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Radiation-induced degradation and subsequent hydrolysis of waste cellulose materials
Radiation-Induced Degradation and Subsequent Hydrolysis of Waste Cellulose Materials MINORU
KUMAKURA
and ISA0
Takasakl Radiation Chemistry Research
Establishment, Takasaki. Gunma,
Japan Japan
KAETSU
Atomic
Energy
Research
Instilutc.
(Ruceir~tl 17 Frhrutrr! 1978: in rrt~ised fhrm 21 April 1978) The effect of y-pre-irradiation of cellulose m cellulose containing waste plants was investigated through enzymatic and acidic hydrolysis reaction. Pre-irradiation of waste rice straw. chaff and saw dust accelerated the enzymatic hydrolysis by cellulase. Reducing sugar and glucose yields were higher with an increasing radiation dose in these materials. The required dose for effective acceleration of enzymatic hydrolysis was much reduced by the addition of chlorine during radiation. On the other hand. the reducing sugar and glucose yields in the subsequent acidic hydrolysis of waste products rather decreased through pre-irradiation treatment. This was attributed to an acceleration effect of a secondary acidic decomposition of sugar to lower molecular weight-products through pre-irradiation.
INTRODUCTION CELLULOSE materials
are important natural resources. Utilization of waste cellulose raw materials such as waste agricultural and forestry products will be one of the interesting problems in the near future. The immobilization of various enzymes including cellulase by means of radiation-induced polymerization at low temperatures was studied!” It is natural to apply these results to the utilization of various waste cellulose materials through enzymatic hydrolysis by immobilizing enzymes or microbial cells. By our method, a considerable part of the enzymes was trapped on the surface of the polymer carrier which was very beneficial to the enzyme reaction with waterinsoluble high molecular weight substrates such as cellulose. However, some kind of preliminary treatment of cellulose raw materials is necessary for their effective hydrolysis because cellulose material as a substrate should be more hydrophilic. In this report, preliminary degradation of cellulose in waste plants by radiation and its effect on the subsequent hydrolysis was investigated as a fundamental study of the hydrolysis process through immobilized enzymes and microbial cells.
MATERIALS
AND
was determined by the Nelson-Somogyi method (2.3) 3&40 min after preparation. The enzyme reaction was carried out as follows: The sample CO.1g] was mixed with [IOml of 0.1 M] acetic acid buffer solution (pH 4.5) and [25 mg] cellulase (Kinki Yakulut Mfg. Co., Ltd.). After reaction at 4o’C 1 ml of this solution was diluted 4 times and tested by the Nelson-Somogyi method to determine total quantity of reducing sugar. The reducing sugar yield was obtained as ratio of the quantity of reducing sugar to the total quantity of carbohydrate. Potential sugar quantity was determined by the saccharilication method.‘4’ The glucose fraction of the total quantity of reducing sugar was obtained by analysis of solution using glucose quantitative reagent (“GOD-PODLK”: Nagase Sangyo Co., Ltd.).“’
RESULTS
30 3
A
DISCUSSION
As a preliminary treatment to make cellulose raw materials accessive to hydrolysis, radiation-induced degradation of waste plants were tried. The effect of pre-irradiation treatment on enzyme hydrolysis of rice straw by cellulase was studied. The relationship between the reducing sugar and glucose yield and the irradiation dose is shown in Fig. 1 as a function of enzyme hydrolysis time by cellulase. The sugar and glucose yields are higher with increasing irradiation dose and rapidly increase in total dose more than lOa rad. It is obviouk that pre-irradiation is effective on the subsequent enzyme hydrolysis reaction. Similar results were obtained in case of other waste materials such as chaff and saw dust.
METHODS
Rice straw (Wheat: Norine No, 61), chaff (Rice husk: Nippon masari), and saw dust (Japan cedar) were used as materials. Rice straw was cut in 1 cm length, washed with water and dried. Chaff and saw dust of 32 mesh were used in our sample. Irradiation was carried out firstly by y-rays (60Co) and secondly an electron beam (EBG electron beam) accelerator with 2 MeV and a current of 2 mA. The irradiated sample CO.1g] was mixed with [lOml] distilled water, stirred and in the decanted solution the reduced sugar A.“.,.
