluloses 
for food and feed production. 
It is another object of the present invention to provide an inoculum of 
Trichoderma reesei on hemicelluloses for the production of an enzyme 
comprising a cellulase-system. 
It is another object of the present invention to provide a process for 
treating biomass which comprises any suitable combination of the following 
steps: 
a) pretreating said biomass to enable it to be fractionated into cellulose, 
lignin and hemicelluloses; 
b) separating pretreated biomass into said cellulose, lignin and 
hemicelluloses. 
c) providing inocula of Chaetomium cellulolyticum IAF-101 (NRRL) 18756), 
species of Pleurotus including Pleurotus sajor-caju, Aspergillus sp. 
IAF-201, (NRRL 18758 ) Penecillum sp. IAF-603 (NRRL 18759) Trichoderma 
reesei QMY-1, (NRRL 18760) yeast and bacteria by growing same on 
hemicellulose fraction obtained by separation from pretreated biomass; 
d) innoculating another hemicellulose fraction obtained by separation from 
pretreated biomass with an inoculum selected from the group consisting of 
inocula of Chaetomium cellulolytium IAF-101, (NRRL 18756), Pleurotus, 
Aspergillus sp. IAF-201 (NRRL 18758) and Penicillum sp. IAF-603 (NRRL 
18759) under aerobic fermentation conditions effective to produce 
protein-rich animal feed; 
e) providing a fraction of pretreated biomass not subjected to separating 
step (b) and inoculating same th an inoculum of Chaetomium cellulolyticum 
IAF-101 (NRRL 18756), species of Pleurotus, Aspergillus sp. IAF-201 (NRRL 
18758) and Penicillum sp. IAF-603 (NRRL 18759) under aerobic fermentation 
conditions effective to produce protein-rich animal feed; 
f) treating a lignin fraction obtained in step (b) to produce adhesives and 
chemical products or burning same to produce heat or energy; 
g) inoculating a cellulose fraction obtained by separation from pretreated 
biomass with an inoculum selected from the group consisting of inocula of 
Chaetomium cellulolyticum IAF-101 (NRRL 18756), species of Pleurotus, 
Aspergillus sp. IAF-201 (NRRL 18758) and Penicillum sp. IAF-603 (NRRL 
18759) under aerobic fermentation conditions effective to produce 
protein-rich animal feed; 
h) fermenting a fraction of pretreated biomass not subjected to separating 
step (b) or fractionated cellulose under aerobic conditions with 
Trichoderma reesei QMY-1 (NRRL 18760) to give a cellulase-system, 
hydrolyzing a cellulose fraction obtained in step (b) with said 
cellulase-system to give glucose, and fermenting said glucose with an 
inoculum selected from said inocula of yeasts and bacteria to give 
ethanol. 
The glucose obtained could be used for the production of pharmaceuticals or 
any other fermentation products.

MICROORGANISMS USED IN INTEGRATED PROCESS 
Each of the microorganisms listed herein which are new embodiments and not 
commercially available have been deposited at the Northern Regional 
Research Center, located at the U.S. Department of Agriculture, 1815 North 
University Street, Peoria, Ill. 61604. The accession numbers for each 
strain so deposited are listed along with the name and description of each 
microorganism and are identified by the prefix "NRRL". In addition, each 
such organism has also been deposited at the Institute Armand-Frappier 
located at 531 Boul. Des Prairies, C.P. 100, Laval, Quebec Canada H7N 4Z3. 
The accession numbers for each strain so deposited are identified by the 
prefix "IAF" along with the name and description of each microorganism, 
together with the NRRL numbers. 
1. Chaetomium cellulolyticum IAF-101 (NRRL 18756): A new mutant produced by 
myself which is better than the parent strain (Chaetomium cellulolyticum 
ATCC 32319). 
It does not produce toxic metabolites. 
The new mutant IAF 101 (NRRL 18756) has a higher growth rate and higher 
conversion ability to convert lignocellulosic biomass or their fractions 
(cellulose or hemicelluloses) into protein-rich animal feed or human food 
than any other microorganisms which could be used in such processes. 
