Sugarcane bagasse pith dry pretreatment for single cell protein production

Bioresource Technology 39 (1992) 17-22

Sugarcane Bagasse Pith Dry Pretreatment for Single Cell Protein Production Refugio Rodriguez-Vazquez, Guadalupe Villanueva-Ventura & Elvira Rios-Leal Departamento de Biotecnologia y Bioingenieria, Centro de Investigacion y de Estudios Avanzados, Av. Instituto Politecnico Nacional, No. 2508, Mexico, D.F., C.P. 07000 (Received 15 October 1990; accepted 28 December 1990)

Abstract

The effect of spraying a solution of sodium hydroxide onto sugarcane bagasse pith, in such a low volume that no free liquid was present, was evaluated with respect to Single Cell Protein production (SCP), and biological degradability. This type of processing will be referred to as a dry pretreatment and it is compared with a wet pretreatment. In-vitro digestibility and microbial growth were obtained with a mixed culture of Cellulomonas flavigena and Xanthomonas sp. Maximum digestibility was 76% using dry pretreatment with a NaOH dosage of O'l g per g pith, a temperature of 50°C and moisture content of 80% (based on oven dry weight). A digestibility value of 71% was obtained for wet conditions, with 0"2 g N a O H per g pith, a temperature of 92°C, and a solid: liquid ratio of l: 10. Biomass production was higher in the dry pretreatment than the wet pretreatment, when washing of the treated pith was omitted before fermentation. The spraying was carried out using a rotary system, which permits a better surface area contact between the substrate and the alkali. In this way the N a O H was efficiently used allowing relatively low alkali concentrations. Furthermore, by eliminating the washing stage, the solubilized carbohydrate fraction is retained. Key words: Lignocellulosic residues, alkaline pretreatment, single cell protein, digestibility, lignin, Cellulomonas flavigena and Xanthomonas sp.

INTRODUCTION

In lignocellulosic residues the cellulose and hemicelluloses are potentially available for biological

processing: however, the lack of digestibility of lignocellulosic residues restricts their usefulness. This poor digestibility is due to an apparent association of lignin with carbohydrates; the nature of this association is still under discussion (Sarkanen & Ludwing, 1971; Morrison, 1974; Millet & Baker, 1975; Haw et al., 1985). Thus, pretreatment of lignocellulosic residues is essential to facilitate digestibility (Jung & Fahey, 1983; Puri & Pearce, 1986; Helmling et al., 1989). A variety of pretreatments have been investigated, (Gould, 1985; Khan et aL, 1986; San Martin et al., 1986; Chun et al., 1988; Rolz, 1988; Szczodrak, 1988) and it has been shown that digestibility of residues may be improved by treatments with sodium hydroxide (Jackson, 1977; Lipinsky, 1983; Tanaka et al., 1985; Wanapat et aL, 1985; Lin et aL, 1986). In dilute alkali, the most important chemical reaction is saponification of uronic acid associated with xylans. There is also solubilization of lignin and a marked increase in the swelling, as well as the superficial area of the fiber (Nesse et al., 1977; Hamilton et al., 1984). Wanapat et al. evaluated the effect of alkali on biodegradability of wheat straw, and observed that the biodegradability was markedly enhanced by a wet pretreatment with NaOH, compared to the values obtained by other methods. Since a dry method is potentially less expensive than a wet method, we have investigated a dry treatment and compared it with a wet treatment. The research reported here is part of the development of technology for an economical process in the production of single cell protein (Rodriguez, 1988). The dry method developed in this research involved a rotary system using low volumes of alkaline solution. The treated lignocellulosic residue was then fermented and divided into two

17 Bioresource Technology 0960-8524/92/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain

18

Refugio Rodriguez- Vazquez, Guadalupe Villanueva-Ventura, Elvira Rios-Leal

groups, one of which was washed. Since the solubilized carbohydrate fraction is retained in the non-washed material, this process alleviates both economic and pollution problems associated with the washing method. However, the effect of residual lignin derivatives and sodium ions on microbial product needs to be evaluated.

