Process for upgrading cereal milling by-products into protein-rich food products

The invention provides a process for the microbial bioconversion of cereal milling by-products into proteinaceous material for human consumption. The by-products are aerobically fermented in a culture of the fungus Neurospora sitophila in the presence of suitable temperature, pH and nutrient conditions, for a time sufficient to grow microbial biomass protein.

The present invention relates to microbial bioconversion of cereal milling 
by-products to form a protein rich product for human or animal 
consumption. 
Cereal (and in particular wheat) milling by-products contain parts of the 
cereal grain left over after recovery of flour and germ. The outer layers 
of the seed which include the pericarp (bran) and seed coat comprise the 
largest components of milling by-products. Cereal milling by-products 
amount to approximately one-fourth (by weight) of the original raw 
material. The wheat milling industry is therefore a source of very large 
quantities of wheat milling by-products. Such cereal milling by-products, 
by their very nature, are non-digestible by humans and many animals, and 
furthermore, are generally resistant to microbial degradation even though 
they usually contain approximately 14% protein and 35-40% carbohydrates 
(by weight). Although cereal milling by-products have heretofore been 
utilized as an animal feed ingredient, most of this material is 
under-utilized and represents a potentially valuable feedstock for 
fermentation processes in the production of microbial biomass 
intrinsically useful a generic human food or animal feed product. 
It is known to utilize by-products from the petroleum industry and organic 
solvents for microbial biomass production for animal or human food. 
It is also known (Canadian Pat. No. 1,129,709, issued Aug. 17, 1982, 
Moo-Young) to utilize agricultural cellulosic waste products to form 
proteinaceous product for animal consumption. This process comprises 
effecting fermentation of non-food grade cellulosic material by the 
non-food grade fungus Chaetomium cellulolyticum at an optimum growth 
temperature of 37.degree. C. 
It is further known to recover protein from cereal milling by-products by 
way of alkali extraction followed by acid precipitation or other protein 
recovery techniques. 
The present invention is concerned with the utilization of cereal milling 
by-products, especially wheat cereal millfeeds, in which the food-grade 
fungal organism, Neurospora sitophila, is mass cultivated in a 
fermentation bioconversion process to produce a protein rich product. The 
resulting microbial biomass product has a relatively high content of 
protein, dietary fibre, ergosterol, natural flavour compounds and B 
vitamins. The biomass lacks animal fat and cholesterol. As animal feed, 
this product appears to be competitive with soy meal and fish meal. As a 
human food, this product appears suitable for special health conscious 
groups who seek vegetarian, high-fibre and/or low cholesterol diets. 
The present invention overcomes the following disadvantages existing in the 
prior art. 
The present invention uses a food-grade organism which does not contravene 
the U.S., Food & Drug Administration guidelines, the so-called GRAS list 
(Generally Regarded As Safe). The present invention uses as a raw 
material, food-grade feedstock which is derived from an acceptable and 
known agro-food resource as distinct from non-food industrial materials 
such as petroleum based hydrocarbons and alcohols, or forestry-based wood 
and paper. The present invention also utilizes technology which is based 
on solid-substrate fermentation as distinct from gaseous and liquid 
materials. A solid-substrate is easier to work with. The present invention 
also uses a multicellular fungal organism as distinct from unicellular 
organisms such as bacteria or yeasts, which cannot be easily filtered due 
to their size. The present invention further utilizes a microorganism 
which has an optimum growth temperature of approximately 26.degree. C. 
Such a lower temperature reduces the risk of opportunistic infections in 
humans (body temperature is 37.degree. C.), providing a safer environment 
for personnel involved in utilizing the invention. Furthermore, in 
addition to being able to utilize readily digestible carbohydrates such as 
starches and hydrolyzates, Neurospora sitophila possesses cellulolytic 
enzymes enabling it to utilize the recalcitrant cellulose component 
present in the majority of cereal milling by-products. 
The present invention provides a process for the production of a 
proteinaceous material comprising: aerobically fermenting, the by-products 
of cereal milling, in a culture of the fungus Neurospora sitophila; at a 
pH of 5.5-7.5; with agitation; in the presence of a suitable nutrient 
broth; at a temperature of 20.degree.-40.degree. C.; and for a time 
sufficient to grow microbial biomass protein. 