3. Pleurotus sajor-caju and other Pleurotus spp.; Aspergillus sp. IAF-201 
(NRRL 18758) and Penicillium sp. IAF-603 (NRRL 18759) which I have 
developed. These are also used for the conversion of lignocellulosic 
biomass into protein-rich feed for animals or food for humans as described 
above. 
4. Trichoderma reesei QMY-1(NRRL 18760): A new mutant developed by myself 
from its parent strain Trichoderma reesei QM94l4. It is used to produce a 
complete cellulase-system by growing on lignocellulosic biomass in solid 
state fermentation (SSF) or in liquid state fermentation (LSF) . This 
cellulase-system is able to convert 80-90% of lignocelluloses or pure 
cellulose into simple sugars. 
5. Other microorganisms used in this process include Saccharomyces 
cerevisiae, Kluyveromyces sp. and Zymomonas mobilis for the fermentation 
of hydrolysate obtained from enzymatic hydrolysis with the above 
cellulase-system into ethanol. 
As used herein, the term "biomass" includes materials of plant origin. i.e. 
agricultural residues, wood and forest residues (generally called 
ligno-celluloses), affluents from wood and pulping and paper industries; 
manures of animal origin i.e. cattle, swines, poultry, human etc.; and 
affluents from food factories. All these materials contain cellulose, 
hemicelluloses, lignin, starch, protein, lipids and some minerals 
depending on their origin. 
DESCRIPTION OF PREFERRED EMBODIMENTS 
Lignin in the biomass of plant origin is bonded physically and chemically 
to the plant polysaccharides, therefore, limited action is performed by 
microbial hemicellulases and cellulases due to the metabolic blocks of 
lignin-carbohydrate bonds. Hence, lignin has to be depolymerized, 
solubilized or removed in order to allow celluloses and hemicellulases to 
hydrolyse the thus exposed cellulose and hemicelluloses. According to the 
invention, four different pretreatments can be used, and these will be 
discussed later. 
With reference to the drawings, it will be seen that one ton of biomass 1 
essentially made of lignocellulose derived from crop residues, wood or 
wood residues is pretreated in a manner known per se 2 to be fractionated 
into approximately 500 kg cellulose 3, 250 kg lignin 4, and 250 kg 
hemicelluloses 5. About 450 kg of the fractionated cellulose is subjected 
to enzymatic hydrolysis by treatment 16 with a cellulase enzyme which will 
be described later to give about 445 kg of a glucose in the hydrolysate at 
17. Anaerobic fermentation of the hydrolysate in known manner with yeasts 
(Sacchoromyces spp. or Kluyveromyces spp.) or bacteria (Zymomonas spp.) at 
18 gives about 214.8 kg or 272 liters of ethanol 19. Of course, instead of 
converting the glucose obtained through enzymatic hydrolysis at 18, into 
ethanol, glucose could also be converted by fermentation into 
pharmaceutical products or any other known commercial products. 
Separately, a portion of the 250 kg of hemicelluloses 5 is inoculated with 
Trichoderma reesei QMY-1 (NRRL 18760) to grow an inoculum 9 of Trichoderma 
reesei QMY-1 (NRRL 18760) which can be combined at 14 with 50 kg of the 
cellulose obtained at 3 to give a cellulase enzyme which will be used for 
the cellulose hydrolysis 16 to give glucose, all as mentioned above. 
The inoculum of Trichoderma reesei OMY-1 (NRRL 18760) produced at 9 is also 
used to inoculate protracted bioman at 2 in solid state or liquid state 
fermentation for cellulase duction for hydrolysis of cellulose into 
glucose. 
Instead of inoculating the above-mentioned portion of 250 kg of 
hemicellulose with Trichoderna reesei, one may elect to inoculate the 
latter with yeasts (e.g. Saccharomyces spp. Kluyveromyces spp.) or 
bacteria (e.g. Zymomonas spp ) to produce inocula 9 for the direct 
fermentation of glucose 18 into ethanol 19. 
Yet another possibility is to inoculate another portion of 250 kg of 
hemicellulose with the following microorganisms: 
Chaetomium cellulolyticum IAF-101 (NRRL 18756) 
Aspergillus sp. IAF-201 (NRRL 18758) 
Pleurotus sajor-caju and other Pleurotus spp. 
