Fermentation products and plant cell wall-degrading enzymes produced by monocentric and polycentric anaerobic ruminal fungi.

Five anaerobic fungal isolates from the bovine rumen were grown on Coastal Bermuda grass (CBG) leaf blades and monitored over a 9-day period for substrate utilization, fermentation products, cellulase, and xylanase activities. Two of the fungal isolates showed monocentric growth patterns; one (isolate MC-1) had monoflagellated zoospores and morphologically resembled members of the genus Piromyces; the other (isolate MC-2) had multiflagellated zoospores and resembled members of the genus Neocallimastix. Three other isolates (PC-1, PC-2, and PC-3) exhibited polycentric growth and have not yet been described in the literature; these isolates were characterized by differences in morphology. All of the isolates degraded CBG to approximately the same extent (70% [dry weight]) in 9 days. Fermentation product accumulation was concurrent with substrate utilization. The major fermentation products for all isolates were formate, acetate, D-(-)-lactate, L-(+)-lactate, ethanol, carbon dioxide, and hydrogen. Succinate was produced by all cultures, with the exception of MC-1. Fermentation balances revealed different profiles for each isolate. As a group, monocentric isolates produced a greater ratio of oxidized to reduced products when grown on glucose or CBG than did the polycentric isolates, which produced a nearly equal ratio of these products. All isolates exhibited cellulolytic and xylanolytic activities, including endoglucanase, exoglucanase, beta-glucosidase, xylanase, and beta-xylosidase activities. Increasing enzyme activity correlated with the accumulation of fermentation products and substrate utilization. The optimum pH for the enzymatic activity of polycentric isolates was within a more narrow range (pH 6.4 to 7.0) than that of the monocentric isolates (pH 5.5 to 7.5). Activity toward cellulosic substrates was not detected until after the disappearance of reducing sugars. Xylanase activity was found to be five to seven times that of carboxymethyl cellulase activity for all cultures grown on CBG.     

Comparison of morphological and enzyme characteristics of anaerobic fungi isolated from Cervus dama

Seven anaerobic fungal isolates from Cervus dama (domesticated and free living) were grown on carboxymethyl cellulose (CMC) and avicel, and monitored over a five day period for substrate utilization and cellulase activities. All fungal isolates showed monocentric growth patterns; four of them had polyflagellated zoospores and morphologically resembled members of the genus Neocallimastix; the other three had monoflagellated zoospores and resembled members of the genus Piromyces. All of the isolates degraded CMC and avicel, and exhibited cellulolytic activities (carboxymethyl cellulase-(CMC-ase) and avicelase).     

Feruloyl and p-coumaroyl esterase from anaerobic fungi in relation to plant cell wall degradation

Summary Trans-feruloyl and trans-p-coumaroyl esterases were found in the culture filtrates of two monocentric (Piromyces MC-1, Neocallimastix MC-2) and three polycentric (Orpinomyces PC-2, Orpinomyces PC-3, and PC-1, an unnamed genus with uniflagellated zoospores) isolates of anaerobic rumen fungi. Treatment of cell walls of Coastal bermudagrass shoots with the filtrates released the trans isomers of ferulic and p-coumaric acids; results of microscopic observations indicated that fungal isolates degraded primarily unlignified cell walls in leaf blades and stems. A greater proportion of ferulic than p-coumaric acid was released by this treatment when compared with the amounts of the acids released by saponification of the walls with 1 M NaOH. The filtrates also showed esterase activities against the trans isomers of methyl ferulate and methyl p-coumarate, with ferulic acid being released at a faster rate than p-coumaric acid. Assays for other cell-wall-degrading enzymes (xylanase, -xylosidase, -l-arabinosidase, cellulase, -glucosidase) indicated that only -xylosidase correlated with ferulate and p-coumarate esterase activities. The monocentric isolate MC-2 had the highest esterase activity against both the plant cell wall and methyl ester substrates and the highest specific activities of acetyl esterase, -xylosidase, -l-arabinosidase, cellulase and -glucosidase. Isolate MC-2 produced substantially greater amounts of feruloyl and p-coumaroyl esterase when the growth substrate contained higher levels of saponifiable ferulic and p-coumaric acids. Offprint requests to: W. S. Borneman     

