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1.
Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification
and fermentation (SSF) system comprised of Trichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation by Saccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised of Clostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H 2SO 4) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H 2SO 4). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference
particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts
of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed
continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC.
The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time.
At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system
(77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF
carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration
in the range of 5–60 g/L cellulose in the feed. 相似文献
2.
Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42°C,
using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate at a
cellulase loading of 37 filter paper units/g of cellulose, and a β-glucosidase loading of 38 IU/gof cellulose. The detoxification
of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol
yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37°C, owing to the cessation of ethanol fermentation after the first 10 h. 相似文献
3.
Simultaneous saccharification and fermentation (SSF) of switchgrass was performed following aqueous ammonia pretreatment.
Switchgrass was soaked in aqueous ammonium hydroxide (30%) with different liquid–solid ratios (5 and 10 ml/g) for either 5
or 10 days. The pretreatment was carried out at atmospheric conditions without agitation. A 40–50% delignification (Klason
lignin basis) was achieved, whereas cellulose content remained unchanged and hemicellulose content decreased by approximately
50%. The Sacccharomyces cerevisiae (D 5A)-mediated SSF of ammonia-treated switchgrass was investigated at two glucan loadings (3 and 6%) and three enzyme loadings
(26, 38.5, and 77 FPU/g cellulose), using Spezyme CP. The percentage of maximum theoretical ethanol yield achieved was 72.
Liquid–solid ratio and steeping time affected lignin removal slightly, but did not cause a significant change in overall ethanol
conversion yields at sufficiently high enzyme loadings. These results suggest that ammonia steeping may be an effective method
of pretreatment for lignocellulosic feedstocks. 相似文献
4.
The Pol6 mutant of Penicillium occitanis fungus is of great biotechnological interest since it possesses a high capacity of cellulases and β-glucosidase production
with high cellulose degradation efficiency (Jain et al., Enzyme Microb Technol, 12:691–696, 1990; Hadj-Taieb et al., Appl Microbiol Biotechnol, 37:197–201, 1992; Ellouz Chaabouni et al., Enzyme Microb Technol, 16:538–542, 1994; Ellouz Chaabouni et al., Appl Microbiol Biotechnol, 43:267–269, 1995). In this work, two forms of β-glucosidase (β-glu 1 and β-glu 2) were purified from the culture supernatant
of the Pol6 strain by gel filtration, ion exchange chromatography, and preparative anionic native electrophoresis. These enzymes
were eluted as two distinct species from the diethylamino ethanol Sepharose CL6B and anionic native electrophoresis. However,
both behaved identically on sodium dodecyl sulfate polyacrylamide gel electrophoresis (MW, 98 kDa), shared the same amino
acid composition, carbohydrate content (8%), and kinetic properties. Moreover, they strongly cross-reacted immunologically.
They were active on cellobiose and pNPG with Km values of 1.43 and 0.37 mM, respectively. β-glu 1 and β-glu 2 were competitively inhibited by 1 mM of glucose and 0.03 mM
of δ-gluconolactone. They were also significantly inhibited by Hg 2+ and Cu 2 at 2 mM. The addition of purified enzymes to the poor β-glucosidase crude extract of Trichoderma reesei increased its hydrolytic efficiency on H 3P0 4 swollen cellulose but had no effect with P. occitanis crude extract. Besides their hydrolytic activities, β-glu 1 and β-glu 2 were endowed with trans-glycosidase activity at high
concentration of glucose. 相似文献
5.
The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the
fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth
and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred
in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase
production measured as product yield ( Y
P/S
) and volumetric productivity ( Q
P
) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on
cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost
all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The
enzyme was substantially stable at 60°C and may find application in some industrial processes. 相似文献
6.