Penicillum sp. IAF-603 (NRRL 18759) 
To produce inocula 9 which can be used in step 10 to produce protein-rich 
animal feed, 11. 
The remaining portion of the 250 kg of hemicelluloses is inoculated with 
any of the five microorganisms 10 for the conversion of the hemicelluloses 
into protein rich animal feed 11, or food for humans, hereinafter referred 
to as single-cell protein (SCP). 
Any residual mycelial biomass of Trichoderma reesei 15 obtained after the 
production of the cellulase-system at step 14 is used as animal feed 11. 
Similarly, any residual microbial biomass of yeasts or bacteria which 
remains after the ethanol fermentation 18 can be used as animal feed 11. 
It will be realized that the bioconversion of hemicelluloses into various 
inocula can save a lot of glucose produced at 17 otherwise used for such 
purposes and consequently reducing yields of the final products, for 
example ethanol 19, or other products. 
Pretreated biomass 2 can also be used as such to produce an inoculum of 
Trichoderma reesei 9 to give the cellulase-system 14 or to ferment with 
inoculum 9 of any fungus to produce directly at 7 protein-rich animal feed 
8. 
Finally, a portion of cellulose 3 can also be treated directly by aerobic 
fermentation at 12 with inoculum 9 of any fungus to produce protein-rich 
animal feed 13. 
Pretreatment 2 may be selected among the following: 
1. Alkali Pretreatment 
About 70% solubilization of solubilizable matters of a biomass of plant 
origin is achieved by treating the substrate with 5-20% sodium hydroxide 
(NaOH) (wt/wt of the substrate) with substrate: water ratio of 1:2 to 1:20 
at 80.degree.-121.degree. C. for 30 minutes to 2 hours depending on the 
nature of the substrate. This treatment solubilizes most of the 
hemicelluloses and lignin and also makes the cellulose most susceptible 
for the growth of the microorganisms for the production of protein-rich 
food/feed or for enzymatic hydrolysis into glucose or for cellulases 
production. 
Pretreated biomass is used as obtained or it can be fractionated into a 
soluble fraction which contains solubilized hemicelluloses and lignin, and 
insoluble fraction which contains mostly cellulose and a little lignin and 
hemicelluloses. The lignin from the soluble fraction is treated by 
acidification. 
2. Chemithermomecanical pulp (CTMP) 
Regular size (2-4 cm wide and 0.5-1.0 cm thick) industrial wood chips are 
used. All chips are screened and washed with water before use. Pulping is 
carried out in a Sunds Defibrator pilot-plant unit, type 300 CD, using a 
single-stage pressurized refining. 
Wood chips are chemically treated in an impregnator built inside the 
pilot-plant unit. The thus chemically treated chips are then steamed in 
the preheater of the unit for five minutes after which they are fed to the 
refiner for refining. 
The conditions used in the pulping process are as follows: 
1) Input of chips to the pilot-plant=0.8 kg (on oven-dry weight) per 
minute. 
2) Chemical charge for the treatment of chips, on oven-dry weight of wood: 
(a) 5 to 10% NaOH and (b) 5% NaOH and 5% Na.sub.2 SO 
3) Ratio of chemical solution fed to the impregnator to the oven-dry weight 
of chips fed to the unit: 1:1 (wt/wt). 
4) Steam pressure in the preheater: 238 kPa (20 psig) which corresponds to 
126.degree. C. 
5) Steam pressure for refining 260 kPa (23 psig) which corresponds to 
129.degree. C. 
6) Clearance between the two flat refining discs: 0.2 mm. 
7) Input of water to the flat disc refining zone: 2.3 liters/minute. 
8) Clearance between the two cone refining discs: 0.1 mm. 
9) Input of water to the cone refining zone: 0.38 liter/minute. 
Under the above conditions, the pulp produced has a consistency of 
approximately 25%. The yield of pulp is about 90% and the resulting pulp 
has an average size, in terms of Canadian standard freeness, of 100 ml. 
The pretreated mass is used as obtained. 
3. Steam Pretreatment 
In this process steam is added to a digestor where a 1 to 5 minutes 
residence time at a temperature comprised between 200.degree.-230.degree. 
