Fermentation of Cellulose

This study is concerned primarily with two aspects of submerged fermentation of solid cellulose1 the effects of foaming and pure oxygen utilization (in place of air) upon growth and enzyme production of the organism T. viride QM9414. Pure oxygen flow rates are varied, and growth and enzyme production data are compared to results obtained during air fermentation runs (with flow rates five times those of pure oxygen for comparison purposes and still to meet oxygen requirements of the organism for growth). ' At an initial cellulose concentration of 1.0% for all fermentation runs, aeration with pure oxygen resulted in an enhancing effect upon enzyme production rate (about 1.5-2 times as fast as with air). It appeared that better oxygen utilization was associated with the pure oxygen fermentations as indicated by slightly lower average DO levels. This was thought to be attributed to better oxygen mass transfer during the oxygen fermentations, which would favor protein synthesis. Foaming was a problem common to both air and oxygen systems, but more so to air runs, especially during the first J-4 days. It was determined that enzyme was present in the foam, which would account in part for lower levels in solution. Cell autolysis, which favors good enzyme production, occurred at earlier times during the oxygen fermentations, which would also help to explain better enzyme production rates with pure

QM9414. Pure oxygen flow rates are varied, and growth and enzyme production data are compared to results obtained during air fermentation runs (with flow rates five times those of pure oxygen for comparison purposes and still to meet oxygen requirements of the organism for growth).