SPRAYING SYSTEM INDUCTION MOTOR

FLOW METER HEATER

ALKALINE SOLUTION

BATH

METHODS The raw material both for fermentation and chemical pretreatment studies was sugarcane bagasse pith from the Emiliano Zapata sugar mill, Morelos, Mexico. The pith was ground to pass a 2 nun screen and dried at room temperature prior to pretreatment. The air-dried moisture content was 7%, based on oven dry weight (ODWT). For chemical analysis the treated pith was washed, dried, and milled.

Alkali pretreatment

Dry method The dry method was conducted on a laboratory scale in a rotary system (Rotavapor Buchler Instruments, Fort Lee, New Jersey, USA), equipped with a 2 liter volumetric flask and an electrical heater with temperature control (Fig. 1 ). The Rotavapor rotation speed was 96 rpm, the N a O H solution to air ratio was 13 : 1. Thirty grams of prescreened pith was placed in the flask. Then the appropriate alkali solution was sprayed on the material. The concentration of the alkaline solution used depended upon the amount of alkali required for each experiment and the desired moisture content. The pith was kept at the required temperature for 24 h in a stopped volumetric flask before sampling for chemical analysis. A 23 experimental design without replication was used with three independent variables: alkali concentration (5% and 10%)= a, moisture content (40% and 80%, ODWT)=b, and temperature (30°C and 5 0 ° C ) = c. Wet method The wet method was performed using 0"2 g N a O H per g pith, with a solid :liquid ratio of 1 : 10. The temperature was controlled at 92°C for 30 min and then the pith was washed and dried at 80°C for 24 h before chemical analysis and fermentation.

FILTER

Fig. 1. Diagram of rotary system for the 'dry' pretreatment.

General analysis Treated pith samples were taken to determine the lignin content by the TAPPI method T-222 (TAPPI, 1974), and ash content by TAPPI T-211 m-58 standard method. Solubilized lignin was estimated by measuring absorbance at 280 nm in a Perkin-Elmer Lambda 3A spectrophotometer (Perkin-Elmer, Dallas, Texas), as described by Areyzaga and Parada (1985). Phenolic monomers were determined using a high pressure liquid chromatography system (HPLC), phase inverse, column lichrosorb RP-18 (Galindo, 1987). The standard compounds used were: p-coumaric acid, ferulic acid, p-hydroxybenzaldehyde, vanillin, and vanillic acid with detection at 280 nm. The fermented product was harvested by quantitatively transferring the fibre and cell mass to a tarred No. 1 Whatman filter paper. Cells passed through the filter and cell concentration was measured turbidimetrically at 560 nm. Corrections were made with a standard curve that related cell absorption to cell concentration of the mixed culture. The material on the filter paper was dried in an oven at 80°C for 24 h, and the substrate weight loss was determined by weight difference between final weight and the initial weight before fermentation. Fermentation A mixed culture of Cellulomonas flavigena and Xanthomonas sp., culture collection designates CDBB-B-532 and ATCC 31920, was maintained in tubes containing carboxymethyl cellulose (CMC). The mineral nutrient medium was 5.5 g NaC1, 2-5 g (NH4) 2 SO4, 3.5 g H3PO 4, 0.1 g CaCI2, and 0"1 g MgSO 4 per liter. W h e n refrigerated at 4°C the mixed culture can be stored for up to four

Sugarcane bagasse pith dry pretreatment

weeks. The inoculum was prepared by growth in a two-step transfer process in a liquid medium. First, cells were removed from the storage tube. The suspension was added to an Erlenmeyer flask containing 1 g pith and 50 ml of nutrient medium, and then incubated for 48 h at 37°C, and at 110 rpm in a reciprocating shaker. Ten percent of the inoculum was transferred into an Erlenmeyer flask containing 1 g pith and 100 ml of the nutrient medium and then incubated for 24 h under the previous conditions. The resultant broth culture was used at a level of 10% (v/v) as inoculum for fermentation, and incubated for 48 h under the same conditions as for the inoculum.

20

•

19

In-vitro digestibility was analyzed according to the method described by Lin et al. ( 1985). RESULTS AND DISCUSSION The effect of alkali concentration, temperature and moisture content, on lignin solubilization during the dry pretreatment is presented in Fig. 2 for each experiment. Data were analyzed by linear regression. It was observed that the lignin solubilization increased in the first 4 h, after which it was constant. Maximum solubilization of 2"1% (w/w) was obtained for Experiment 8. Delignification and solubilized lignin results are shown in Table 1.