Carbohydrate fermentation by three species of polycentric ruminal fungi from cattle and water buffalo in tropical Australia

Fructose, glucose and xylose were the only monosaccharides to be fermented by the polycentric fungi, Orpinomyces joyonii (three cattle isolates) and O. intercalaris (two cattle isolates) and Anaeromyces spp. (four cattle isolates and two water buffalo isolates). Both Orpinomyces spp. utilised a similar range of oligosaccharides and polysaccharides by fermenting cellobiose, gentiobiose, lactose, maltose, sucrose, cellulose, glycogen, starch and xylan. In contrast, there was considerable variation in carbohydrate fermentation amongst Anaeromyces spp., with only cellobiose, gentiobiose and cellulose being fermented by all strains. Formate, acetate and ethanol were the major fermentation end-products formed from glucose by all polycentric fungi. In addition, Anaeromyces spp. produced considerable amounts of lactate, although only small amounts were formed by Orpinomyces spp. This difference was explained by the low specific activity for lactate dehydrogenase in Orpinomyces spp. Several Anaeromyces spp. also produced malate as a significant end-product of glucose fermentation. Fermentation of specifically-labelled Z14C]glucose molecules by polycentric fungi showed that hexose was catabolised by both polycentric and monocentric fungi via the glycolysis pathway with end-products being derived from the following carbon atoms: lactate and malate (C1-C3; C4-C6), acetate and ethanol (C1-C2; C5-C6), CO2 and formate (C3; C4). The results were compared to those obtained for monocentric and polycentric fungi isolated from temperate climate ruminants.     

Rumen fungi: morphological types from Georgia cattle and the attack on forage cell walls

Fungal colonies developing in anaerobic media from zoospores in rumen fluid from cows eating Cynodon dactylon or Medicago sativa included types showing monocentric and polycentric growth. High energy supplements added to diets of Sorghum bicolor silage increased fungal numbers in the rumen, but increases were also affected by the history and predisposition of the animal. Mixed fungal types in rumen fluid and pure cultures of isolates showing monocentric and polycentric growth degraded and weakened lignocellulosic tissues and penetrated the cuticle of C. dactylon leaf blades. By weakening or degrading recalcitrant structures in forages, rumen fungi may alter physical parameters of plants that influence utilization of fibre by ruminants.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.     

Freeze tolerance of soil chytrids from temperate climates in Australia

Very little is known about the capacity of soil chytrids to withstand freezing in the field. Tolerance to freezing was tested in 21 chytrids isolated from cropping and undisturbed soils in temperate Australia. Samples of thalli grown on peptone–yeast–glucose (PYG) agar were incubated for seven days at −15 °C. Recovery of growth after thawing and transferring to fresh medium at 20 °C indicated survival. All isolates in the Blastocladiales and Spizellomycetales survived freezing in all tests. All isolates in the Chytridiales also survived freezing in some tests. None of the isolates in the Rhizophydiales survived freezing in any of the tests. However, some isolates in the Rhizophydiales recovered growth after freezing if they were grown on PYG agar supplemented with either 1 % sodium chloride or 1 % glycerol prior to freezing. After freezing, the morphology of the thalli of all isolates was observed under LM. In those isolates that recovered growth after transfer to fresh media, mature zoosporangia were observed in the monocentric isolates and resistant sporangia or resting spores in the polycentric isolates. Encysted zoospores in some monocentric isolates also survived freezing. In some of the experiments the freezing and thawing process caused visible structural damage to the thalli. The production of zoospores after freezing and thawing was also used as an indicator of freeze tolerance. The chytrids in this study responded differently to freezing. These data add significantly to our limited knowledge of freeze tolerance in chytrids but leave many questions unanswered.     