C. forces to break the cellulose-hemicellulose and hemicellulose-lignin 
cleavages. Flash decompression of the treated material forces the needed 
ultrastructure modifications which frees large fractions of hemicelluloses 
(75%) and the lignin (80%). The pretreated mass is ready to use as 
follows: 
i) as obtained, 
ii) washed with water (hemicelluloses-free, i.e. cellulose-lignin complex) 
iii) extracted with ethanol, alkali or other solvents (lignin-free i.e. 
cellulose). 
iv) water-solubles (hemicelluloses and a little water soluble lignin). 
4. Thermomechanical Pretreatment of Aqueous Suspension 
A 12-14% solids aqueous suspension is treated at temperatures of 
150.degree.-230.degree. C. while being homogenized. Only ground biomass 
(0.5 mm) can be treated via this method. The residence time can be 
controlled more precisely than in the so-called stream explosion methods 
and is typically comprised between 0-2 minutes. As a consequence of this 
treatment, the hemicelluloses are solubilized together with one third of 
the lignin. The cellulosic residue has a DP 800-1400. 
Removal of the lignin from the residue can be done via simple 
organo-solvent method, ethanol, water being a convenient system. From this 
pretreatment the following fractions are available: 
i) the liquid hemicellulose-rich filtrate, 
ii) the residue containing cellulose and lignin, 
iii) the lignin-free residue, cellulose. 
By way of examples the compositions of five nutrient media which may be 
used in the integrated process of the invention will now be given: 
______________________________________ 
Medium I 
(All the requirements are for biomass containing 
10 g of glucose equivalent) 
(NH.sub.4).sub.2 SO*.sub.4 = 
1.88-2.357 g 
(KH.sub.2 PO.sub.4 = 
0.75-1.5 g 
MgSO.sub.4.7H.sub.2 O = 
0.25-0.5 g 
FeSO.sub.4.7H.sub.2 O = 
0.25-5 mg 
ZnSO.sub.4.7H.sub.2 O = 
0.25-5 mg 
Trace element solution* = 
0-1 ml 
water = 1 liter pH = 4-7.5 
Boric acid = 114 mg 
Amonium molybdate = 480 mg 
cuperic sulphate = 780 mg 
Manganese chloride = 
144 mg 
Medium II 
Same as medium I plus 
CaCl.sub.2 = 0.3 g 
MnSO.sub.4.7H.sub.2 O = 
1.56 mg 
Proteose peptone/yeast extract = 
0-0.5 g 
pH = 4-7.5 
Medium III 
(For Yeasts) 
Same as Medium I plus 
Yeast Extract = 0.5 g 
Water = 1 liter 
pH 4-7.5 
Medium IV 
(For Zymomonas mobilis) 
Same as Medium I plus 
Yeast Extract = 1.0 g 
Water = 1 liter 
pH 4-7.5 
Medium V 
(For Fermentation of Ethanol) 
Sugars equivalent to = 
80-160 g glucose/L 
KH.sub.2 PO.sub.4 = 
1.0 g/L 
NH.sub.4 Cl = 1.5 g/L 
MgSO.sub.4.7H.sub.2 O = 
0.16 g/L 
CaCl.sub.2 = 0.08 g/L 
Yeast Extract = 1.0 g/L 
______________________________________ 
*(or equivalent nitrogen in any other form or in combination thereof or i 
combination with phosphoric acid which will also eliminate KH.sub.2 
PO.sub.4 from the medium) 
*Trace element solution in one liter 
Each ton of biomass used for the production of ethanol will generate 
250-290 kg of hemicellulose sugars. However, some toxic compounds 
(furfural, hydroxymethyl furfural and phenolic compounds) are produced 
during pretreatments of lignocelluloses. During the separation of 
cellulose, all the toxic compounds become part of solubilized 
hemicelluloses. Therefore, these toxic compounds become a problem for 
further utilization of hemicelluloses by most microorganisms. The 
solubilized hemicelluloses contain mostly pentoses (xylose and arabinose), 
some hexoses (glucose, mannose, galactose), some uronic acids, some 
solubilized lignin and lignin components. 
This hemicellulose fraction is not utilized in almost all the processes for 
production of protein-rich food/feed. This fraction has also not been 
economically converted into ethanol because of the presence of toxic 
compounds. Therefore, hemicellulose fraction ends up as a waste which 
creates a lot of pollution problem because of its very high BOD. 