The organism, Neurospora sitophila has been consumed by mankind in 
Indonesia for hundreds of years, as part of oncom or ontjom an oriental 
food prepared by the solid state fermentation of peanut presscake and 
solid waste from tapioca manufacture. The fermentation is carried out at 
room temperature for 36 to 48 hours. The initial pH of the presscake is 
approximately 4.5 and it gradually rises as the fermentation progresses. 
Moderate aeration and humidity of approximately 90% is required during the 
process. The principal microogranisms involved in the fermentation of 
peanut presscake belong to the Genus Neurospora with several species 
involved: N. sitophila, N. crassa and N. intermedia. However, since the 
fermentation is not carried out under controlled monoculture conditions, 
other microorganisms such as yeasts and bacteria are present. The 
essential role of Neurospora in this process is the enzymatic hydrolysis 
of protein and starches of the substrate. Such action on the high protein 
presscake produces easily digestible and more flavourful food due to the 
formation of shorter protein peptides and amino acids. Consequently, the 
food-grade safety of this fungus has, to some extent been tested. As a 
result of experimentation, the present inventors have discovered that 
Neurospora sitophila can be cellulolytic and that it has good growth 
characteristics on both insoluble lignocellulosic and soluble carbohydrate 
based media. The inventors have also found that Neurospora sitophila 
demonstrates surprisingly significant speed of growth and diversity of 
advantages over other microorganisms known in the prior art in relation to 
the production of microbial biomass. 
The present invention is a controlled monoculture fermentation process of 
cereal milling by-products. In addition to the utilization of starch and 
easily digestible carbohydrates, the recalcitrant lignocellulosic 
component of the by-products is chemically and enzymatically broken down 
by the Neurospora sitophila. This is achieved in the invention process by 
softening and loosening the bonds between lignin and cellulose through an 
alkali pretreatment followed by enzymatic hydrolysis of cellulose by the 
various cellulases produced by the fungus during fermentation. The product 
of such hydrolysis are in turn used for the production of additional 
fungal biomass, thus substantially increasing the protein content of the 
final product of the fermentation. 
The cereal milling by-products are utilized in the form of a solid-liquid 
slurry, as is or after deproteination with caustic leaching. The 
fermentation medium is seeded with an inoculum of the fungus N. sitophila 
prepared according to well-established standard protocols. Preferably the 
cereal is wheat. 
The aerobic fermentation should occur in the environment of means for 
creating low shear aeration-agitation conditions. The aerobic fermentation 
broth should contain dissolved oxygen equal to approximately 50% air 
saturation. Levels below 30% will result in significant reduction in 
yields. 
Neurospora sitophila is grown until the fermentation broth consists of over 
50% (on a dry weight basis) of the fungal biomass, or the fungus reaches a 
"stationary growth phase" according to the principles of fermentation 
technology. A suitable nutrient broth should be used, which preferably has 
a nitrogen content of 50-75% (by weight) of the "basic nutrient 
composition" preferably in the form of ammonium sulphate and urea. The 
fungal biomass may be separated from the fermentation broth by filtration 
or other separation techniques known to those skilled in the art. It may 
also be dried for storage and/or transportation purposes. 
Table 1 below shows the nutritional quality of the resulting fungal protein 
product based on its composition of "essential amino acids", compared with 
fodder yeast (Candida utilis), soymeal and the guidelines on human food of 
the United Nation's Food and Agriculture Organization (FAO). It should be 
noted that the generic microbial protein product of the present invention 
compares well to the other amino acid distribution profiles (the important 
guideline for nutritional protein quality). In particular, the sulfur 
containing amino acids (cystine and methionine) are present in comparable 
or higher amounts than other microbial proteins such as fodder yeast. 