=c

•

--

x e-

~"

15

O')

g ~ N m

1.0

r.,D Z

0.5

-q

2 0

5

I

I

I

10

15

20

PRETREATMENT

TIME(H)

Fig. 2. Effect of reaction conditions on the extent of lignin solubilization. Samples were treated at the following alkali concentration (% O D W T ) , t e m p e r a t u r e (°C), and moisture content (% O D W T ) , respectively: 1 0 : 8 0 : 5 0 (e e), 10:40:50 (V--V), 1 0 : 8 0 : 3 0 (<> o), 1 0 : 4 0 : 3 0 (/x zx), 5 : 4 0 : 3 0 (O----O), 5 : 4 0 : 5 0 (n r~), 5 : 8 0 : 5 0 ( I ; ) , and

5:80:30(0

o). Table 1. Effect of alkali concentration, moisture content, and t e m p e r a t u r e on lignin solubilization and delignification

Experiment

Treatmenta OH

1 2 3 4 5 6 7 8 9

+ + + +

H20

Delignification (% w/w)

T

+ + + + + + + + Wet m e t h o d

aOH, Alkali c o n c e n t r a t i o n (% w/w) - :5; + :10. H 2 0 , M o i s t u r e content (% O D W T ) - :40; + :80.

T, T e m p e r a t u r e (°C) - : 3 0 ; + :50.

Solubilized l i g n i n ((g/gpith) × 100) 0'65 1"28 0"36 1"53 0"64 1"51 0'66 2"05 -

4"74 15"15 25"90 26"46 16"81 26"89 15"00 40'00 72.44

20

Refugio Rodriguez- Vazquez, Guadalupe Villanueva- Ventura, Elvira Rios-Leal

From the 23 factorial plan, it appears that the most important factors are N a O H concentration and temperature, while the initial moisture content is not as important. This implies that the concentration of the alkali in the 'dry' solid could be an important consideration. T h e r e is also a significant alkali/temperature interaction in the analysis of the fermentation experiments (Table 2), in which the microbial growth is analyzed by a 24 factorial plan,where cell growth in washed pret r e a t m e n t = & O n e finds that the important p > 0.001 significance level effects are a, ab, c. At the p > 0"01 level the effects are b, bc, and bcd, while the ac effect is only significant at the 0-1 level. It is interesting that once again, the concentration of N a O H in the dry solid appears to be a significant factor, while the moisture content alone is not as important. Since no further changes in lignin solubilization occurred after 24 h of treatment, fermentation of material with the mixed culture was performed. Results of fermentation are presented in Table 2. Values of cell growth and digestibility of unwashed pretreated material were slightly higher than when washed material and wet pretreatment

Table 2. Results of fermentation Experiment

Treatment" OH

1 2 3 4 5 6 7 8 9 10

H20

Cell growth (g cell~liter)

Digestibility (% w /w)

2'06 2"56 1"01 2"75 2"02 2-89 2'20 4"50 3'71 1"90

49"47 68"47 44"66 67"50 48"44 73'58 41"60 78"43 71'77 30"00

T

+ + + + + + + + + + + Wet method Control

~Alkali concentration, moisture content and temperature conditions as in Table 1.

Table3. Phenolic compounds detected by HPLC in extracted liquors

Method

Dry Wet

Compound&g~pu~ ~ p-CA

FA

p-H

V

VA

800 1857

397 512

58 76

21 43

20 40

ap-CA, p-coumaric acid; FA, ferulic acid; p-H, phydroxybenzaldehyde; V, vanillin;VA, vanillic acid.