Keeping in mind that large quantities (250-290 kg/ton of biomass of plant 
origin) of hemicelluloses are released during pretreatments, the present 
invention intends to overcome all the prior problems for the utilization 
of the hemicellulose fraction. 
According to the present invention, the hemicellulose fraction is utilized 
inter alia to produce inocula. The inocula of various microorganisms 
required in the process according to the present invention are grown in 
the nutrient media described above. For the production of inocula of 
Chaetomium cellulolyticum IAF-101 (a new mutant), Pleurotus sajor-caju and 
other species of Pleurotus, Aspergillus sp. IAF-201 (NRRL 18758) and 
Penicillium sp. IAF-6O3, (NRRL 18759) the microorganisms are grown on 
hemicellulose fraction fortified with nutrient medium I, whereas the 
inoculum of Trichoderma reesei QMY-1 (NRRL 18760) is produced on 
hemicellulose fraction fortified with nutrient medium II. The inocula of 
yeasts and bacteria (Zymomonas mobilis) are produced on hemicelluloses and 
glucose fortified with medium III and medium IV, respectively. 
EXAMPLE I 
PRODUCTION OF PROTEIN-RICH FOOD/FEED 
1. On Substrate Obtained after Pretreatment 
The substrate obtained after pretreatment in a concentration of 1-6% 
(wt/vol) is fortified with sterile nutrient medium I. As the substrate is 
sterilized during pretreatment and sterile nutrients are used, there is no 
need of autoclaving the medium again. The mixture is inoculated with 5-10% 
(vol/vol) with an inoculum (produced with medium I with a hemicellulose 
fraction or glucose) of either of the following fungi: Chaetomium 
cellulolyticum IAF-101 (NRRL 18756) species of Pleurotus, Aspergillus Sp. 
IAF-201 (NRRL 18758) and Penicillium sp. IAF-603 (NRRL 18759). The pH of 
fermentation is maintained at 4-7.5, preferably at 6. The incubation time 
varies from 12-72 hours depending upon the concentration of the 
carbohydrate, the nature of the substrate and its pretreatment. The 
end-product, protein-rich feed is a mixture of biomass of the above fungi 
and unutilized cellulose and the unsolubilized lignin and contains 23-50% 
(dry wt basis) crude protein depending on the substrate and the fungus 
used. 
2. On Hemicellulose Fraction 
A hemicellulose fraction containing 1-6% (wt/vol) carbohydrates is 
fortified with nutrient medium I, autoclaved at 121.degree. C. for 20-30 
minutes, and cooled at 25.degree.-40.degree. C. It is inoculated with 
5-10% (vol/vol) inoculum (produced as described above) of either of the 
following fungi: Chaetomium cellulolyticum IAF-101 (NRRL 18756), Pleurotus 
sajor-caju or other species of Pleurotus, Aspergillus sp. IAF-201 (NRRL 
18758) and Penicillium sp. IAF-603 (NRRL 18759). The pH of fermentation is 
maintained at 4-7.5, preferably at 6. The incubation time varies from 
12-72 hours depending upon the concentration of the carbohydrates and the 
nature of the substrate used to obtain hemicellulose fraction. The 
end-product, protein-rich food/feed is a biomass of the above fungi 
containing 37-50% (dry wt basis) crude protein. 
3. On Cellulose Fraction 
A cellulose fraction whose concentration is from 1 to 6% (wt/vol) is 
fortified with nutrient medium I, autoclaved at 121.degree. C. for 30 
minutes to one hour depending on the concentration of cellulose in the 
medium, and cooled to 25.degree.-40.degree. C. It is inoculated with an 
inoculum produced on a hemicellulose fraction as described above) of 
either of the following fungi: Chaetomium cellulolyticum IAF-101 (NRRL 
18756), species of Pleurotus, Aspergillus sp. IAF-201 (NRRL 18758) and 
Penicillium sp. IAF-603 (NRRL 18759). The pH of fermentation is maintained 
at 4-7.5, preferably between 5.5 and 6.0. The incubation time varies from 
20 to 72 hours depending on the concentration of the cellulose in the 
medium. The end-product, protein-rich food/feed is a biomass of the above 
fungi containing 37-50% (dry wt basis) crude protein. 