TABLE 1 
______________________________________ 
Essential amino acid compositions (as % by weight 
total protein) for various protein products and the 
microbial biomass of the present invention 
FAO Soybean Fodder 
N. sitophila 
Amino Acid 
Reference Meal Yeast S/P W/R T/R 
______________________________________ 
Isoleucine 
4.2 4.3 5.3 5.59 4.96 5.07 
Leucine 4.8 7.6 7.0 9.26 7.96 8.17 
Lysine 4.2 5.7 6.7 5.09 5.32 5.30 
Phenylalanine 
2.8 4.8 4.3 4.02 4.15 4.14 
Tyrosine 2.8 3.6 3.3 1.93 2.11 2.09 
Cystine 2.0 1.98 0.7 0.44 0.75 0.71 
Methionine 
2.2 1.14 1.2 1.06 1.81 1.70 
Threonine 
2.8 3.8 5.5 3.26 3.81 3.74 
Tryptophan 
1.4 1.6 1.2 2.46 2.94 2.88 
Valine 4.2 4.4 6.3 7.43 6.94 7.03 
______________________________________ 
S/P produced from starch permeate. 
W/R produced from wet residue. 
T/R produced from tota1 residue. 
Table 2 below provides proximate values of the key food parameters for the 
generic product (microbial biomass per se). Compared to animal meat such 
as beef, the generic product has desirably higher proportions of protein 
and dietary fibre. In addition, it contains no cholesterol thus making it 
a healthier food, in theory, than traditional livestock meat products. 
TABLE 2 
______________________________________ 
Proximate composition (% dry weight) 
of microbial biomass produced from cereal 
milling by-products utilizing N. sitophila 
Component Amount 
______________________________________ 
Crude Protein 30-60%* 
Total Dietary Fibre 40%* 
Fat 5% 
Cholesterol 0% 
______________________________________ 
*The amount of protein and total dietary fibre varies with the substrate 
used. 
The good quality of the microiial biomass product from this microorganism 
has been confirmed in feeding trials using rats. In these studies, final 
products from starch permeate and wet residue fermentations were evaluated 
for palatability and protein efficiency ratio against casein standard 
diets with encouraging results. 
In the process of the present invention, cereal milling by-products may be 
utilized in their existing form, or in the form of either a "wet residue", 
the combined "starch and permeate" or "total residue". Cereal milling 
by-products may be subjected to aqueous alkaline extraction in order to 
extract protein. The separation of the proteinaceous extract from the 
solid residue results in a "wet residue". Applying further physical 
separation techniques to the proteinaceous extract yields "starch" and a 
"permeate". The wet residue, the combined starch and permeate or a 
combination of the two: "total residue", represent three streams of the 
extraction process which may be subjected to fermentation with N. 
sitophila for the production of microbial biomass protein. The raw 
by-products of cereal milling prior to extraction (alone or combined with 
the extracts and residues) may also be subjected to fermentation by the 
same fungus. 
Feedstocks or streams which contain ligno-cellulosic materials require 
pretreatment prior to utilization by the microorganism. These streams are 
the total residue, wet residue or any cereal milling by-products which are 
not deproteinated. 
Pre-treatment of the wet residue with heat in the presence of alkali 
demonstrated an improved rate of bioconversion. In a lignocellulosic 
material such as wheat milling by-products, the ratio of cellulose to 
lignin is very important since the lignin content directly affects the 
availability of the cellulose. In typical structural and protective 
elements in plants, lignin impregnates the cellulose of the cell-wall, 
where it acts as a protective cement. In cereal milling by-product raw 
material, the ratio of cellulose to lignin was found to be 1:1 which is 
extremely high and indicates that any bioconversion process would be 
slowed. In the insoluble lignocellulosic residue (wet residue) remaining 
after deproteination, the cellulose to lignin ratio was found to be 2:1. 
Biodegradability of most lignocellulosic materials will increase if the 
lignin is either partially or fully removed. This may be achieved by the 
use of a caustic solution in conjunction with high temperatures Such 
pretreatment solublizes the lignin and swells the cellulose which allows 
for greater penetration of the cellulaze enzymes (present in the fungus) 
and henc greater degradation of the cellulose. 
In the process of the present invention pretreatment was carried out in a 
solution of 0.25-1% NaOH W/V and slurry concentrations of 7-10% w/v 
solids. The pretreatment temperature can range from 121.degree. C. to 
165.degree. C. under the appropriate pressure and retention times of 4 to 
30 minutes. The mixture is then diluted to an appropriate concentration 
for the required fermentation and the pH adjusted prior to sterilization. 