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At an initial cellulose concentration of 1.0% for all fermentation runs, aeration with pure oxygen resulted in an enhancing effect upon enzyme production rate (about 1.5-2 times as fast as with air). It appeared that better oxygen utilization was associated with the pure oxygen fermentations as indicated by slightly lower average DO levels. This was thought to be attributed to better oxygen mass transfer during the oxygen fermentations, which would favor protein synthesis. Foaming was a problem common to both air and oxygen systems, but more so to air runs, especially during the first J-4 days. It was determined that enzyme was present in the foam, which would account in part for lower levels in solution. Cell autolysis, which favors good enzyme production, occurred at earlier times during the oxygen fermentations, which would also help to explain better enzyme production rates with pure    A persistent problem of importance and concern to both the scientific community and general public is world food provision. Although much scientific research has been devoted to alleviating this problem, it is only recently that cellulose, a traditional waste material, has been recognized as a potential source of foodstuffs. Cellulose is the most abundant carbohydrate in the plant kingdom, and comprises between 40-60% of all the waste generated annually in the United States (1). In view of the plentiful existence 6f this compound, it would seem both fruitful and imperative that we expand our efforts toward reprocessing and developing new uses for cellulose. The development of technology to utilize waste cellulose as a fermentation or hydrolysis substrate would afford several opportunities1 it could help mitigate the waste problem, provide a valuable raw material supply, and aid in providing potential food products for an ever increasing world population {2,J,4,5). Figure 1 schematically depicts some of the potential methods of cellulose utilization.
Degradation of cellulose, aside from being investigated for possible future industrial applications, is of great importance to most all living systems in nature.
Microorganisms are capable of degrading cellulose into soluble oligosaccharides and monosaccharides, which are then recycled as nutrients (6). Furthermore, animals such as horses, cows, goats, and rabbits, as well as certain insects, also possess the ability to digest cellulose to some extent (7).
Of particular concern here are methods of cellulose degradation suitable for industrial applications. Acid hydrolysis, oxidation, thermal degradation, visible or u.v. radiative destruction, pyrolysis, alcoholysi~, and acetolysis have been attempted in the past, but have exhibited several drawbacks. Unfortunately, these reactions typically produce so great a diversity of products that elaborate separation techniques are required. Furthermore, many of the above reactions depend upon rather harsh reaction conditions for their success, and thus would directly increase energy requirements. Enzymatic degradation, on the other hand, is more specific in attack, would result in fewer undesired products, and at the same time, could be carried out under milder reaction conditions (8).
One problem with microbial degradation of cellulose is the lengthy time required for hydrolysis. Any improvement in enzyme production rate and yield would brighten the economic outlook of the process (9). Because of the highly crystalline complex nature and insolubility of the cellulose substrate, intimate contact between enzyme and substrate is impeded. This contact, which is required for successful hydrolysis, can only be achieved by diffusion of J the extracellular enzymes from the organisms into the highly resistant cellulose matrix (10). Such substrate characteristics have prompted investigation into possible pretreatment of the cellulosic material to produce a more reactive cellulose (11). It has been suggested that because of the foaming problem associated with cellulose fermentation, there is the possibility of the enzyme complex being swept into the foam with subsequent deactivation resulting in reduced enzyme levels for substrate degradation ' in the liquid phase (12).
In light of the latter information, this study sought to investigate the effect of foaming upon enzyme production, with the intention of controlling foam levels to enhance enzyme levels. To reduce foam levels pure oxygen was substituted for air. Oxygen requirements for organism growth were satisfied by maintaining pure oxygen flow rates at one fifth the value of air, Thus, the effects of pure oxygen upon enzyme productivity rates were also demonstrated, The variables of interest which affect organism growth rate and its production of the desired enzyme system were temperature, agitation, pH, dissolved oxygen level, and pure oxygen flow rate. Although many of the variables have yet to be optimized (12), some parameter operation ranges have been proposed and have resulted in better enzyme yields (lJ).
Other approaches to the problem have been suggested by Sternberg (lJ) and M andels (14). These include generation and isolation of hyperproducing mutant strains of organisms by irradiation, and additional work in optimization of the fermentation medium. However, approaches fall more naturally into the realm of microbiology. Finally, it was also the intent of this study to determine whether fermentation data obtained here could be more properly described by one of the few existing kinetic models for enzymatic degradation such as "tilat of Suga, Van Dedem, and  During enzymatic hydrolysis, the susceptibility of the cellulose substrate to enzyme attack is largely de-J>endent upon the accessibility of the extracellular yrne s secreted by or bound to the microorganisms. Inenz . te contact is mandatory for effective hydrolysis. t 1ma Because cellulose is an insoluble structurally complex substrate , it is necessary for the cellulase enzymes to diffuse through the cellulose matrix to achieve the nece ssary contact between enzyme and substrate. Accordingly, there are certain structural influences which will affect this diffusion process and the susceptibility of the cellulose substrate to attack. The moisture content of the fiber is significant because of the possible swelling effec t which opens the pathway for enzymes, and also acts as a medium for transport. The size and diffusivity of Additional irradiation of this strain produced a second mutant strain, QM9414, capable of even greater enzyme ievels (1,35), For this reason, this latter strain has been selected for this present study. T. viride has very simple growth requirements and will produce cellulase in a solution of nutrient salts with no special additives.
Increased yields of enzyme are evident if small quantities of soluble carbon and nitrogen sources are added to such a solution. Nitrogen containing compounds, such as peptone and other protein derivatives, have proven more effective than non-nitrogeneous substances with regard to ~nhancement of enzyme yields. Cellulose substrate concentrations between .5 and 1.0%, together with peptone concentrations in the .1 to .2% (13) range seem to yield optimum enzyme quantities, while peptone concentrations greater than .5% are known to be inhibitory to enzyme production (J4). Surfactants have also been added in the .05 to .2% concentration range and have proven successful. · Increased yields of enzyme of over 50% were noted with the addition of a surfactant, Tween 80 (polyoxyethylene sorbitan mono-oleate) (J?). The enhancing mechanism of the Tween 80 is presently not well understood a however, it could be related to the increased permeability of the cell membrane which would allow more rapid secretion of the enzyme system necessary for the increased enzyme synthesis (lJ).
Because of the difficulty associated with the breakdown of native cellulose through the production lJ of the enzyme cellulase, it has been proposed that the cellulase enzyme system is multicomponent in nature (JS).
The enzyme scheme is represented in the following diagrama b-l endo-/3 -1-4 glucanases which preferentially attack internal portions of the cellulose linkages.
The cellobiose is subsequently hydrolyzed to glucose units by the action of the S -glucosidase enzyme compont Although the action of the ~~glucosidase enzyme is en • highly specific for cellobiose, it is capable of hydrolyzing some of the otner oligomers derived from cellulose.
A precise description as to the actual kinetics of the T. viride-cellulase system is not completely known at this ti~e. M uch ambiguity st~ll lies in the exact mechanism of the cellulose hydrolysis. There are several theories which approach the problem with limiting assumptions and simplifications to the model (13,16,17,41,42). While important questions still remain to be answered in order to fully understand T. viride-cellulose kinetics of cellulase ~roduction, these simplified theories do help to point out and explain the existence of certain phenomena during cellulose degradation.
In the T. viride-cellulose fermentation scheme, the enzyme production phase is coincidental with the growth or trophophase (lJ). This can readily be seen upon examination of the reduction in dry weight versus enzyme production curves. While this trend differs from many systems where the metabolites and enzymes are produced in the idiophase which lags behind the growth phase, this phenomenon can be rationalized on the basis that, because of the insolubility of the substrate cellulose, the activity of the cellulase enzyme system is lower than the other glucosidases which hydrolze soluble substrates.
Relatively large amounts of enzyme are required to liberate metabolizable sugar from cellulose for growth. Thus, cellulase production and growth are tightly coupled. although it has been suggested that the cell will compensate somewhat for the diffusional resistnace by increasing either the quantity of permease or internal enzyme systems involved with glucose metabolism, Generally, the lower the pH (or greater the hydrogen ion concentration), the slower the metabolic activity with relatively little effect upon the stoichiometry or detrimental effect upon cell structure (41).
There are several detectable phenomena which occur during _the fermentation operation which undoubtedly have some effect upon cellulase production rate. Huang has observed that adsorption of the cellulase system can occur rapidly onto the cellulose substrate during the initial st!ges of the reaction (16). As the reaction proceeds, the enzyme is subsequently released back into the liquid phase because of lesser availability of the substrate. The entire scheme is indicated by an initial drop in protein concentration followed by a gradual increase as the reaction proceeds. In a similar manner, soluble sugars such as cellobiose and glucose are also capable of being adsorbed onto the cellulose substrate material. This woul. d consequently reduce hydrolysis rates because of the blocking effect of the sugar, preventing the necessary intimate contact of substrate and enzyme.
It has been determined in the hydrolysis reaction that the true inducers of cellulase for a fungus growing upon cellulose are the soluble products of hydrolysis, specifically cellobiose (44). However, cellobiose can also be instrumental in repressing cellulase formation if present in excessive amounts, usually .5-1.0%. The following diagram is useful in demonstrating the complex role of cellobiose (45)r This dual nature is also true of other rapidly metabo• lized carbon sources such as glucose and glycerol, This "catabolic repression" (sometimes referred to as the "glucose effect"), occurs when the rate of carbohydrate catabolism exceeds that required for growth.
The soluble sugars become plentiful, and the organism preferentially utilizes them for growth. Cellulase synthesis then decreases or stops (13,44,45), It has also been reported that product inhibition of cellobiose and glucose combining with the enzyme system to form inactive complexes is also possible (16).
The fermentation operation has been characterized by Mandels (J4) as havimg certain reproducible trends.
The growth requirements of the organism are simple, and when grown in an agitated vessel produces threadlike mycelium whose large surface area would be desirable for growth scale-up or continuous cultivation. There is an initial lag phase which generally continues from 12 to 24 hours, after which the pH rapidly drops with the pro- tions (JJ). Vacuum evaporation and ultrafiltration have been utilized to concentrate enzyme levels in culture filtrates to as much as 4 to 8-fold (46,48 to use because of an initial moisture content of nearly 5%. The particle size distribution was 149 -19.4%, 149 -80.5%, and 53 -11.6% (49).