The nutrients given in the medium I are for a substrate equivalent to 10 g 
glucose. The quantities of nutrients are increased with the increase of 
the concentration of the substrate in the medium. 
The concentrations of the substrates (as described in 1, 2 and 3 above) are 
increased by a fed-batch method i.e. starting the fermentation initially 
with 2-3% and then adding the rest of the substrate at various intervals 
according to the growth rate of the particular fungus. The interval varies 
from 5-12 hours. 
The temperature of fermentation is 30.degree.-40.degree. C. preferably at 
37.degree. C. for Chaetomium cellulolyticum IAF-101(NRRL 18756), 
Aspergillus sp. IAF-201 (NRRL 18758), but for species of Pleurotus and 
Penicillium.sp. IAF-603 (NRRL 18759) it is 25.degree.-37.degree. C. 
preferably 30.degree. C. 
4. On Manures 
The manures are fractionated into two phases: (i) a liquid phase containing 
soluble carbohydrates, starch, protein, lipids, etc. and (ii) a solid 
phase contains cellulose, hemicelluloses and lignin. The solid phase is 
composed of cellulose, hemicelluloses and lignin and is converted into 
protein-rich feed as described above under 1, 2, and 3. 
The liquid phase is fortified with 0 to 0.12% (wt/vol) (NH.sub.4).sub.2 
SO.sub.4 as desired and is autoclaved at 121.degree. C. 30 minutes. It is 
converted into protein-rich feed by inoculation with the following fungi 
(grown on hemicellulose fraction): Chaetomium cellulolyticum IAF-101 (NRRL 
18756), spacies of Pleurotus, Aspergillus. sp IAF-201 (NRRL 18758), and 
Penicillium sp IAF-603 (NRRL 18759). The end-product is a biomass of these 
fungi containing 30-37% crude protein. 
The liquid phase is mixed with a hemicellulose fraction or a cellulose 
fraction in various ratio 1:1 to 1:10 and is fortified with 0 to 0.12% 
(wt/vol) (NH.sub.4)SO.sub.4 as desired. The mixture is autoclaved at 
121.degree. C. for 30 minutes. It is inoculated with the following fungi 
(grown on hemicellulose fraction): Chaetomium Cellulolyticum IAF-101 (NRRL 
18756), species of Pleurotus, Aspergillus sp. IAF-201 (NRRL 18758), and 
Penicillium sp. IAF-603 (NRRL 18759). The end-product is a biomass of 
these fungi containing 37-50% crude protein. 
The end-product is harvested by passing through 3 layers of cheese cloth, 
pressed and dried to about 10% moisture. During harvesting all the 
solubilized lignin and toxic compounds become the part of the filtrate 
which can be precipitated by acidifying the filtrate to pH 3.5. The latter 
can be used for making adhesive and other chemicals or could be used as 
fuel for burning. Therefore, at the end of this process there are no 
affluents which could cause any pollution problems. 
EXAMPLE II 
Production of Ethanol 
1. Production of Cellulase-System 
The cellulase-system is produced on pretreated biomase which has not been 
fractionated or on fractionated cellulose. The pretreated biomass or 
fractionated cellulose is fermented aerobically with Trichoderma reesei 
QMY-1 (NRRL 18760). 
Fifty Kg of cellulose or 100 Kg of pretreated biomass (FIG. 1) is required 
for producing sufficient enzyme-system to hydrolyze 450 Kg of cellulose 
(FIG. 1). 
The cellulase-system can be produced by solid state fermentation (SSF) or 
liquid state fermentation (LSF). According to the invention, the 
hemicelluloses and lignin are preferably kept along with the cellulose, 
thus there is no fractionation of pretreated biomass. In SSF the nutrient 
medium II is mixed well and the moisture is maintained from 60-85% (wt/wt) 
so that there is no free water. The pH is adjusted between 5-7. It is 
autoclaved at 121.degree. C. for 30 minutes to 1 hour depending on the 
depth of the substrate. The preferable depth is 2-3 cm. The substrate is 
spread in a steel tray of any convenient size with a 40 mesh screen at the 
bottom for aeration. The tray is covered with a steel lid to avoid 
contamination. The cultures are inoculated with Trichoderma reesei QMY-1 
(NRRL 18760) grown on hemicellulose fraction. The inoculum is used in an 
amount of 50-100 ml (containing about 0.5-1.0 g (dry wt) mycelium for 100 
g (dry wt) of substrate. 