The drawing attached hereto as FIG. 1 is a flow chart which illustrates 
four embodiments of the present invention. Referring to the flow chart, 
cereal milling by-products 2 may be deproteinated by alkali extraction 4 
which may be followed by acid precipitation and filtration (or other means 
of isolation) to produce wet residue 6 and starch and permeate 
(hereinafter "starch permeate") 8. The starch permeate may be admixed with 
the wet residue to form a total residue 10. Alternatively, the process of 
the present invention may be applied to untreated cereal milling 
by-products as at 7. Four embodiments of the present process are reflected 
in Flow Chart 1 as the process streams, namely, wet residue, starch 
permeate, total residue and untreated by-products. Pretreatment 12 of the 
untreated by-products 7, the wet residue 6 or the total residue 10 as 
aforesaid will partially delignify the solid particulates therein. Table 3 
shows the utilization of cellulose in the wet residue as related to the 
amount of sodium hydroxide in the pretreatment. 
TABLE 3 
______________________________________ 
Utilization of Cellulose in Wet Residue by N. 
sitophila Under Pretreatment Conditions 
Containing Various Amounts of NaOH 
Cellulose Concentration in 
Level of NaOH 
Fermentation Broth g/L 
% Cellulose 
% w/v 0 Time 24 Hours Utilization 
______________________________________ 
0 1.60 1.45 9.4 
0.10 1.58 1.47 7.0 
0.25 1.49 0.94 37.0 
0.50 1.53 0.48 68.6 
*1.00 2.25 0.25 88.9 
______________________________________ 
Shake flask experiments. 
*Pilot scale 1,000 L fermentor. 
Following the pretreatment 12 an appropriate nutrient broth (basic nutrient 
composition) is added. The pretreated solids are supplemented with 
chemicals in the form of a nutrient broth 14 according to known standard 
procedures such that the available carbon, nitrogen, phosphorus and 
potassium are in appropriate ratios to form a fermentation medium or broth 
16. The solid-liquid slurry of cereal milling by-products used as a 
fermentation medium is diluted with water to a concentration in the range 
of 0.5% to 3.0% (w/v) solids and supplemented with the appropriate 
nutrients in a mixing tank. The nutrients may have the following 
composition per 1,000 litres of fermentation medium: Ammonium sulphate 
(NH.sub.4) SO.sub.4 :472 grams, Urea CO (NH.sub.2)2:856 grams, potassium 
phosphate monobasic KH.sub.2 PO.sub.4 : 2,000 grams, magnesium sulfate 
seven hydrate MgSO.sub.4. 7H.sub.2 O:200 grams, Calcium chloride 
CaCl.sub.2 : 200 grams, Zinc sulfate seven hydrate ZnSO.sub.4.7H.sub.2 
O:4.4 grams, ferric chloride six hydrate Fe Cl.sub.3.6H.sub.2 O:3.2 grams, 
Boric acid BH.sub.3 O.sub.3 :0.114 grams, Ammonium molybdate four hydrate 
(NH.sub.4).sub.6 Mo.sub.7 0.sub.24.4H.sub.2 O:0.489 grams, Cupric sulfate 
five hydrate CuSO.sub.4.5H.sub.2 O:0.789 grams, manganese chloride four 
hydrate:0.144 grams. These nutrients comprise the "basic nutrient 
composition" and may be used in full or in part, individual compounds may 
be varied or omitted, or additional ones may be used depending on the 
nature of the fermentation medium. The scope of such variation will be 
apparent to those skilled in the art. 
Nitrogen in the nutrient broth may be supplied as (NH.sub.4).sub.2 SO.sub.4 
and urea. In the case of wet residue, optimal nitrogen and substrate 
utilization occurs when the nitrogen content of the fermentation broth is 
50% to 75% (by weight) of the basic nutrient composition. Experimentation 
further indicated, that Neurospora sitophila grown on starch permeate 
requires at least 75% (by weight) nitrogen of the same composition for 
maximum growth rate and protein production. 
In the case of both wet residue and starch permeate, it was determined 
there is no requirement for added phosphorus. Growth at the zero addition 
level was not significantly different from growth in the full phosphorus 
treatment. Consequently, naturally occurring phosphorus in the substrate 
is adequate. 