AND
Acceleration of’irradiation efSect by addition
of chlorine
It was found that pre-irradiation was effective on enzyme hydrolysis of waste materials. However. large 139
Enzymorlc
hydrolysis
flme
ctlr*
/
FIG. I. Effect of pre-irradiation on the subsequent cn~ymc hydrolysis of rice straw. Irradiation bq ;‘-raq: at 25 C’ m air: (0) reducing sugar yield in I x IOHrad; (A) reducing sugar yield in I x IO“ rad: (El) reducing sugar yield in zero rad: (0) glucose yield in 1 x IO” rad: (A) glucose yield in I x IO” rad: (m) glucose yield in xro rad.
irradiation dose more than IOx rad was required to give a noticeable effect on those cellulose materials. To reduce the required irradiation dose for economic reasons, on one of the trials, the effect of addition of various chemical additives on acceleration in cellulose degradation was studied. Water and alcohol gave no significant acceleration effect on radiolysis of cellulose raw materials. However, it was found that the addition of halogen molecules such as chlorine and bromine accelerated the degradation of those materials noticeably (see Figs. 2 and 3). As seen in Fig. 2. the reducing sugar yield in irradiated rice straw after an irradiation with IO’rad increased in the presence of chlorine more than eight times compared to the same irradiation in the absence of chlorine. Furthermore, the reducing sugar yield after enzyme reaction of rice straw irradiated with IO’ rad in the presence of chlorine increased more than six times over irradiated rice straw in the absence of chlorine. It was
FIG 3. I:ffect of chlorine addition in Irradiation on the subsequent hydrolysis of irradiated rlcc stra\\. (0) Irr:tdlated bq ;‘-ray at 25 C in the prcxnce of chlorine ~a\ (I atm) and hydrol!\ed hl cellulnsc at 30 C for 60 mln (cellulase 2S me. l-ice ‘Itram SO mg in 0. I M acetIc acid buffer solution): (A) irradiated sh! ;‘-r‘t! at 25 ( 111 the presence of chlorine $aa (I atm) and p! rely/cd at IO0 (’ (in water) for 6Omin. (0) irradiated hq ;‘+a) at 75 C‘ 111 air and hvdrolksed hy celluavz at 40 (’ for 60 mln (cellular 25 mg. rice strau SO rn$ in 0.1 M acetic ;rcld hull’cr Wlutlon). that halogen compounds accelerated the radiolysis of degradative type polymer such as polymethylmethacrylate, probably owing to the incrcasc of radical formation in polymer by active hydrogen pulling out of halogen radicals. Morco\er. hydrogen halide as a result of that reaction might cause a secondary chemical degradation of ccllulosc materials. Hydrolysis decomposition of irradiated \am@s in the presence of chlorine could also hc achieved by thermal heating (see Fig. 2). This suggests the formation and hydrolysis action of hydrochloric a&. It can he concluded that preliminary degradation hq irradiation of waste materials such as rice strah i\ accelerated and the required dose can he reduced markedly by the addition of halogens
known
Ol.--.‘.L. A\cldK
FIG. 2. Effect of chlorine addition on reducing sugar yield in irradiated rice straw. Irradiation by ;-ray: at 25 C: (@) in the presence of chlorine gas (I atm); (0) in the presence of oxygen gas (I atm): (0) in air.
120
60
0
hydroly51P
rime
irrs,
FIG. 4. EtTect of pre-irradiation on acidx hydrolysis of \a~ dust. Irradiation: at 25 C in air: acidic hydrolysis: OX”,, H,SO, solution at I80 C. Pre-irradiation condition: (0) 5 x 10” rad by ;-ray; (0) 5 x IO8 rad by electron beam: (A) 5 x IO rad by electron beam: (0) I x IO’ rad hy elcctron heam. (n) /era rad.
