producton of single-cell protein from cellulose by aspergillus terreus

Producton of Single-Cell Protein from Cellulose by AspelgiZZus Terreus

T. FERRELL MILLER* and V. R. SRINIVASAN, Department of Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803

summary

Cellulose fermentation studies were conducted with a thermotolerant strain of Aspergillus terreus. Batch cultivation of A. terreus using purified or complex cellulose showed that 8O-88% of the available cellulose was utilized in 30-36 h with an average doubling time of 7.5-8.3 h. The protein content in the biomass ranged from 23 to 38%. Semicontinuous cultivation studies, in which 90% of the biomass was withdrawn at the end of the growth cycle, indicated that 84% of added cellulose was utilized with the biomass containing 32% crude protein. No loss in cellulose consumption, growth rate, or protein production occurred through two growth cycles. Con- tinuous cultivation of A. terre-us showed that 78-84% cellulose consumption occurred over growth temperatures ranging from 35 to 45°C. Maximum specific growth rates (0.14 h-') occurred at 40 and 45°C with a minimum doubling time of 4.9 h.

INTRODUCTION

Cellulose is the most abundant renewable biopolymer produced in the world. The tremendous volume of cellulosic products produced by modern so- cieties, and subsequently generated as wastes, causes serious disposal prob- lems. Estimates have shown that one-half of the total solid waste generated by America each year are cellulosic in nature and amount to over 500 million tons.' Production of urban wastes has been quantitated on an individual basis as two to three pounds of cellulosic biodegradables per person per day.2

Microbial fermentation has been studied as a possible solution for disposal of these wastes as well as a means of generating valuable by-products such as food or feed supplements. With worldwide increases in population and chronic food shortages, the bioconversion of cellulose into a highly proteinaceous product (single cell protein or SCP) is an attractive approach for providing food for humans or animals. We report in this article the results of fermentation studies conducted with a thermotolerant, cellulolytic strain of Aspergillus terreus usin purified or complex cellulose as sole carbon source.

*Present address: Diamond Shamrock Corporation, Painesville, OH 44077.

Biotechnologyand Bioengineering Vol. XXV. Pp, 1509-1519 (1983)

1510 MILLER AND SRINIVASAN

MATERIALS AND METHODS

Organism

A fungal isolate identified as Aspergillus terreus (ATCC 20514) was used in all experiments. The organism was maintained on potato dextrose agar (PDA) slants and transferred monthly.

Inoculum

Spore suspensions were prepared from PDA plates containing well- sporulated mycelia. Several drops of 1 % Tween 80 were added as a wetting agent to obtain uniform suspensions. The suspensions were stored in sterile screw cap tubes at 4°C. Spore inocula were added at a concentration of 1-2 mL/L of growth medium.

In experiments requiring a more purified spore suspension, crude spore suspensions were filtered through a sterile sintered glass funnel (coarse) to remove mycelial filaments and collected on millipore filters (0.22 pm Type GS, Millipore Corp.) Spores were washed three times with sterile water, re- suspended in sterile water, and stored at 4°C. Viable counts of purified spore suspensions were determined by plating on PDA containing 0.08% sodium deoxycholate.

Medium Composition

The growth medium employed in cultivation of the fungus was developed from medium optimization studies where A. terreus was grown in continuous culture under glucose limitation. These studies were carried out using a modification of the method of Mateles and B a t t a ~ ~ The composition of the medium is shown in Table I . Each nutrient was proportionately changed with

TABLE I Nutrients Used in the Experiments

Nutrient Amount (g/L)

Cellulose or glucos (NHd)2SO4 KlHPOd NaH2P04 . H 2 0 MgC12.6H20 CaCIz.2H20 FeS04. 7Hz0 ZnS04.7H20 MnClz.4H20 CoC12 .6H20 CUSO, .5H,O

;e 10.0 2.5 4.65 X lo- ' 1.55 X lo-' 2.50 X lo- ' 2.0 x 10-2 5.0 x 1 0 - ~ 2.4 x 1 0 - ~ 6.0 X 10K5 4.0 X 1.0 x l o r h

SINGLE CELL PROTEIN PRODUCTION FROM CELLULOSE 1511

alteration of the concentration of the carbohydrate source. All experiments were conducted at pH 4.