Similarly, tests showed that trace elements present in the pretreated wet 
residue and starch permeate are sufficient to support the growth of 
Neurospora sitophila without supplementation. This is a further economic 
advantage present in the use of the present process. 
The mixture 16, after the addition of nutrients 14, is then adjusted to an 
appropriate pH and passed through batch mode sterilization (l21.degree. C. 
and necessary time) or a continuous sterilizer at approximately 
165.degree. C. with retention time of 0.25-3.0 minutes. It is then 
diverted into the fermentor vessel where the temperature of the medium 17 
is adjusted to 20.degree.-40.degree. C. and preferably 26.degree. C., and 
seeded with a precultivated inoculum 18 of N. sitophila. The level of the 
inoculum may vary from 3% to 10% of the fermentor working volume. Process 
conditions 20 include oxygen, agitation, temperature, pH and time. The 
mixture is subjected to special low-shear aeration-agitation conditions at 
an air supply of 0.5 to 1.0 VVM (Volume of air per Volume of medium per 
Minute) at a temperature 20.degree.-40.degree. C. and preferably 
26.degree. C. and a pH range of 5.5 to 7.5, depending on the type of 
by-product or stream to be fermented. The said fermentor vessel is 
equipped with sensors and controls for pH (acid or alkali addition), 
antifoam, oxygen consumption and carbon dioxide evolution. It is 
specifically designed to supply aeration and agitation which is conducive 
to the requirements of N. sitophila which was found to be shear sensitive. 
An air lift fermentor device is ideally suited. It has low local shear 
conditions combined with excellent mixing capabilities and has the added 
benefit of reduced power requirements for operation. Further, with no need 
for impellers, the risk of contamination through a physical failure of the 
drive shaft seals is greatly reduced insuring continuity of operation. 
The biomass of the fungus should be grown until the final product consists 
of over 50% of the fungus on a dry weight basis, or the fungus reaches a 
stationary growth phase. At this point harvesting will take place (batch 
fermentation) or continuous fermentation will be initiated. 
Once the fermentation is completed and a sufficient biomass of fungus has 
been grown, it may be isolated from the fermentation broth by filtration 
(or other suitable separation technique) as at 22. This produces a solid 
microbial biomass product 24 which thereafter may be dried by appropriate 
means known to those skilled in the art. 
The end fermentation product may be further processed as follows: The broth 
from the fermentor vessel can be removed by level control or a pumping 
device and concentrated by removal of the liquid by filtration, 
centrifugation or other means of separation. The liquid waste is discarded 
and the concentrated slurry of microbial biomass can be further treated by 
drying using various means; freeze dried preserved frozen or further wet 
processed, to fit its end use. 
Various experiments were carried out to optimize the systems and operating 
variables of the fermentation process. Table 4 below shows typical results 
of the process based on typical values of the following system variables: 
slurry concentration of the fermentation medium =2% weight per volume 
basis; temperature =26.degree. C.; pH =6.7; dissolved oxygen in the 
medium=50% of air saturation. The table reveals that for the two types of 
by-product residue (namely wet residue and total residue), microbial 
biomass in the range of 64 to 72% (dry weight basis) was formed by the 
process invention, from the original material which contained virtually no 
protein. Table 4 also reveals that comparable degrees of bioconversion in 
terms of microbial biomass composition of the product are obtained in 
relatively shorter fermentation periods when the process is conducted in 
relatively low-shear bioreactor devices of the air-lift type, than in the 
relatively high-shear bioreactor devices of the more conventional 
mechanically-stirred type. 
TABLE 4 
______________________________________ 
Microbial Biomass Production 
in Different Bioreactor Types 
Mechanically Stirred 
Bioreactor Air-Lift 
% Biomass Bioreactor 
(dry weight Time Time 
Residue Type 
basis) (hours) % Biomass (hours) 
______________________________________ 
Wet residue 
64 40 72 16 
Total residue 
72 28 -- -- 
______________________________________ 
The results of Table 5 below, further illustrate the beneficial effect of 
the low-shear conditions of the fermentation bioreactor in microbial 
biomass formation. In this case, the starch permeate liquid mixture was 
used to cultivate the fungus. 