(b)
FIG. 5. ElTect
of pre-irradiation on acidic hydrolysis of chaff and rice straw. (a) hydrolysis of chaff: (0) 5 x IO’ rad by electron beam: (A) 5 x 10’ rad by electron beam; (0) nonirradiated. (b) hydrolysis of rice straw: (0) 5 x 10” rad by electron beam; (A) 5 x IO’ rad by electron beam: (U) non-irradiated.
Acidic
Other conditions
hpfrolysis
Preliminary
irradiation and acidic arc same as in Fig. 4.
of‘ irruditrfed
irradiated
waste
wuste
plants
plants
hydrolysis
in comparison
were
with
the
tested
for
enzyme hydrolysis results. Reducing sugar yields in acidic hydrolysis of irradiated rice straw, chaff and saw dust under various irradiation dose are plotted against the hydrolysis time (Figs. 4 and 5). According to those results, pre-irradiation of these materials affected clearly the acidic hydrolysis, but the irradiation effect was quite different from that on enzyme hydrolysis. In non-irradiated samples or irradiated samples with a smaller dose than lOBrad, reducing sugar yield first increased with time during early stages of hydrolysis and then gradually decreased with time in later stages of hydrolysis. On the other hand. in samples irradiated with a bigger total dose (more than lo8 rad), reducing sugar yield continuously decreased during the whole hydrolysis stage. However, the initial sugar yield was higher in irradiated samples with larger irradiation dose. These facts suggest that formed reducing sugar is further decomposed to lower molecular weight products by acid. This secondary decomposition was faster in irradiated samples with larger dose. Figure 6 showed the change in the glucose yield against hydrolysis time of irradiated samples. The glucose yield continuously increased with increasing time in non-irradiated sample and irradiated samples with a smaller dose than IO’ rad, but the glucose yield decreased with time after reaching a maximum in samples irradiated with a dose of 5 x IO’rad. The glucose yield showed a continuous decrease with time in samples irradiated with a dose of 5 x IO8 rad. acidic
60
Acidic
hydrolysis
hydrolysis Time
120
(minutes)
FIG 6. Effect of pre-irradiation on potential sugar and glucose yield in acidic hydrolysis of saw dust. (a) remained potential sugar yield: (0) 5 x IO” rad by electron beam: (A) 5 x IO” rad by electron beam: (0) I x IO’ rad by clcctron beam: (0) non-irradiated. (b) glucose yield: (0) 5 x IO8 rad by electron beam: (A) 5 x IO’ rad by electron beam: (W) I x IO’ rad by electron hcam; (0) non-irradiated. Other irradlatlon and acidic hydrolysis conditions are same as in Fig. 4. These results are similar in time-yield behaviour relating to the reducing sugar yield but show more clearly the acceleration effect of pre-irradiation on secondary induced decomposition of sugars by acid to lower molecular weight products. The change of the remaining potential sugar against hydrolysis time is shown in Fig. 6. The potential sugar decreased with the hydrolysis time, but decreased more quickly in irradiated samples with larger doses. From this, it is evident that overall reaction rates in the acidic hydrolysis process including hydrolysis of glucose to other sugars and lower molecular weight products are accelerated by pre-irradiation. It can be concluded that the pre-irradiation treatment of waste plants as cellulose raw materials has a significant effect on the glucose or reducing sugar yield in the following enzyme hydrolysis, whilst it has a negative effect on sugar yields in the following acidic hydrolysis owing to the excessively accelerated decomposition of sugar products. REFERENCES A.. YOSHIDA M..
ASANO M. and KAETSV I. J. Solid-Phase Biochem In -press. SOMOGUl M., J. hiol. Chem. 195, 19 (1952). NELSON N.. J. biol. Chem. 153, 375 (1944). SAEMAN J. F., BUBI_ J. I. and HARRIS E. E. Ind. Eng. Chem. Anal. Ed. 17, 35. (1945). SALOMAN L. L. and JOHNSON J. E. Anal. Chem. 31, KUMARL,KA
453 (1959).
