Exposure to pretreatment hypothermia as a determinant of heat killing

When Chinese hamster ovary (CHO) cells were exposed to 22 degrees C for 2 hr prior to 42.4 degrees C hyperthermia, neither the shoulder region of the survival curve nor the characteristic development of thermotolerance after 3-4 hr of heating were observed. Absolute cell survival after 4 hr at 42.4 degrees C was decreased by a factor of between 10 and 100 (depending on the rate of heating of nonprecooled controls). Conditioning at 30 degrees C for 2 hr, 26 degrees C for 2 hr, or 22 degrees C for 20 min followed by heating to 42.4 degrees C over 30 min did not result in sensitization. Prolonged (16 hr) conditioning at 30 degrees C, however, increased the cytotoxicity of immediate exposure to 41.4 or 45 degrees C with maximum sensitization to 45 degrees C occurring after 6 hr at 30 degrees C. Both 3- and 18-hr pretreatments at 30 degrees C similarly increased the cytotoxicity of 45-41.5 degrees C step-down heating (D0 = 28 min in precooled versus 40 min in nonprecooled cells).     

Metabolic alterations induced by hypoxia in the tortoise heart. Comparison between spongy and compact myocardium

Experiments were performed to check the tolerance to severe hypoxia of the tissue layers (compact and spongy) of the tortoise heart. The animals were subjected to hypoxia (7% O2) at 18 degrees C, 28 degrees C and 38 degrees C for 30, 6 and 2 hr respectively, or to anoxia for 30 hr at 18 degrees C and 2 hr at 38 degrees C. At 18 degrees C the metabolic alterations caused by a 30 hr hypoxia were mild whereas at 28 degrees C and 38 degrees C the cardiac glycogen was depleted, lactate had accumulated and the phosphate creatine and ATP content had decreased. The extent of these metabolic changes was similar in the compact and in the spongy layers of the heart.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

Temperature ranges over which rainbow trout fibroblasts survive and synthesize heat-shock proteins

Cultures of the rainbow trout fibroblast cell line RTG-2 withstood temperatures from 0 degrees C to 28 degrees C. At 0 degrees C and 28 degrees C, no proliferation occurred, but cells persisted for at least 7 days. If the cultures were placed back at 22 degrees C, proliferation returned to normal in those that had been kept at 0 degrees C but was reduced in cultures that had been kept at 28 degrees C. Above 28 degrees C, cultures survived for only short periods. If RTG-2 cells that were grown routinely at 22 degrees C were shifted to 26, 28, and 30 degrees C, heat shock proteins (hsps) of 100, 87, 70, 68, 60, 39, 27, and 19 kilodaltons were synthesized. Synthesis was most pronounced at 28 degrees C, and at this temperature hsp synthesis was maximal by 2 hr and had returned to control levels by 36 hr. Individual hsps were synthesized maximally at slightly different times and temperatures, but under all conditions hsps 87 and 70 were most abundant. If cultures were shifted to 24 degrees C or 32 degrees C, hsp synthesis was not observed. Neither the placement of cultures at 5 degrees C nor the shift of cultures that had been maintained at 0 degrees C or 5 degrees C back to 22 degrees C induced the synthesis of hsps. However, cultures incubated at 5 degrees C for 24 hr did synthesize hsps at 26 degrees C, 28 degrees C, and 30 degrees C.     