Morphology of three polycentric rumen fungi and description of a procedure for the induction of zoosporogenesis and release of zoospores in cultures

Three polycentric rumen fungi, LL, LC2 and Ruminomyces elegans (C2), isolated from the rumen of cattle were grown in six culture media. LL and LC2 were morphologically similar. Their characteristics resembled those of Orpinomyces and Neocallimastix joyonii, and they grew well and produced sporangia after 3-4 d growth in all the media. R. elegans differed morphologically from LL and LC2, but although it also grew well in all media, abundant sporangia occurred only after 2-3 d growth in media containing cellulose. Undifferentiated sporangia were produced by all three isolates; differentiation of the sporangia did not occur in the spent growth media. However, if thalli possessing recently-formed sporangia were transferred to, or flooded with, fresh liquid medium or rumen fluid, zoosporogenesis and liberation of zoospores occurred within 17-20 min for isolates LL and LC2 and 30 min for R. elegans. Procedures for inducing zoosporogenesis by polycentric anaerobic fungi are described.     

Production and biochemical characterization of a novel cellulase-poor alkali-thermo-tolerant xylanase from <Emphasis Type="Italic">Coprinellus disseminatus</Emphasis> SW-1 NTCC 1165

Fungi producing xylanases are plentiful but alkali-thermo-tolerant fungi producing cellulase-poor xylanase are rare. Out of 12 fungal strains isolated from various sources, Coprinellus disseminatus SW-1 NTCC 1165 yielded the highest xylanase activity (362.1 IU/ml) with minimal cellulase contamination (0.64 IU/ml). The solid state fermentation was more effective yielding 88.59% higher xylanase activity than that of submerged fermentation. An incubation period of 7 days at 37°C and pH 6.4 accelerated the xylanase production up to the maximum level. Among various inexpensive agro-residues used as carbon source, wheat bran induced the maximum xylanase titres (469.45 IU/ml) while soya bean meal was the best nitrogen source (478.5 IU/ml). A solid substrate to moisture content ratio of 1:3 was suitable for xylanase production while xylanase titre was repressed with the addition of glucose and lactose. The xylanase and laccase activities under optimized conditions were 499.60 and 25.5 IU/ml, respectively along with negligible cellulase contamination (0.86 IU/ml). Biochemical characterization revealed that optimal xylanase activity was observed at pH 6.4 and temperature 55°C and xylanase is active up to pH 9 (40.33 IU/ml) and temperature 85°C (48.81 IU/ml). SDS–PAGE and zymogram analysis indicated that molecular weight of alkali-thermo-tolerant xylanase produced by C. disseminatus SW-1 NTCC 1165 was 43 kDa.     

A Bifunctional Endoglucanase/Endoxylanase from <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> with Potential Use in Industrial Processes at Different pH

Cellulomonas flavigena CDBB-531 was found to secrete a bifunctional cellulase/xylanase with a molecular mass of 49 kDa and pI 4.3. This enzyme was active on Remazol brilliant blue-carboxymethylcellulose (RBB-CMC) and Remazol brilliant blue-xylan (RBB-X). Based on thin-layer chromatographic analysis of the degradation products, the cellulase activity produced glucose, cellobiose, cellotriose, and cellotetraose from CMC as the substrate. When xylan from birchwood was used, end products were xylose, arabinose, and xylobiose. The bifunctional enzyme showed a pH optimum of 6 for cellulase activity and 9 for xylanase activity, which pointed out that this enzyme had separate sites for each activity. In both cases, the apparent optimum temperature was 50 degrees C. The predicted amino acid sequence of purified protein showed similarity with the catalytic domain of several glycosyl hydrolases of family 10.     

The enzymatic hydrolysis and fermentation of pretreated wood substrates

Aspenwood chips were pretreated by steam explosion. The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species. Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates. When T. harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production. When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1%. Highest xylanase activities were detected after growth of T. reesei C30 and T. harianum E58 on xylan and solka floc respectively. All of the steam exploded fractions were at least partially hydrolyzed by the T. harzianum E58 cellulase system. The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol. About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.     