TABLE 5 
______________________________________ 
Cultivation Times for Equal Conversion Levels 
of Starch Permeate Substrate to Microbial 
Biomass in Two Bioreactor Types 
TYPE TIME 
______________________________________ 
Mechanically stirred Bioreactor: 
31 hours 
Air-Lift Bioreactor: 18 hours 
______________________________________ 
The process of the present invention may be performed as a batch or 
continuous run process. In addition, the protein quality may be improved 
by genetic techniques dependent upon the presence of a sexual stage which 
Neurospora sitophila has as an ascomycete. The use of the optimal form of 
the fungus may further improve protein quality.

Illustrative Examples 
Example 1: Batch Fermentation of Total Residue: 
Following the general protocol described earlier, a fermentation was 
carried out using total residue as the substrate. The wet residue 
component of the total residue was pretreated at 10% (w/v) slurry at l2loC 
for 30 minutes with 1% (w/v) NaOH. The starch permeate and pretreated wet 
residue were then combined and added to a l5L fermentor (MBR Switzerland), 
so that the final concentration of solids was 2% w/v. Appropriate 
nutrients (described earlier) were then added in full and the pH of the 
mixture adjusted to 6.0. The medium was then sterilized in situ, at 
121.degree. C. for 15 minutes, cooled to 26.degree. C. and inoculated with 
precultivated N. sitophila at a level of 10% (v/v) of the fermentor 
working volume. The fermentor was equipped with pH control (addition of 
H.sub.2 SO.sub.4 or NaOH accordingly) and antifoam control. The 
temperature was maintained at 26.degree. C. and the dissolved oxygen at 
more than 50% of saturation by sparging air and agitation. Carbon dioxide, 
dissolved oxygen, pH and temperature were monitored throughout the 
fermentation. Samples of fermentation broth were taken at regular 
intervals and subjected to the following analysis: total solids, crude 
protein, reducing sugars cellulose and total carbohydrates. At the end of 
fermentation (36 hours) the broth contained 2.24 g/L protein; an increase 
from 0.50 g/L. The harvested broth was then concentrated by filtration and 
dried at temperatures of about 60.degree.-70.degree. C. The dried product 
contained 34% (dry weight) crude protein. 
Example 2: Batch Fermentation of Wet Residue Pilot Plant Scale: 
Fermentation was carried out in a 1,000 L pilot plant using wet residue as 
a substrate. The wet residue was pretreated as described in the previous 
example. Water was then added to the fermentor so that the medium reached 
a solids concentration of 1.6% w/v. A full compliment of nutrients was 
added and the pH of the broth was adjusted to 6.0. Sterilization was 
carried out in situ as described in the previous example, and following 
cooling to 26.degree. C., the medium was inoculated with precultivated N. 
sitophila using a 3% (v/v) level of inoculum. Parameters were monitored 
and controls were carried out as in the previous example. No mechanical 
agitation was carried out, but air was sparged from an annular ring at the 
base of the fermentor vessel at the rate of 0.8 VVM. 
The fermentation was completed in 46 hours and at termination, the protein 
level in the broth was 2.6 g/L. The broth was concentrated to 15 to 20% 
(w/v) solids using a centrifuge and dried to 4-10% moisture level at 
60.degree.-70.degree. C. The final product contained 32% w/v crude 
protein. 
Example 3: Continuous Fermentation in Pilot 
Scale Plant Using Starch Permeate 
The total available substrate for fermentation was (starch permeate 
mixture) 53.3l5kg solids in 5270 L of water (1.01% w/v). To further extend 
the fermentation time, the fermentor was operated below capacity at 790 L 
which enabled 7 volumes to pass through it and thus achieve steady state 
conditions. 
Operating conditions for the fermentor were as follows: air supply 0.8 VVM, 
pH 6.7, temperature 26.degree. C. and dilution rate 0.235 h.sup.-1. The 
continuous sterilizer was operated at 165.degree. C. with a 2.08 minute 
retention time. The temperature of the feedstock at the fermentor inlet 
head was 35.degree. C. Additional cooling was required from the fermentor 
cooling coils during the entire fermentation. 
Harvest of the microbial biomass protein was accomplished by feeding a 
continuous centrifuge (10,000 rpm) from a Moyno (trade mark) pump at the 
desired rate. Immediately after harvesting, the microbial biomass protein 
product was frozen. The frozen material was later oven dried at 60.degree. 