Substrate and Pretreatment

Solka-Floc (Brown Paper Company) or sugar-cane bagasse was used as the cellulose source in all experiments. Each cellulose source was initially ground in a Wiley Mill to a 1-mm particle size and then alkali treated. Alkali treat- ment involved autoclaving 50 g cellulose suspended in 1 L of W sodium hydroxide for 15 min at 121°C. The swollen fibers were filtered on a sintered glass funnel (coarse), washed with distilled water to remove the alkali, and stored in distilled water as a slurry. The slurries were sterilized and added to the fermentor in bulk quantities.

Culture Conditions

Batch culture (glucose)

Specific growth rates of glucose-grown A. terreus were measured by grow- ing the organism on 0.5% glucose-salts medium. Sterile medium was dis- pensed in 10-mL aliquots into 50-mL Erlenmeyer flasks. Each flask was in- oculated with (1-5) X lo6 spores and incubated at the desired temperature in a water-batch shaker (New Brunswick Scientific Company). -The agitation rate was 200 rpm. Duplicate flasks were removed from the shaker at various times after inoculation. The mycelium was collected on tared sintered glass funnels, washed, and dried to constant weight at 90°C. From the growth- curve data, maximum specific growth rates were determined.

Batch culture (cellulose)

Batch culture experiments were carried out in a 7-L continuous culture ap- paratus (New Brunswick Scientific Company) having suitable temperature, agitation, pH, and aeration controls. Growth studies were carried out using the gradient feed technique of Srinivasan, Fleenor, and summer^.^ Gra- dients were constructed such that nutrients were provided for a final volume of 4.5 L containing 10 g cellulose/L. The total volume of the gradients pumped into the fermentor was 1 L.

The starter culture was grown in 1 L of 0.5% Solka-Floc/salts medium containing 0.05% yeast extract at 35°C for 36 h. The culture was filtered into a sterile 350-mL sintered glass funnel (coarse). The mycelial pad was aseptic- ally removed and suspended in 500 mL sterile distilled water. The suspension was then added to the fermentor containing 2.0 L of 0.05% Solka-floc/salts (based on 4.5 L) medium. Sterile slurries were added to the fermentor at prescribed times during the fermentation. The total combined volume of the cellulose slurries was approximately 1 L.

After equilibration of the inoculum, duplicate 50-mL samples were withdrawn, filtered, and washed. The biomass pad was analyzed for cellulose and


protein content. In order to obtain representative samples from the fermentor, approximately 50 mL of culture fluid was withdrawn and discarded before samples were taken. The agitation rate, initially set at 400 rpm, was increased to 600 rpm during the latter stages of the fermentation. The aeration rate was gradually increased as the substrate level increased. Aeration rates ranged from 1.1 to 3.1 v/v/m. The growth temperature varied with the experiment and ranged from 35 to 45°C. The pH was held constant at pH 4 k 0.1 with sterile 0.5N sodium hydroxide. Biomass, collecting on fermentor walls, was scraped into the fermentor using a sterile glass rod. At the end of the fermentation period, duplicate 100-mL samples were withdrawn for dryweight analysis. Triplicate 50-mL samples were collected and assayed for protein and cellulose content. The final volume of the fermentor was also measured. Specific growth rates were calculated from the relative increase in biomass protein over the entire growth cycle.

When pretreated bagasse was used as substrate, the loading schedule was based on the fact that bagasse was composed of 35% cellulose. Bagasse slurries were made up such that a final cellulose concentration of 10 g/L was achieved.

Semicontinuous Culture

Semicontinuous cultivaton studies were conducted in a similar fashion as in batch culture. However, at the end of the initial fermentation cycle, 90% of the biomass was harvested with the remaining 10% serving as the inoculum for the next cycle. The sampling schedule was similar to procedures described in the batch culture case.