Effects of temperature upon capacitation of guinea pig spermatozoa

Spermatogenesis in many mammalian species requires a temperature a few degrees below body core temperature. Upon ascent through the male tract and deposition in the female tract, the temperature of spermatozoa is increased to body core temperature. This report investigates the effects of temperatures above or below normal body core temperature, which is also the usual temperature of in vitro gamete incubations and fertilization, upon sperm acrosome reacting ability and fertility. Epididymal guinea pig spermatozoa were preincubated in a Ca2+-free medium at temperatures of 15 degrees C, 25 degrees C, 37 degrees C, or 44 degrees C for increasing periods of time. At 15 degrees C or 25 degrees C, no or very few spermatozoa acquired the ability to acrosome react upon exposure to Ca2+ even after 18 hr of culture or warming up to 37 degrees C. A known stimulator of acrosome-reacting ability, lysophosphatidylcholine, was ineffective in promoting acrosome-reacting ability in spermatozoa incubated at 15 degrees C or 25 degrees C. At 37 degrees C the percentage of acrosome reaction increased steadily over time, reaching about 65% after 18 hr. At 44 degrees C the time course of acquisition of acrosome-reacting ability was greatly accelerated with a percentage at 2 hr comparable to that achieved at 37 degrees C only after 18 hr of preincubation. This effect of incubation at 44 degrees C could be reversed by cooling the spermatozoa to 37 degrees C before they were exposed to Ca2+. Spermatozoa induced to undergo the acrosome reaction after preincubation at 44 degrees C were fully capable of fertilizing intact guinea pig eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cooling and warming rate on glycerolized rabbit kidneys

Cooling and warming rates are known to be important determinants of viability for cryopreserved cells, but optimal rates have not previously been determined for any whole organ. In this study, rabbit kidneys, permeated with 2 M glycerol were cooled to -80 degrees C at four rates varying from 1 degrees C/hr to 3.1 degrees C/min and then rewarmed at four rates from 1 degrees C/hr to 4.2 degrees C/min, giving 16 experimental treatments. After gradual deglycerolization at 10 degrees C, each kidney was autografted and observed for 30 min. Assessment was by measurement of vascular resistance, immediate post-thaw lactate dehydrogenase (LDH) release, gross appearance, light- and electron microscopy, and tissue K+/Na+ ratio 30 min after transplantation. The best results were obtained after cooling at 1 degrees C/hr; warming rate had little apparent influence on the criteria used to assess function with the exception of LDH release, which indicated a preferred warming rate around 1 degrees C/min. Histological studies revealed extensive vascular damage, notably to the glomerular capillaries, that was minimized by very slow cooling. Freeze substitution, carried out on samples removed at -80 degrees C, demonstrated extensive ice formation in the interstitial space and, at the faster cooling rates, in the glomerular capillaries. Intracapillary ice formation was reduced in the kidneys cooled at 1 degrees C/hr.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.     

Catalatic capacities in heat-shocked Euglena cells

1. Various heat treatments were applied to the wild strain Z. Klebs. of Euglena gracilis. 2. Samples of cells were taken at day 1 of the culture at 26 degrees C in a 33 mM lactate medium, when the catalatic capacities of the catalase were highest. 3. They were either submitted to heat treatments (36 and 38 degrees C), or heat-shocks (40, 42 degrees C) or non-permissive heat stress (45 degrees C) for 15 min, 1 and 2 hr. 4. After a 2-hr 45 degrees C treatment the cells were unable to recover normal physiological functions. 5. Heat treatments between 36 and 38 degrees C decreased the catalatic capacities of cells, while heat-shocks at 40 and 42 degrees C strongly reinforced these capacities of hydrogen peroxide dismutation. 6. Having been heat-shocked at 42 degrees C for 2 hr, the cells became different from control cells: (a) after several months of culture, they displayed catalatic capacities increased by 65%; (b) they were able from now on to survive a 2 hr heat shock at 45 degrees C.     

Action of shear on enzymes: studies with alcohol dehydrogenase.