were used. T h e r e does not appear to be any fermentation inhibition due to either lignin or phenolic c o m p o u n d s present in the dry pretreated material. Relative quantities of both lignin and phenolic c o m p o u n d s are smaller than those obtained in the wet method, as can be seen in Tables 1 and 3. T h e high a m o u n t of phenolic c o m p o u n d s obtained in the wet process is due to the use of elevated temperature. It has been reported that under such conditions large quantities of phenolic m o n o m e r s are p r o d u c e d (Jung & Fahey, 1983). Microbial growth and digestibility increased slightly as residual lignin content decreased in the range from 83% to 27% delignification. Furthermore, 78-43% digestibility was obtained with 40% of delignification for Experiment 8, this compares favorably with the wet process in which the degradation was 71.77% at a lignin removal of 73%. These results show that the lignin content is not the unique parameter involved in the conversion, as was m e n t i o n e d by Holtzapple and H u m p h r e y (1984). T h e y presented the relationship between the extent of ultimate conversion of cellulose and lignin removal, in which the ultimate conversion decreased above 65% of lignin removal. These results were obtained from poplar wood treated under different conditions, and hydrolyzed by thermophylic bacterium cellulases. T h e ultimate conversion decreased above 65% of lignin removal. Therefore, other factors are involved during this dry process that permit the carbohydrates to be accessible. O n e possible explanation may be the increase in the surface area due to swelling of the material rather than the extent of delignification which is also associated with hemicellulose removal (Holtzapple and Humphrey, 1984). There are some other factors which have been reported to have a high effect on increasing the accessibility of the carbohydrate fraction (Gharpuray et al., 1983; Cowling, 1985). CONCLUSIONS This research establishes the advantages of a dry pretreatment versus a wet pretreatment in the p r o d u c t i o n of single cell protein. - T h e dry process does not include washing or neutralization, and consequently the solubilized carbohydrates are kept for the biomass production, reducing equipment and

Sugarcane bagasse pith dry pretreatment recovery costs, as well as avoiding waste products. Similar values of biomass and digestibility are obtained on unwashed and washed material, when the dry method is used. In addition a smaller quantity of N a O H and liquid is used. The quantity of N a O H could be reduced to 5% to obtain the same cell mass concentration as the wet method, and the pretreatment time can be shortened to 4h. -The mixed rotary system permits a fine spray injection for a uniform wetting, with a low volume of solution. In this way the possible problems of drying are avoided. This system might be appealing for development on an industrial scale. More research needs to be carried out with respect to waste treatment, such as sodium ions and lignin derivatives in SCP production. ACKNOWLEDGEMENTS

The authors are indebted to Dr B. Dale and Dr H. A. Schroeder, Colorado State University, Fort Collins, Colorado, for their advice and comments on the work. This work was carried out with the support of Consejo Nacional de Ciencia y Tecnologia (CONACyT), Mexico. REFERENCES

Areyzaga, M. & Parada, A. (1985). Caracterizacion de la lignina extraida de la cascarilla del arroz (Oryza sativa) por medio de dioxano. Undergraduage thesis, ESIQIE - Instituto Politecnico Nacional, UPALM, Mexico. Chun, H. L., Johnson, D. K., Black, S., Baker, J., Grohmann, K., Sarkanen, K. V., Wallace, K. & Schroeder, H. A. (1988). Organosolv pretreatment for enzymatic hydrolysis of poplars: I. Enzyme hydrolysis of cellulosic residues. Biotechnol. bioengng, 31,643-9. Cowling, E. B. (1975). Physical and chemical constraints in the hydrolysis of cellulose and lignocellulosic materials. Biotechnol. Bioengng. Symp., 5, 163-81. Galindo, A. (1987). Determinacion de compuestos fenolicos en los licores de pretratamiento alcalino. Undergraduate thesis, UNAM, Mexico. Gharpuray, M. M., Lee, Y. -H. & Fan, L. T. (1983). Structural modification of lignocellulosics by pretreatments to enhance enzymatic hydrolysis. BiotechnoL Bioengng, 25, 157-72. Gould, J. M. (1985). Studies on the mechanism of alkaline peroxide delignification of agricultural residues. Biotechnol. Bioengng, 27, 225-31. Hamilton, T. J., Dale, B. E., Ladisch, M. R. & Tsao, G. T. (1984). Effect of ferric tartrate/sodium hydroxide solvent pretreatment on enzyme hydrolysis of cellulose in corn residue. Biotechnol. Bioengng, 26, 781-7.