Continuous Culture

Continuous cultivation of cellulose-grown A. terreus was carried out in a seven liter stirred tank fermentor with a 5-L working volume. The influent cellulose concentration was 1.5 g/L and was discontinuously pumped to avoid clogging of the feed lines. The substrate was maintained in a 10-L car- boy and kept in a uniform suspension by continuous stirring with a magnetic stir bar. The agitation rate was 400 rpm while the aeration rate was held constant at 1.2 v/v/m. The growth temperature varied with the experiment and ranged from 35 to 45°C. The pH was held constant at 4 k 0.1 with 0.2N sodium hydroxide. The myceiia collecting on the walls of the fermentor was routinely scraped into the fermentor twice daily with a sterile glass rod.

The starter culture was grown in 1 L 0.5% Solka-Floc/salts medium containing 0.05% yeast extract for 36 h at 35°C. The culture was added to the fermentor which contained 4 L of 0.05% Solka-Floc/salts medium. The flow rate was initially set at a 15-h residence time and the culture allowed to equilibrate until microscopic examination of the culture fluid showed that maximum cellulose utilization had occurred. The flow rate was then increased in a step-wise manner until the desired growth rate was reached. The culture


was allowed to reach steady state for six to eight residence times. Duplicate 50-mL samples were then withdrawn for analysis of dry weight, residual cellulose, and biomass protein content.

Assays

True protein

Test samples were filtered on sintered glass funnels (coarse). The biomass pad was washed three times with distilled water. The washed mycelial pads were extracted three times in 5 mL of sodium hydroxide in a boiling water bath. A final extraction was made with 5 mL distilled water. Suitable dilu- tions of these extracts were measured individually or after being pooled for true protein content. Protein content was measured by the method of Lowry et aL5 Bovine serum albumin (Sigma Chemical Co.) was used as the protein standard.

Crude protein

Crude protein was measured by the Kjeldahl method of total nitrogen analysis as described by Hiller, Plazin, and Van Slyke.6 In experiments where sugar cane bagasse was used as substrate, test samples were extracted as indicated in true protein analysis. The extracts were pooled, analyzed for total nitrogen content, and converted to crude protein values (N X 6.25). Crude protein was also calculated from true protein values by multiplying by a calibration factor (1.54). The factor was determined from data obtained during continuous cultivation studies of glucose-limited A . terreus.

Cellulose Analysis

Cellulose was measured by the method of Updegraff.' Solka-Floc was used as the cellulose standard.

Dry Weight Analysis

Samples collected for dry weight determination were filtered on sintered glass funnels (coarse) and washed three times with distilled water. The washed biomass was placed in tared aluminum weighing pans and dried to constant weight at 90°C.

RESULTS

Early studies on the effects of temperature on the growth response of A . terreus indicated that this organism grew over a wide range of temperatures. A summary of these experiments with glucose-grown fungi is shown in Figure 1. The optimal growth temperature was 35OC (pLmax = 0.35 h-*) although specific growth rates greater than 0.20 h-' were found for the entire


I I I I

SP. GROWTH o.30

0.20

0.10

30 35 40 45

TEMP. (C ) Fig. 1. Effect of temperature on the specific growth rate of glucose-grown Aspergillus terreus

in batch culture.

temperature range (30-45°C) tested. Such rapid growth rates at elevated temperatures indicate that this strain of A. terreus is thermotolerant.

Specific growth rates of A. terreus, when grown on purified or complex cellulose in batch culture, are shown in Table 11. At 35"C, A. terreus grew at an average doubling time of 7.5 h ( p = 0.093 h-l), (Table I1 experiment 2). When the growth temperature was increased to 45"C, the growth rate was somewhat slower with an average doubling time of 8.1 h, (Table 11, experiment 3) . Bagasse-grown A. terreus exhibited a 10% slower growth rate than cultures grown on purified cellulose at the same temperature (Table 11, experiments 2 and 4).