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70–650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.     

Cellulases and xylanase of an anaerobic rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50 degrees C, whereas FPase had an optimum of 45 degrees C. The pH and temperature optima for xylanase activity were pH 6.0 and 50 degrees C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the R1 isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of R1 on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellulases and xylanase of an anaerobic rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan.

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50 degrees C, whereas FPase had an optimum of 45 degrees C. The pH and temperature optima for xylanase activity were pH 6.0 and 50 degrees C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the R1 isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of R1 on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an anaerobic fungus from llama faeces.

An anaerobic fungus was isolated from llama faeces. Based on its morphological characteristics, polyflagellated zoospores, extensive rhizoid system and the formation of monocentric colonies, the fungus is assigned to the genus Neocallimastix. Neocallimastix sp. L2 is able to grow on several poly-, oligo- and monosaccharides. It differs from other Neocallimastix isolates in its inability to ferment inulin. Neocallimastix sp. L2 requires CO2 for growth. In the presence of 100% CO2 in the gas phase glucose is fermented to H2, CO2, formate, acetate, lactate, succinate and ethanol (33.8, 15.4, 74.3, 69.2, 26.7, 8.2, and 28.7 mmol per 100 mmol glucose, respectively). Reduced sulphur compounds can be used as sulphur source and ammonium or amino acids as nitrogen source. The temperature range for glucose fermentation is from 37 to 42 degrees C with an optimum of around 38 degrees C. The pH range for glucose fermentation is from pH 6 to pH 8 with a broad optimum between pH 6.5 and pH 7.5. The zoospores of Neocallimastix sp. L2 contain ribosomal 'globules' and hydrogenosomes. In the kinetosomes of the zoospores spurs, scoops and skirts are visible. In both the rhizoids and the sporangia 'crystal bodies' and hydrogenosomes are present. Mitochondria were not detected in either of these life stages.     

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.     

Isolation, characterization and fibre degradation potential of anaerobic rumen fungi from cattle

A total of 20 fungal cultures were isolated from the rumen of cattle fed a high fibre-containing diet. All of the isolates showed polycentric growth patterns and were identified as different strains of Orpinomyces and Anaeromyces. Enzyme assays of most of the isolates showed the highest carboxymethylcellulase (CMCase) and xylanase activities after 96 h of growth and highest avicelase activity after 120 h. Among all enzymes tested, xylanase activity was the highest, followed by CMCase and avicelase. The results of the in vitro fibre digestibility and rumen fermentation analyses revealed that the addition of fungal cultures significantly increased acetate, in vitro dry matter digestibility, partition factor values and microbial biomass synthesis levels. Overall, Orpinomyces spp. were found to be the better enzyme producers and fibre degraders than Anaeromyces spp.     

Laccase, cellulase and xylanase activities during growth ofPleurotus sajor-caju on sagohampas

Sagohampas, the fibrous pith residue left after starch extraction from sago palm, is abundant at sago-processing factories and can be used as a substrate for the production of laccase by solid substrate fermentation (SSF) withPleurotus sajorcaju, an edible mushroom. The fungus grown onhampas with an adjusted carbon : nitrogen ratio of 35:1, exhibited high laccase activity together with variable cellulase (0.3-2.8 U/g) and xylanase (0.9-10.1 U/g) activity. The maximum amount of laccase produced was approximately 17.7 U/g after 6 days of SSF using 4-week-old inoculum at a density of 10%. With the mature four-week inoculum, laccase activity increased 12-fold compared to that achieved with two-week-old inoculum. The optimum pH and temperature of the crude laccase were 6.0 and 50‡C, respectively. The apparent K_m and V_max values obtained were 0.073 mM and 0.962 U/min, respectively. The maximum laccase activity could be almost doubled after 6 days of fermentation by addition of 0.2 mM vanillin or ferulic acid; the cellulose to lignin ratio increased significantly during the 12 days of SSF, from 2.74 in the control to 3.3, when 0.2 mM of either vanillin or ferulic acid was added to the substrate.