Yeast alcohol dehydrogenase (ADH) solutions (approximately 1 mg/ml, pH 7) were sheared in a coaxial cylindrical viscometer. This was fitted with a lid sealing the contents from the atmosphere and preventing evaporation. At 30 degrees C after a total of 5 hr intermittent shearing at 683 sec-1 no losses of activity were observed. No losses were found after 5 hr continuous shearing and in a no-shear control. At 40 degrees C and 683 sec-1 there were only small activity losses in 5 hr. Shearing at 3440 sec-1 no measurable losses of activity were found with a 1.03 mg/ml solution in 5 hr at 30 degrees C, a 1.03 mg/ml solution in 8 hr at 5 degrees C, and with a 3.89 mg/ml solution in 3 hr at 5 degrees C. In all these cases, however, a white precipitate formed that was not observed in zero shear control experiments. The sheared 3.89 mg/ml solution was clarified by centrifugation. It was shown that there were no ADH aggregates in the supernatant and that the precipitate was less than 2% of the original protein. At 30 degrees C under adverse pH conditions (pH 8.8) there was no significant difference in activity losses of an approximately 1 mg/ml solution sheared at 65 and 744 sec-1. An approximately 0.5 mg/ml ADH solution, pH 7, was agitated in a small reactor with no free air-liquid interface. Peak shear rates near the impeller were estimated to be about 9000 sec-1. Only a small decrease in specific activity was observed until over 15 hr total running at 5 degrees C.     

Metabolism and evaporative heat loss in the dik-dik antelope (Rhynchotragus kirki)

1. Under controlled conditions, the rate of oxygen consumption (VO2) respiratory frequency, evaporative water loss, heat balance, rectal (Trec) and surface temperatures were determined in the dik-dik antelopes at ambient temperatures (Ta) ranging from 1 to 44 degrees C. 2. The thermal neutral zone was found to be between 24 and 35 degrees C. 3. Respiratory frequency ranged between 27 and 630 breaths/min. 4. At a Ta of 44 degrees C, 95% of the heat produced by the dik-dik was lost via respiratory evaporation. Despite an increase in Trec, cutaneous evaporation did not increase. 5. During panting, VO2 increased in accordance with the expected Q10 effect, contrary to earlier findings. 6. Measurements of circadian rhythm [LD 12:12 (7-19) CT26 degrees C] in VO2 showed that the minimum VO2 (0.42 ml O2/g/hr) occurred at midnight while the maximum (0.78 ml O2/g/hr) occurred at midday. The 24 hr mean VO2 was 0.61 ml O2/g/hr. 7. These measurements suggest that in nature, determinants other than light may be responsible for triggering the variations observed in VO2.     

Thermotolerance induced by heat, sodium arsenite, or puromycin: its inhibition and differences between 43 degrees C and 45 degrees C

When CHO cells were treated either for 10 min at 45-45.5 degrees C or for 1 hr with 100 microM sodium arsenite (ARS) or for 2 hr with 20 micrograms/ml puromycin (PUR-20), they became thermotolerant to a heat treatment at 45-45.5 degrees C administered 4-14 hr later, with thermotolerance ratios at 10(-3) isosurvival of 4-6, 2-3.2, and 1.7, respectively. These treatments caused an increase in synthesis of HSP families (70, 87, and 110 kDa) relative to total protein synthesis. However, for a given amount of thermotolerance, the ARS and PUR-20 treatments induced 4 times more synthesis than the heat treatment. This decreased effectiveness of the ARS treatment may occur because ARS has been reported to stimulate minimal redistribution of HSP-70 to the nucleus and nucleolus. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) or PUR (100 micrograms/ml) after the initial treatments greatly inhibited thermotolerance to 45-45.5 degrees C in all cases. However, for a challenge at 43 degrees C, thermotolerance was inhibited only for the ARS and PUR-20 treatments. CHM did not suppress heat-induced thermotolerance to 43 degrees C, which was the same as heat protection observed when CHM was added before and during heating at 43 degrees C without the initial heat treatment. These differences between the initial treatments and between 43 and 45 degrees C may possibly be explained by reports that show that heat causes more redistribution of HSP-70 to the nucleus and nucleolus than ARS and that redistribution of HSP-70 can occur during heating at 42 degrees C with or without the presence of CHM. Heating cells at 43 degrees C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45 degrees C immediately after heating at 43 degrees C. Compared to the nonthermotolerant cells, thermotolerance ratios were 10 for the ARS treatment and 8.5 for the initial heat treatment. Adding CHM (10 micrograms/ml) or PUR (100 micrograms/ml) to inhibit protein synthesis during heating at 43 degrees C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43 degrees C, protein synthesis was required during 43 degrees C for the development of additional thermotolerance to 45 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hyperthermic enhancement of antibody-complement cytotoxicity against normal mouse B lymphocytes and its relation to capping