Analysis of the dried product for cellulose and protein content from fer-

TABLE I1 Specific Growth Rates of Cellulose-Grown Aspergillus terreus in Batch Culture

Duration of Specific Doubling Cellulose Temperature fermentation growth rate time

Experiment conc. (g/L) ("C) (h) 0 - l ) (h)

1 10 35 48 0.057 12.20 2 10 35 30 0.093 7.45 3 10 45 30 0.086 8.10 4 10 35 36 0.084 8.25

Experiments 1, 2, and 3 were conducted using alkali-treated Solka-Floc as the cellulose source. Alkali-treated bagasse was used as the cellulose source in experiment 4. Bagasse contains 35% cellulose on a dry weight basis and the loading of bagasse into the fermentor was based on this value.


mentation studies described in Table I1 are shown in Table 111. Comparison of experiments 1 and 2 indicates that extension of the fermentation time from 30 to 48 h resulted in only a 6% increase in cellulose consumption. Results showed that 88% of the added cellulose was utilized in 30 h, (Table 111, experiment 2). At 45"C, over 73% of the available cellulose was utilized, ap- parently a result of slower growth rates noted in Table 11, (Tables I1 and 111, experiments 3). Analysis of residual cellulose in bagasse-grown cultures indicated that 80% of the available cellulose was utilized but required somewhat longer fermentation periods (36 h). The protein content of the dried biomass ranged from 23 to 38%.

Results from semicontinuous cultivation of cellulose-grown A. terreus are summarized in Tables IV and V. Comparsion of average doubling times in batch (Table IV, experiment 1) or semicontinuous cultivatioa modes (Table IV, experiments 2 and 3), indicated that doubling times were relatively constant ranging from 7.3 to 7.6 h.

Analysis of cellulose and protein content of biomass produced from these fermentations indicated little difference in protein content or cellulose consumption even though the time of fermentation was decreased by 20% in the semicontinuous mode (Table V, experiments 2 and 3). Approximately 84% of added cellulose was utilized with the protein content ranging from 30 to 33%.

Data obtained from continuous cultivation experiments are summarized in Tables VI and VII. Cellulose utilization values ranged from 78 to 84% over a temperature range of 35-45OC. At 40 and 45OC, the maximum specific growth rate achieved was 0.14 h-' (4.9 h doubling time). Attempts to achieve steady state at higher specific growth rates resulted in rapid washout of the culture.

Table VII shows results of protein analysis of the dried biomass produced from fermentation experiments described in Table V. The crude protein con-

TABLE 111 Utilization of Cellulose and Protein Content of the Dried Product of Cellulose-Grown

Aspergillus terreus in Batch Culture

Duration Total Percent

Percent protein of cellulose Residual cellulose dried in

Experi- Temperature fermentation added cellulose consumed product ment ("C) (h) (g) (g)

1 35 48 49.0 3.20 93.5 37.8 2 35 30 49.3 5.85 88.1 32.9 3 45 30 49.1 13.10 73.3 23.0 4 35 36 48.8 9.10 80.1 29.4

~

Experiments 1, 2, and 3 were conducted using alkali-treated Solka-Floc as the cellulose source. Experiment 4 was conducted with alkali-treated bagasse. Protein content of the dried product in experiment 4 was measured by the Kjeldahl method of total nitrogen determination and converted to crude protein values.


TABLE IV Specific Growth Rates of Cellulose-Grown Aspergillus terreus during Semicontinuous

Cultivation with a Modified Gradient Feed

Cellulose Duration of Specific Doubling conc. Temperature fermentation growth rate time

Experiment (g/L) ("C) (h) (h-') (h)

1 10 35 30 0.094 7.4 2 10 35 24 0.095 7.3 3 10 35 24 0.091 7.6

~

Experiments 1, 2, and 3 were conducted using alkali-treated Solka-Floc as the cellulose source. In experiments 2 and 3, 10% of the biomass was used as the inoculum from the previous experiment.

TABLE V Utilization of Cellulose and Protein Content of the Dried Product of Cellulose-Grown Aspergillus terreus during Semicontinuous Cultivation with a Modified Gradient Feed

Percent

of cellulose Residual cellulose in dried Experi- Temperature fermentation added cellulose consumed product

Duration Total Percent protein in

ment ("C) (h) (g) (g)

1 35 30 49.2 7.7 84.3 30.0 2 35 24 45.5 7.4 83.7 32.0 3 35 24 45.8 7.4 83.8 32.5

Experiments 1, 2, and 3 were conducted using alkali-treated Solka-Floc as the cellulose source. In experiments 2 and 3, 10% of the biomass was used as the inoculum from the previous experiment.