The trays are kept in a humidified (about 80% relative humidity) incubator 
maintained at 25.degree.-37.degree. C., preferably 30.degree. C. 
After 2-6 weeks growth depending on the nature of the substrate used, the 
fermented substrate is shaken with water in a ratio of 1:20 for about 1/2 
hour. It is centrifuged, after which the supernatant serves as a 
cellulase-system for the hydrolysis of cellulose to give a glucose 
solution. The cellulase system is also utilized to hydrolyze pretreated 
biomass without fractionation to give a mixture of glucose, xylose, 
galactose, mannose, arabinose, etc. 
The cellulase-system can also be prepared in LSF on pure cellulose or 
pretreated unfractionated biomass at a concentration of 2-16 with nutrient 
medium II. The concentration of the substrate is increased steadily by the 
fed-batch method. The enzyme broth is separated as explained above. 
2. Hydrolysis of Cellulose 
The enzyme-system produced in SSF on pretreated but unfractionated biomass 
contains: cellulase 5-20 IU/ml, B-glucosidase 5-20 IU/ml, and xylanases 
300-800 IU/ml. Higher concentrations of enzymes can be obtained by 
extracting the cellulase-system with a small quantity of water. This 
enzyme system can hydrolyze 80-90% of cellulose. The hydrolyzate contains 
9-15% glucose syrup with very little cellobiose. In this cellulase-system 
there is no need of addition of extra .beta.-glycosidase or xylanases as 
is done in a number of known processes of the prior art. Similarly the 
enzyme-system produced in LSF has almost the same ratios of various 
enzymes except that the enzyme activity may be a little less. 
3. Ethanol Fermentation 
The inocula of yeasts and bacteria, required for the fermentation of the 
hydrolysate are grown on hemicellulose fraction or glucose fortified with 
the nutrient medium III and nutrient medium IV, respectively. 
The hydrolysate obtained from the enzymatic hydrolysis of cellulose 
containing 10-15% (wt/vol) glucose is fortified with nutrient medium V. 
Fermentation is carried out at 25.degree.-30.degree. C. for 24-72 hours 
depending on the concentration of glucose in the medium, the type of 
organism used and the amount of inoculum of yeast or bacteria used. 
The fermentation of glucose into ethanol could be carried out even without 
the addition of any nutrients (Medium V) as some residual nutrients are 
brought in during hydrolysis from the medium in which the cellulase-system 
was produced and also some from the medium in which the inocula of yeasts 
or bacteria were produced. 
4. 
Hydrolysis of Hemicelluloses 
The hemicellulose fractions obtained by various pretreatments (1,2,3 and 4) 
still contain polymers, oligomers or even dimers of various sugars 
(xylose, mannose, galactose, arabinose and glucose). Most of the 
microorganisms cannot metabolize for microbial protein production or 
cannot catabolize for ethanol or other solvent production. However, the 
enzyme-system produced here in SSF or LSF contains very high amount of 
hemicellulases (measured as xylanase in this case) and can be used to 
hydrolyze the hemicellulose fraction into its monomer sugars. The 
microorganisms being used in this case do not need any such hydrolysis of 
hemicelluloses for the production of protein-rich feed because these 
microorganisms already have the ability to hydrolyze the hemicelluloses 
into simple sugars (monomers). 
The production of food, feed and fuel by the integrated process according 
to the invention leaves nothing unutilized, which means that there is no 
effluent disposal problem. In addition, because of the complete 
utilization of hemicelluloses, the process is more economical than those 
presently known. 
It is recommended that a forest biomass especially one derived from poplar 
trees be used as a major feedstock, although other wood species and other 
lignocellulosic materials are used. 
Preliminary feeding trials on rats indicated that up to 20-40% of the total 
protein requirement can be replaced by the protein produced according to 
the present invention without any pathological symptoms.