Normal mouse B lymphocytes were exposed to water-bath hyperthermia in vitro and examined for susceptibility to antibody-complement (Ab-C) cytotoxicity. Enhancement of Ab-C cytotoxicity was observed during heat treatment at 42 or 43 degrees C. Sensitivity to Ab-C cytotoxicity returned to normal levels by 2-3 hr post exposure to 42 degrees C. No such recovery was observed when cells were preheated at 43 degrees C for 40 min. The mechanism responsible for heat-induced enhancement of Ab-C cytotoxicity may be related to the way heat affects the redistribution of membrane-bound antigen-antibody (Ag-Ab) complexes. To investigate this possibility, cells were preheated at 37, 42, or 43 degrees C. The Ab-C assay was then performed at 37 degrees C immediately or 2.5 hr after hyperthermia. The distribution of Ag-Ab complexes was evaluated by immunofluorescence. A direct correlation was found between the hyperthermic enhancement of Ab-C cytotoxicity and the hyperthermic inhibition of capping, a process where membrane-bound Ag-Ab complexes coalesce into a polar cap on the cell surface. Sensitivity to Ab-C cytotoxicity returned to normal levels when cells restored the ability to cap Ag-Ab complexes following 42 degrees C hyperthermia. Cells heated at 43 degrees C were still sensitive to Ab-C cytotoxicity and did not recover the capping ability even 2.5 hr after heat treatment.     

Effects of time and temperature during attachment of sections to microscope slides on immunohistochemical detection of antigens.

To study the effects of time and temperature on attachment of tissue sections to microscope slides, we examined the intensity of immunohistochemical staining of selected antigens in nine different neoplastic and normal tissues after attaching sections at different times and temperatures. Typically, both the temperature and time are minimized when tissue sections attached to slides; however, suboptimal times and temperatures during attachment may result in either loss of tissue due to poor attachment or the necessity for inconvenient staining regimens. Using standard immunohistochemical techniques, 5 microm tissue sections were attached at 58 degrees C for 1, 4 and 24 hr. In a separate study, 5 microm tissue sections were attached for 16 hr at 58, 68 and 80 degrees C. The intensity of staining decreased slightly when the tissue sections were heated at 80 degrees C for 16 hr, but there was little or no decrease when tissues were heated at 68 degrees C or lower for 16 hr, or at 58 degrees C for up to 24 hr.     

Induction and decay of thermotolerance in rainbow trout fibroblasts

Thermotolerance was studied in the rainbow trout fibroblast cell line RTG-2. RTG-2 cultures that had been incubated at 28 degrees C for 24 h were better able to withstand ultimately lethal temperatures above 28 degrees C than RTG-2 cultures that had been maintained at the routine growth temperature of 22 degrees C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28 degrees C. After development for 24 hr at 28 degrees C, thermotolerance showed little change over 72 h at 0 and 5 degrees C but approximately a 40 and 60% reduction at 10 and 22 degrees C, respectively. This is the first demonstration of heat-induced thermal resistance in the cells of a poikilothermic vertebrate.     

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--I. Influence of preincubation length, medium composition and temperature

In the absence of HCG, production of testosterone by whole testes superfused in vitro was quite constant during the 5-hr superfusion period. Addition of 23-184 mIU/ml HCG caused a significant increase of testosterone production which was apparent from 30 min after start of superfusion. Basal and HCG-stimulated testosterone production by whole testes was significantly higher (400, 1950 ng/testis/5 hr, without and with 100 mIU HCG) than by isolated cells (200, 1350 ng/testis/5 hr). Incubation of isolated interstitial cells in medium 199 supplemented with fetal calf serum (FCS), (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid, HEPES) and 3-isobutyl-methylxanthine (MIX), and in medium 199 without FCS, HEPES or MIX, gave similar testosterone responses. While centrifugation at 8000 g for 2 min drastically diminished testosterone formation by isolated interstitial cells, production was similar by cells incubated in either 0.5, 1.0 or 1.5 ml medium. A significant decrease of testosterone synthesis by isolated interstitial cells was found when cells were stored at 4 degrees C for 2 days and then were incubated at 35 degrees C for 6 hr without or with 1-1000 microIU HCG. While isolated interstitial cells incubated at 5 degrees C did not produce testosterone at all, testosterone production increased to 49.5 +/- 3.9 ng/10(5) cells (30 degrees C) and 24.1 +/- 1.1 ng/10(5) cells (40 degrees C), respectively. HCG-stimulated testosterone production was maximal when interstitial cells were incubated at 34 degrees C.     