Medium
The fermentation medium was that proposed by Mandels and Weber (J4) with a slight modification in replacing urea with dibasic ammonium phosphate which was reported to further enhance cellulase production (50). Bacto-Peptone was added to the above mineral solution at a value of 10% of the substrate level because of its reported beneficial effect upon cellulase complex yields ()4). Prior to sterilization the pH of the medium was 5-5.5.   NaOH was then sprayed onto the pads to wash off any remaining solids from the filter paper. 6. All samples were run in duplicate, and results were reported in mg/ml of glucose equivalent.

Cellulase Activity
The assay for enzyme activity was the filter paper

IV. Submerged Fermentation Results
The variables of interest and results of six batch fermentations are given in table 1.
This study was conducted using 1% cellulose for all the runs because of reported success in obtaining good enzyme yields (34). pH was allowed to fall from initial values of nearly five to values of about 2,8-3.0 before pH control was implemented to maintain the pH between 2.8-3,4 for good cellulase yields (lG). DO levels were on the average lower in the pure oxygen runs than in the air runs as shown in figure 6 and 7, Since agitation rate was constant in all runs, DO level differences were primarily dependent upon gas flow rate and rate of uptake by the organism. In all cases, the initial rapid decreases in DO level were attributed to the unrestricted growth of the microorganism. All runs were were carried out at temperatures between 28.5-30 c.      Foam levels, while not actually measured because of the lack of uniformity in foam height, were observed and qualitatively noted, Foam level comparisons between either pure oxygen or air runs alone were inconclusive.
However, it appeared that during the first J or 4 days of the runs, the foam levels produced by air sparging were higher (touching the head plate of the fermenter, about 6 inches from the liquid level) than those produced by pure oxygen sparging (on the average, 1.5 to 2. 0 inches)\,