21

Haw, J. E, Maciel, G. E., Linden, J. C. & Murphy, V. G. (1985). Nuclear magnetic resonance study of autohydrolyzed and organosolv-treated lodgepole pinewood using carbon-13 with cross polarization and magic-angle spinning. Holzforschung, 39, 99-107. Helmling, O., Arnold, G., Rzehak, H., Fahey, G. C., Jr., Berger, L. L. & Merchen, N. R. (1989). Improving the nutritive value of lignocelluiosics: The synergistic effects between alkaline hydrogen peroxide and extrusion treatments. Biotechnol. Bioengng, 33(2), 237- 341. Holtzapple, M. T. & Humphrey, A. E. (1984). The effect of organosolv pretreatment on the enzymatic hydrolysis of poplar. Biotechnol. Bioengng, 26, 670-6. Jackson, M. G. (1977). Review article: The alkali treatment of straws. Anita. Feed Sci. Technol., 2, 105-30. Jung, H. G. & Fahey, G. C. Jr (1983). Nutritional implications of phenolic monomers and lignin: A review. J. Anita. Sci., 57(1), 206-19. Khan, A. W., Labrie, J. P. & McKeown, J. (1986). Effect of electron-beam irradiation pretreatment on the enzymatic hydrolysis of softwood. Biotechnol. Bioengng. 28, 1449-53. Lin, K. W., Ladish, M. R., Voloch, M., Patterson, J. A. & Noller, C. H. (1985). Effect of pretreatment and fermentation on pore size in cellulosic materials. Biotechnol. Bioengng, 27, 1427-33. Lin, K. W., Schaefer, D. m., Ladisch, M. R., Patterson. J. A. & Noller, C. H. (1986). In-vitro anaerobic fermentation of alkali-treated corn stover by rumen microbes. J. Anim. Sci., 62, 822-9. Lipinsky, E. S. (1983). The cellulose challenge: Where do we go from here? TAPPI, 66 (10), 47-9. Millet, M. A. & Baker, A. J. (1975). Pretreatments to enhance chemical, enzymatic, and microbiological attack of cellulosic materials. Biotechnol. Bioengng Syrup., 5, 193-219. Morrison, I. M. (1974). Structural investigations on the lignin-carbohydrate complexes of Lolium perenne. Biochem. J., 139-97. Nesse, N., Wallick, J. & Harper, J. M. (1977). Pretreatment of cellulosic wastes to increase enzyme reactivity. BiotechnoL Bioengng, 19,323-36. Puri, V. P. & Pearce, G. R. (1986). Alkali-explosion pretreatment of straw and bagasse for enzymic hydrolysis. Biotechnol. Bioengng, 28, 480-5. Rodriguez, R. (1988). Project. PVT/NAL/85/3171CONACyT-CINVESTAV, Mexico, D.F. Report. Rolz, C. (1988). Review on effects of some physical and chemical pretreatments on composition, enzymatic hydrolysis and digestibility of lemongrass, citronella and sugarcane bagasse. In Horizons of Biochemical Engineering, ed. S. Aiba. Oxford University Press, New York, p. 349. San Martin, R., Blanch, H. W., Wilke, C. R. & Sciamanna, A. E (1986). Production of cellulase enzymes and hydrolysis of steam-exploded wood. Biotechnol. Bioengng, 28, 564-9. Lai, Y. Z. & Sarkanen, K. V. (1971). Isolation and structural studies. In Lignins: Occurrence, Formation, Structure and Reactions, eds K. V. Sarkanen & C. H. Ludwing. J. Wiley & Sons, New York, pp. 220-4. Szczodrak, J. (1988). The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol. Biotechnol. Bioengng, 32, 771-6. Tanaka, M., Robinson, C. W. & Moo-Young, M. (1985). Chemical and enzymic pretreatment of corn stover to produce soluble fermentation substrates. Biotechnol. Bioengng, 27,362-8. TAPPI (1974). Standard method T-222, Insoluble lignin determination.

22

Refugio Rodriguez- Vazquez, Guadalupe Villanueva- Ventura, Elvira Rios-Leal

Wanapat, M., Sundstol, E & Garmo, T. H. (1985). A comparison of alkali treatment methods to improve the nutritive value of straw. I. Digestibility and metabolizability. Anim. Feed Sci. Technol., 12,295-309.

Wanapat, M., Sunstol, F. & Hall, J. M. R. (1986). A comparison of alkali treatment methods used to improve the nutritive value of straw. II. In sacco and in vitro degradation relative to in vivo digestibility. Anim. Feed Sci. Technol., 14,215-20.