Tolerance of chick embryos to low temperatures in reference to the heart rate

Ten-day-old embryos were exposed to 28, 18 and 8 degrees C environments and their electrocardiograms (ECG) monitored. Embryos in 28 and 18 degrees C environments maintained a constant heart rate averaging 97 and 25 beats/min, respectively, followed by arrhythmias and cardiac arrest at 101 and 59 hr. Embryos in an 8 degrees C environment went into cardiac arrest after 2-4 hr, but recovered 20 hr later upon rewarming to 38 degrees C. Six to 20-day-old embryos exposed to 8 degrees C were examined for tolerance time after cardiac arrest. The younger the embryo the longer its tolerance to prolonged cardiac arrest.     

Cryopreservation of dog platelets with dimethyl sulfoxide: therapeutic effectiveness of cryopreserved platelets in the treatment of thrombocytopenic dogs, and the effect of platelet storage at -80 degrees C

Dog platelets were frozen with 6% dimethyl sulfoxide at 2-3 degrees C per minute in a -80 degrees C mechanical freezer. The frozen platelets were stored at -80 degrees C for as long as 39 months. After storage at -80 degrees C for less than 1 year, platelet in vitro freeze-thaw-wash recovery values were 70%, and in vivo survival values 1 to 2 hr after transfusion were 40% those of fresh platelets. After 2 years or longer storage, in vitro freeze-thaw-wash recovery values were 60%, and in vivo survival values 1 to 2 hr after transfusion were 20% those of fresh platelets. These results indicate that significant deterioration of the dog platelets occurred between the first and second year of storage at -80 degrees C. Platelets that were stored frozen at -80 degrees C for less than 1 year and washed before transfusion into lethally irradiated thrombocytopenic dogs were hemostatically effective.     

Sugar production from agricultural woody wastes by saccharification with Trichoderma viride cellulase.

The saccharification of agricultural woody wastes was studied using a commercial enzyme preparation, Cellulase onozuka, derived from Trichoderma viride or the solid culture extracts of the fungus. With the intention of producing sugar at low cost, a simple procedure of enzymatic saccharification of rice straw, bagasse, and sawdust was studied. Delignifying methods of these wastes were investigated using dilute sodium hydroxide solution and dilute peracetic acid. Rice straw and bagasse were effectively delignified by boiling in a 1% sodium hydroxide solution for 3 hr or by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a broad leaved tree (Machilus thunbergii) was delignified by autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr and by subsequent boiling in diluted 1/5 peracetic acid for 1 hr. This type of sawdust was also delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr. The sawdust from a coniferous tree (Cryptomeria japonica) was delignified by boiling in 1/5 peracetic acid for 1 hr and by subsequent autoclaving at 120 degrees C in a 1% sodium hydroxide solution for 1 hr; however, the successive treatment by autoclaving with alkali solution and subsequent boiling with diluted peracetic acid failed to bring about the desired effect. The saccharification of delignified rice straw, bagasse, and sawdust was examined using Cellulase onozuka, wheat bran or rice straw solid culture at various substrate concentrations, resulting in the formation of 5 to 10% sugar solutions after incubation at pH 5.0, 45 degrees C for 48 hr. The optimum substrate concentration existed at around 10%. Reuse of cellulase solution and resaccharification of residual sawdust were considered to be inadequate.