KUMAKURA
and ISA0
Takasakl Radiation Chemistry Research
Establishment, Takasaki. Gunma,
Japan Japan
KAETSU
Atomic
Energy
Research
Instilutc.
(Ruceir~tl 17 Frhrutrr! 1978: in rrt~ised fhrm 21 April 1978) The effect of y-pre-irradiation of cellulose m cellulose containing waste plants was investigated through enzymatic and acidic hydrolysis reaction. Pre-irradiation of waste rice straw. chaff and saw dust accelerated the enzymatic hydrolysis by cellulase. Reducing sugar and glucose yields were higher with an increasing radiation dose in these materials. The required dose for effective acceleration of enzymatic hydrolysis was much reduced by the addition of chlorine during radiation. On the other hand. the reducing sugar and glucose yields in the subsequent acidic hydrolysis of waste products rather decreased through pre-irradiation treatment. This was attributed to an acceleration effect of a secondary acidic decomposition of sugar to lower molecular weight-products through pre-irradiation.
INTRODUCTION CELLULOSE materials
are important natural resources. Utilization of waste cellulose raw materials such as waste agricultural and forestry products will be one of the interesting problems in the near future. The immobilization of various enzymes including cellulase by means of radiation-induced polymerization at low temperatures was studied!” It is natural to apply these results to the utilization of various waste cellulose materials through enzymatic hydrolysis by immobilizing enzymes or microbial cells. By our method, a considerable part of the enzymes was trapped on the surface of the polymer carrier which was very beneficial to the enzyme reaction with waterinsoluble high molecular weight substrates such as cellulose. However, some kind of preliminary treatment of cellulose raw materials is necessary for their effective hydrolysis because cellulose material as a substrate should be more hydrophilic. In this report, preliminary degradation of cellulose in waste plants by radiation and its effect on the subsequent hydrolysis was investigated as a fundamental study of the hydrolysis process through immobilized enzymes and microbial cells.
MATERIALS
AND
was determined by the Nelson-Somogyi method (2.3) 3&40 min after preparation. The enzyme reaction was carried out as follows: The sample CO.1g] was mixed with [IOml of 0.1 M] acetic acid buffer solution (pH 4.5) and [25 mg] cellulase (Kinki Yakulut Mfg. Co., Ltd.). After reaction at 4o’C 1 ml of this solution was diluted 4 times and tested by the Nelson-Somogyi method to determine total quantity of reducing sugar. The reducing sugar yield was obtained as ratio of the quantity of reducing sugar to the total quantity of carbohydrate. Potential sugar quantity was determined by the saccharilication method.‘4’ The glucose fraction of the total quantity of reducing sugar was obtained by analysis of solution using glucose quantitative reagent (“GOD-PODLK”: Nagase Sangyo Co., Ltd.).“’
RESULTS
30 3
A
DISCUSSION
As a preliminary treatment to make cellulose raw materials accessive to hydrolysis, radiation-induced degradation of waste plants were tried. The effect of pre-irradiation treatment on enzyme hydrolysis of rice straw by cellulase was studied. The relationship between the reducing sugar and glucose yield and the irradiation dose is shown in Fig. 1 as a function of enzyme hydrolysis time by cellulase. The sugar and glucose yields are higher with increasing irradiation dose and rapidly increase in total dose more than lOa rad. It is obviouk that pre-irradiation is effective on the subsequent enzyme hydrolysis reaction. Similar results were obtained in case of other waste materials such as chaff and saw dust.
METHODS
Rice straw (Wheat: Norine No, 61), chaff (Rice husk: Nippon masari), and saw dust (Japan cedar) were used as materials. Rice straw was cut in 1 cm length, washed with water and dried. Chaff and saw dust of 32 mesh were used in our sample. Irradiation was carried out firstly by y-rays (60Co) and secondly an electron beam (EBG electron beam) accelerator with 2 MeV and a current of 2 mA. The irradiated sample CO.1g] was mixed with [lOml] distilled water, stirred and in the decanted solution the reduced sugar A.“.,.