v, Submerged Fermentation-Discussion
The DO aata presented in figures 6 and 7 show that avera~e DO levels for the air runs were slightly higher than those runs with oxygen, This suggests no oxygen limitation upon either air or oxygen systems, Growth patterns were similar, and fungal tissue amounts were in the same ' numerical range as demonstrated in figure   14, Cellulase activity however was unquestionably higher in oxyg~n runs (see fig, 15) which suggests an enhancing effect upon enzyme production by pure oxygen bubbles.
Although oxygen may have been available for utilization by the organism in both systems, it appears that the effective use of oxygen is in question. This implies that the oxygen transfer mechanism is more eff icient for enzyme production using a pure oxygen sparging system, which is not unreasonable when separately considering the transfer possibilities of the oxygen from oxygen and air bubbles. It is quite possible that the nitrogen contained within the air bubbles acts as a resistance to the transfer of oxygen from the inner Portion of the bubble to the medium. This would explain why DO levels were slightly higher in the air runs, although actual oxygen utilization by the organism was less efficient.
Increased enzyme levels with oxygen could further be explained in light of the previous information. In considering protein synthesis alone, we know that the energy rich compound ATP (adenosine-5'-triphosphate) is required. Furthermore, it is known that to furnish the necessary ATP for protein synthesis, oxidative reactions must occur which require molecular oxygen for their successful completion. If the oxygen were made more availab~ for these protein synthesis reactions, increased protein levels and consequently increased enzyme levels should be observed.
Foaming was a problem associated with both air and oxygen fermentations, although it appeared more evident in the air runs during the first three to four days.
This was in part due to the higher air flow rates.
While it was difficult to determine whether or not the enzyme was being lost by gas fractionation and subsequently inactivated by oxygen at the gas-liquid interface (54), it was evident from several enzyme determinations on foam samoles that enzyme was being swept up into the foam during the reaction. This phenomenon was particularly prevalent during the first three to four days when foam levels were highest in both systems.
Higher foam levels were observed during these periods from air runs (see results section), which would imply that a greater quantity of enzyme was being entrapped within the foam at any given sampling time. This would help to explain lower levels of enzyme present in the liquid medium during the air runs.
Another concept relating air content in foam to viscosity reported by Punton (55) assists in explaining higher enzyme yields in the oxygen runs. In figure 2J viscosity is plotted against air content of the foam

VI. Submerged Fermentation-Conclusions
The effect of pure oxygen upon growth and enzyme production for the T. viride-cellulose submerged fermentation system was studied at various gas flow rates and compared to results obtained during air control runs.
It appears, from comparing enzyme production curves from both air and oxygen runs, that oxygen does have an enhancing effect upon enzyme production rate. Reasons for this phen~menon are tangible in some instances, and somewhat speculative in others.
Better utilization of oxygen by the organism in oxygen runs seems to be indicated by lower average DO levels (see figures 6 and ?). This would help to substantiate the concept of differing oxygen mass transfer mechanisms between air and oxygen fermentations, whether this was primarily due to an added gas resistance within the bubbles, or viscosity differences between ~he different fermentation media. With better oxygen utilization, increased protein and therefore enzyme production would be expected because of a greater number of ATP molecules made available for protein synthesis.
Although enzyme test results upon several foam samples indicated that gas fractionation of the enzyme was not occurring, it would be possible for some of the enzyme to be inactivated as it's swept up into the foam (58). This phenomenon would be manifested by lower enzyme activity in the foam than in the liquid phase. In any event, the higher foam levels associated with air runs would mean that greater amounts of enzyme would be contained within these foams at any particular sampling time, Consequently, enzyme levels in the medium would be reduced, This effect seemed to be partially responsible for the lower enzyme activity during the first three to four days.
Diffe~ences in the morphology of the organism during growth in the two systems would provide a possible explanation, involving surface area of the organism available for secretion of the cellulase enzyme system, for better enzyme yields wit.h ·1oxygen. These morphological differences would first need to be ascertained, However• nfrom the growth data accumulated in this study, it was apparent that the cell autolysis which occurred earlier during oxygen runs was in part responsible for better enzyme production in the latter stages of these runs ( 54).
Finally, the catabolic repression role of glucose and cellobiose seemed to be in evidence here, specifically during the initial stages of both air runs. This helps to account for lower enzyme production during these times.  .0600 • 0750 • 0700 • 0450 .0350