TABLE VI Cellulose Utilization during Continuous Cultivation of

Aspergillus terreus on Alkali-Treated Solka-Floc

Influent Residual Percent Dilution cellulose cellulose utilization

rate Temperature conc. conc. of cellulose Experiment (h-') ("C) (mg/L) (mg/L)

1 0.10 35 1500 330 78.0 2 0.11 35 1500 250 83.3 3 0.14 40 1500 270 82.0 4 0.14 45 1500 237 84.2

Samples for analysis were removed from the fermentor six to eight residence times after the culture was adjusted to a given dilution rate.


TABLE VII Protein Content of the Biomass from Continuous Cultivation of

Aspergillus terreus on Alakli-Treated Solka-Floc

Dilution Crude Percent

Dry weight protein protein in rate Temperature of biomass content biomass

Experiment (h-') ("C) (mg/L) (mg/L)

1 0.10 35 868 306 35.3 2 0.11 35 870 280 32.2 3 0.14 40 780 273 35.0 4 0.14 45 873 303 34.7

Samples for analysis were removed from the fermentor six to eight residence times after the culture was adjusted to a given dilution rate.

tent was relatively constant ranging from 32 to 35% at all growth temperatures and specific growth rates tested.

DISCUSSION

Batch cultivation studies of glucose-grown A. terreus showed that this organism grew rapidly over a wide temperature range (30-45°C). Specific growth rates greater than 0.20 h-' were determined at all temperatures tested although longer incubation periods were necessary for spore germina- tion to occur at 45°C. Similar growth responses over this temperature range occurred when cellulose was employed as sole carbon source. Thermotolerant, cellulose-decomposing fungi have been reported by other investigators.8-10

Batch cultivation studies using alkali-treated Solka Floc or sugar-cane bagasse as carbon source indicated that rapid growth occurred over short fermentation periods (30-36 h) with high levels of cellulose utilized (Tables 11-V).

Extending the fermentation by 18 hours resulted in only a 6% increase in cellulose consumption (Table 111, experiments 1 and 2). Growth at 45°C resulted in somewhat lower specific growth rates and cellulose utilization (Tables I1 and 11, experiments 3). The inoculum in this experiment was not preadapted to 45OC prior to addition to the fermentor. A longer adaptation period at elevated temperatures could account for these values. However, once adapted, A. terreus actively grew at 45°C as evidenced by results shown in continuous cultivation experiments (Tables VI and VII).

Batch cultivation studies using purified or complex cellulose as carbon source have been reported by other workers. Investigations by Peitersen showed that up to 71% of available barley straw was utilized in 48-144 h by Trichoderma viride." Crawford et al. demonstrated that 60-65'30 of added cellulose was consumed in 96 h by Thermomonospora fusca.I2 Cellulose fermentation studies conducted by Updegraff with Myrothecium verucarria showed that 62% of added cellulosic substrate was utilized in 144 h.I3 By

1518 MILLER AND SNNIVASAN

comparison, studies with A . terreus show higher levels of cellulose utilized and shorter incubation periods to achieve maximal cellulose consumption.

Our results compare favorably with data reported by Chahal et aL8 and Moo-Young et aL9 in studies with Chaetomium cellulolyticum. These investigators demonstrated approximately 60% of added cellulose was utilized in 36 h. More recent investigations by Chahal et a1 have shown that 7.583% of available cellulose was utilized in 17-20 h when C. cellulolyticum was grown on alkali-treated or steam exploded wood.14 Comparable results were also reported by Humphrey et al., where 75-90% of added cellulose was consumed in 24-42 h by a Thermoactinomyces sp.l

The dried product generated from cellulose-grown A . terreus was highly proteinaceous with values ranging from 29 to 33%. Higher protein values were reported by Chahal et and Moo-Young et (40-4370) although values are dependent upon the method of protein analysis. l5 Investigations by Rogers et al., showed that biomass obtained from cellulose fermentation studies using Aspergillus fumigatus contained 13.3% protein in 96 h.16 Com- parable protein values (30-3570) were obtained by Crawford et al. using T. fusca.12 Other investigations using A . terreus as the source of single cell protein have shown comparable protein production (20-3270) but required significantly longer to generate the product (72-168 h).”J8

Few reports exist in the literature on cellulose fermentation studies conducted in the semicontinuous mode. Our studies have shown that growth rate, cellulose utilization, and protein production by A . terreus were unaf- fected through two cycles in this fermentation mode (Tables IV and V, experiments 2 and-3), even though the duration of fermentation was decreased by 20%.