C. and then ground. 
Samples (750 ml) of the fermentor broth were taken periodically and 
analyzed for total dry weight, crude protein, soluble nitrogen and soluble 
carbohydrates. 
After an initial batch time period of 15 h, continuous operation was 
started and maintained for 25.5. h. The average protein content was 48% 
(dry weight) and the protein concentration was in excess of 1.6 g/L. The 
level of nitrogen in the effluent indicated that the amount supplied was 
in excess of the amount required. After the third volume had passed 
through the fermentor, there was little fluctuation in the nitrogen level 
of the effluent. This indicated that steady state conditions existed in 
the fermentor. This was verified by both the dissolved oxygen in the 
fermentor broth and the CO.sub.2 in the effluent gas which did not vary 
after the third volume. 
The harvested material, with a moisture content of 85% was dried at 
60.degree. C. to avoid damage to the protein. The final product yield was 
16.763 kg (16.713 kg +0.05 kg sampled) of microbial biomass protein from 
53.315 kg substrate or 0.31. 
Example 4: Continuous Fermentation Pilot 
Plant Scale Using Wet Residue 
This fermentation utilized wet residue in the amount of 604.4 kg containing 
29.8% solids (180.1 kg). This material was adequate to run 9 volumes 
through the fermentor on a continuous basis at 2% solids (w/v). This mass 
of material was pretreated in a caustic solution to solubilize the lignin 
and increase cellulose availability. To pretreat the substrate it was 
diluted to 7.5% solids (w/v) with 1% NaOH solution (1795.6 L) and run 
through the continuous sterilizer at l65.degree. C. with a retention time 
of 4.5 minutes. The pretreated wet residue was stored in polyethylene 
tanks at pH 12 until it was required for fermentation. The pretreated wet 
residue was transferred into a suitable vessel and diluted with water to 
contain 2% solids w/v. A full compliment of nutrients was added with the 
exception of nitrogen and phosphorus, which were adjusted to 75% and 10% 
of their original values respectively. The pH was adjusted to 6.0 and the 
slurry passed through the continuous sterilizer at 165.degree. C. with 
retention time of 2.05 minutes. Following an initial 24 hour batch 
operation, subsequent volumes of medium were prepared as above and the 
fermentor was fed in a continuous mode at a 1,000 L operating level, for a 
total of nine volumes. 
Operating conditions for the fermentor were as follows: air supply 0.8 VVM, 
temperature 26.degree. C. and dilution rate 120 L/h. The continuous 
sterilizer operated at 165.degree. C. with a 2.05 minute retention time. 
The feedstock temperature was 40.degree. C. at the fermentor head. 
Dissolved oxygen and CO2 in the effluent gas were monitored throughout the 
experiment. 
Harvest of the microbial biomass protein was accomplished using a 
continuous centrifuge (10,000 rpm). Immediately after harvesting, the 
product was frozen. The frozen material was either oven dried at 
60.degree. C. and ground or it was thawed and spray dried. 
Samples (750 mL) of the fermentor broth were taken periodically and 
analyzed for total dry weight, crude protein, soluble nitrogen and total 
carbohydrates. 
Continuous mode was initiated after 24 h of batch growth during which time 
.sup.u max was calculated to be 0.23lh.sup.-1. The percent protein peaked 
at 40% (2.5 g/L) dry weight eight hours after continuous operation began 
and dwindled to 32% (2 g/L) by the end of the run. The level of residual 
cellulose was at a minimum after 24 h and although it remained low for the 
duration of the experiment, there was an upward trend that matched the 
decline in protein. 
Measurements of total carbohydrates and cellulose indicated that the only 
residual carbohydrate in the solid matter was cellulose and that an 
average, 86-90% of it was utilized during the continuous phase. 
The total mass of microbial biomass protein product generated was estimated 
at 63 kg (dry). This was derived from 180 kg (dry) of substrate for a 
product yield of 0.35, containing approximately 40% (w/w) crude protein. 
Variations of the process described herein and advantages of the present 
invention, beyond those mentioned herein, will be apparent to those 
skilled in the art.