AND
Acceleration of’irradiation efSect by addition
of chlorine
It was found that pre-irradiation was effective on enzyme hydrolysis of waste materials. However. large 139
Enzymorlc
hydrolysis
flme
ctlr*
/
FIG. I. Effect of pre-irradiation on the subsequent cn~ymc hydrolysis of rice straw. Irradiation bq ;‘-raq: at 25 C’ m air: (0) reducing sugar yield in I x IOHrad; (A) reducing sugar yield in I x IO“ rad: (El) reducing sugar yield in zero rad: (0) glucose yield in 1 x IO” rad: (A) glucose yield in I x IO” rad: (m) glucose yield in xro rad.
irradiation dose more than IOx rad was required to give a noticeable effect on those cellulose materials. To reduce the required irradiation dose for economic reasons, on one of the trials, the effect of addition of various chemical additives on acceleration in cellulose degradation was studied. Water and alcohol gave no significant acceleration effect on radiolysis of cellulose raw materials. However, it was found that the addition of halogen molecules such as chlorine and bromine accelerated the degradation of those materials noticeably (see Figs. 2 and 3). As seen in Fig. 2. the reducing sugar yield in irradiated rice straw after an irradiation with IO’rad increased in the presence of chlorine more than eight times compared to the same irradiation in the absence of chlorine. Furthermore, the reducing sugar yield after enzyme reaction of rice straw irradiated with IO’ rad in the presence of chlorine increased more than six times over irradiated rice straw in the absence of chlorine. It was
FIG 3. I:ffect of chlorine addition in Irradiation on the subsequent hydrolysis of irradiated rlcc stra\\. (0) Irr:tdlated bq ;‘-ray at 25 C in the prcxnce of chlorine ~a\ (I atm) and hydrol!\ed hl cellulnsc at 30 C for 60 mln (cellulase 2S me. l-ice ‘Itram SO mg in 0. I M acetIc acid buffer solution): (A) irradiated sh! ;‘-r‘t! at 25 ( 111 the presence of chlorine $aa (I atm) and p! rely/cd at IO0 (’ (in water) for 6Omin. (0) irradiated hq ;‘+a) at 75 C‘ 111 air and hvdrolksed hy celluavz at 40 (’ for 60 mln (cellular 25 mg. rice strau SO rn$ in 0.1 M acetic ;rcld hull’cr Wlutlon). that halogen compounds accelerated the radiolysis of degradative type polymer such as polymethylmethacrylate, probably owing to the incrcasc of radical formation in polymer by active hydrogen pulling out of halogen radicals. Morco\er. hydrogen halide as a result of that reaction might cause a secondary chemical degradation of ccllulosc materials. Hydrolysis decomposition of irradiated \am@s in the presence of chlorine could also hc achieved by thermal heating (see Fig. 2). This suggests the formation and hydrolysis action of hydrochloric a&. It can he concluded that preliminary degradation hq irradiation of waste materials such as rice strah i\ accelerated and the required dose can he reduced markedly by the addition of halogens
known
Ol.--.‘.L. A\cldK
FIG. 2. Effect of chlorine addition on reducing sugar yield in irradiated rice straw. Irradiation by ;-ray: at 25 C: (@) in the presence of chlorine gas (I atm); (0) in the presence of oxygen gas (I atm): (0) in air.
120
60
0
hydroly51P
rime
irrs,
FIG. 4. EtTect of pre-irradiation on acidx hydrolysis of \a~ dust. Irradiation: at 25 C in air: acidic hydrolysis: OX”,, H,SO, solution at I80 C. Pre-irradiation condition: (0) 5 x 10” rad by ;-ray; (0) 5 x IO8 rad by electron beam: (A) 5 x IO rad by electron beam: (0) I x IO’ rad hy elcctron heam. (n) /era rad.