Continuous cultivation of cellulose-grown T. viride was studied by Peitersen.19 The fungus was grown over dilution rates ranging from 0.033 to 0.080 h-’ with values for cellulose utilization ranging from 49 to 76%. By contrast, A . terreus was grown at higher dilution rates (0.10-0.14 h-’) and utilized higher levels of cellulose (78-8470). Varying dilution rates did not af- fect the utilization ofecellulose byA. terreus while studies with T. viride showed that cellulose utilization was inversely affected by increasing dilution rates.

Protein production was generally higher in A . terreus fermentations (32-35%) than values shown for T. viride (16-3470). Comparable protein values, however, were found for T. viride at very low dilution rates.

The ultimate goal of this study was to develop a continuous cellulose fermentation process exhibiting high cellulose utilization and protein production values with rapid growth rates. Results shown in Tables VI and VII indicate that similar cellulose utilization (78-84%) and protein production (32-35%) values were obtained over varying dilution rates (0.10-0.14 h-l) and temperatures (35-45’0.

Continuous cultivation experiments were carried out at low substrate levels (1.5 g/L) to avoid clogging of feed lines. While this cellulose level is too low for development of a continuous fermentation process, results obtained from batch and semicontinuous cultivation studies shown in this report as well as


continuous cultivation studies with glucose-limited fungi (unpublished data) indicate that scaleup to higher cellulose loadings can be achieved with little difficulty.

The high growth rates, protein production, and cellulose utilization values achieved by A. terreus is attributed to an optimized nutrient medium obtained from continuous cultivation studies with this organism.

The minimum doubling time of 4.9 h (0.14 h-I) achieved by A. terreus at 45°C is one of the more rapid growth rates reported for cellulose-grown fungi when grown in the continuous cultivation mode.

These studies have thus shown the feasibility of rapidly producing fungal biomass in different fermentation modes using cellulose as substrate. The fungal biomass may be valuable as an SCP source for use in animal feed if proven to be nontoxic.

Further studies on production of cellulases indicate that this organism pro- duces a potent cellulase. In saccharification studies, up to 39 g/L of reducing sugar was produced in 24 h with over 70% of the cellulose saccharified (unpublished data). These data suggest that A. terreus cellulases may also have considerable value for production of glucose syrups from cellulose.

References

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6. A. Hiller, J. Plazin, and D. D. Van Slyke, J. Biol. Chem., 176, 1401 (1948). 7. D. Updegraff, Anal. Biochem., 32, 420 (1969). 8. D. S. Chahal, J. E. Swan, and M. Moo-Young, Dev. Znd. Microbiol., 18, 433 (1977). 9. M. Moo-Young, D. S. Chahal, J. E. Swan, and C. W. Robinson, Biotechnol. Bioeng.,

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13. D. M. Updegraff, Biotechnol. Bioeng.. 13, 77 (1971). 14. D. S. Chahal. M. Moo-Young, and D. Vlach, Biotechnof. Bioeng., 23, 2417 (1981). 15. C. Christias, C. Couvaraki, S. G. Georgopoulos, B. Macris, and V. Vamvoyani, Appl.

16. C. J. Rogers, E. Coleman, D. F. Spino, T. C. Purcell, and P. V. Scarpino, Environ. Sci.

17. S . K. Garg and S. Neelakantan, Biotechnol. Bioeng., 23, 1653 (1981). 18. B. R. Geethadevi, N. Sitaram, A. Mohammad Kunhi, and T. N. Ramachandra Rao,

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Accepted for Publication November 18, 1982

producton of single-cell protein from cellulose by aspergillus terreus

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