(b)
FIG. 5. ElTect
of pre-irradiation on acidic hydrolysis of chaff and rice straw. (a) hydrolysis of chaff: (0) 5 x IO’ rad by electron beam: (A) 5 x 10’ rad by electron beam; (0) nonirradiated. (b) hydrolysis of rice straw: (0) 5 x 10” rad by electron beam; (A) 5 x IO’ rad by electron beam: (U) non-irradiated.
Acidic
Other conditions
hpfrolysis
Preliminary
irradiation and acidic arc same as in Fig. 4.
of‘ irruditrfed
irradiated
waste
wuste
plants
plants
hydrolysis
in comparison
were
with
the
tested
for
enzyme hydrolysis results. Reducing sugar yields in acidic hydrolysis of irradiated rice straw, chaff and saw dust under various irradiation dose are plotted against the hydrolysis time (Figs. 4 and 5). According to those results, pre-irradiation of these materials affected clearly the acidic hydrolysis, but the irradiation effect was quite different from that on enzyme hydrolysis. In non-irradiated samples or irradiated samples with a smaller dose than lOBrad, reducing sugar yield first increased with time during early stages of hydrolysis and then gradually decreased with time in later stages of hydrolysis. On the other hand. in samples irradiated with a bigger total dose (more than lo8 rad), reducing sugar yield continuously decreased during the whole hydrolysis stage. However, the initial sugar yield was higher in irradiated samples with larger irradiation dose. These facts suggest that formed reducing sugar is further decomposed to lower molecular weight products by acid. This secondary decomposition was faster in irradiated samples with larger dose. Figure 6 showed the change in the glucose yield against hydrolysis time of irradiated samples. The glucose yield continuously increased with increasing time in non-irradiated sample and irradiated samples with a smaller dose than IO’ rad, but the glucose yield decreased with time after reaching a maximum in samples irradiated with a dose of 5 x IO’rad. The glucose yield showed a continuous decrease with time in samples irradiated with a dose of 5 x IO8 rad. acidic
60
Acidic
hydrolysis
hydrolysis Time
120
(minutes)
FIG 6. Effect of pre-irradiation on potential sugar and glucose yield in acidic hydrolysis of saw dust. (a) remained potential sugar yield: (0) 5 x IO” rad by electron beam: (A) 5 x IO” rad by electron beam: (0) I x IO’ rad by clcctron beam: (0) non-irradiated. (b) glucose yield: (0) 5 x IO8 rad by electron beam: (A) 5 x IO’ rad by electron beam: (W) I x IO’ rad by electron hcam; (0) non-irradiated. Other irradlatlon and acidic hydrolysis conditions are same as in Fig. 4. These results are similar in time-yield behaviour relating to the reducing sugar yield but show more clearly the acceleration effect of pre-irradiation on secondary induced decomposition of sugars by acid to lower molecular weight products. The change of the remaining potential sugar against hydrolysis time is shown in Fig. 6. The potential sugar decreased with the hydrolysis time, but decreased more quickly in irradiated samples with larger doses. From this, it is evident that overall reaction rates in the acidic hydrolysis process including hydrolysis of glucose to other sugars and lower molecular weight products are accelerated by pre-irradiation. It can be concluded that the pre-irradiation treatment of waste plants as cellulose raw materials has a significant effect on the glucose or reducing sugar yield in the following enzyme hydrolysis, whilst it has a negative effect on sugar yields in the following acidic hydrolysis owing to the excessively accelerated decomposition of sugar products. REFERENCES A.. YOSHIDA M..
ASANO M. and KAETSV I. J. Solid-Phase Biochem In -press. SOMOGUl M., J. hiol. Chem. 195, 19 (1952). NELSON N.. J. biol. Chem. 153, 375 (1944). SAEMAN J. F., BUBI_ J. I. and HARRIS E. E. Ind. Eng. Chem. Anal. Ed. 17, 35. (1945). SALOMAN L. L. and JOHNSON J. E. Anal. Chem. 31, KUMARL,KA
453 (1959).