Previous shake flask and stirred tank evaluations of temperature tolerant (37–43°C) yeasts in simultaneous saccharification and fermentation (SSF) on Sigmacell-50 cellulose substrates to ethanol have identified several good microorganisms for further SSF studies (27). Of these, the glucose fermenting yeastCandida acidothermophilum, C. brassicae, Saccharomyces cerevisiae, S. uvarum, and a mixed culture of the cellobiose fermenting yeastBrettanomyces clausenii withS. cerevisiae as a control were chosen for shake flask SSF screening experiments with pretreated wheat straw. This study indicates that theSaccharomyces strainscerevisiae anduvarum, give very good performance at high cellulase loadings or when supplemented with Novo-188 β-glucosidase. In fact, with the higher enzyme loadings these yeast will give complete conversion of cellulose to ethanol. Yet at the lower, more economical enzyme loadings, the mixed culture ofBrettanomyces clausenii andS. cerevisiae performs better than any single yeast. 相似文献
7.
Hydrolysis of cellulose by Trichoderma cellulases often results in a mixture of glucose, cellobiose, and low-mol-wt cellodextrins. Cellobiose is nonfermentable
for most yeasts, and therefore it has to be hydrolyzed to glucose by β-glucosidase prior to ethanol fermentation. In the present
study, the β-glucosidase production of one Penicillium and three Aspergillus strains, which were previously selected out of 24 strains, was investigated on steam pretreated willow. Both steam-pretreated
willow and hemicellulose hydrolysate, released during steam explosion of willow, were used as carbon sources. Reference cultivation
runs were performed using prehydrolyzed Solka Floc and glucose: The four strains were compared with Trichoderma reesei regarding sugar consumption and β-glucosidase production. Aspergillus niger and Aspergillus phoenicis proved to be the best enzyme producers on hemicellulose hydrolysate. The maximum β-glucosidase activity, 4.60 IU/mL, was
obtained when A. phoenicis was cultivated on the mixture of hemicellulose hydrolysate and steam-pretreated willow. The maximum yield of enzyme activity,
502 IU/g total carbohydrate, was obtained when Aspergillus foetidus was cultivated on the hemicellulose hydrolysate. 相似文献
8.
Cellulase production was investigated in pH-controlled cultures of Acremonium cellulolyticus. The response to culture pH was investigated for three cellulolytic enzymes, carbomethyl cellulase (CMCase), avicelase, and
β-glucosidase. Avicelase and β-glucosidase showed similar profiles, with maximum activity in cultures at pH 5.5–6. The CMCase
activity was highest in a pH 4 culture. At an acidic pH, the ratios of CMCase and avicelase activity to cellulase activity
defined by filter paper unit were high, but at a neutral pH, the β-glucosidase ratio was high. The pH 6.0 culture showed the
highest cellulase activity within the range of pH 3.5–6.5 cultures. The saccharification activity from A. cellulolyticus was compared to those of the cellulolytic enzymes from other species. The A. cellulolyticus culture broth had a saccharification yield comparable to those of the Trichoderma enzymes GC220 and Cellulosin T2, under conditions with the same cellulase activity. The saccharification yields from Solka
floc, Avicel, and waste paper, measured as the percent of released reducing sugar to dried substrate, were greater than 80%
after 96 h of reaction. The yields were 16% from carboxymethylcellulose and 26% from wood chip refiner. Thus, the A. cellulolyticus enzymes were suitable for converting cellulolytic biomass to reducing sugars for biomass ethanol production. This study is
a step toward the establishment of an efficient system to reutilize cellulolytic biomass. 相似文献
9.
The lactic acid bacterium, Lactobacillus casei, produces an intracellular β-glucosidase when grown on Man-Rogosa-Sharpe (MRS) medium with cellobiose as carbon source. The
β-glucosidase activity is produced intracellulary, and no extracellulary activity was detected. The enzyme was purified by
ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase as estimated
by gel filtration was 480 kDa, consisting of six probably identical subunits. The enzyme exhibited optimum activity at 35°C
and pH 6.3 with citrate-phosphate buffer. The enzyme was active against soluble glycosides with (1→4)-β configuration and
from Lineweaver Burk plots, K m value of 16 mmol/L was found for β-pNPG. The β-glucosidase was competitively inhibited by glucose, and no glycosyl transferase
activity was observed in the presence of ethanol. 相似文献
10.
Lignocellulosic materials pretreated using liquid hot water (LHW) (220°C, 5 MPa, 120 s) were fermented to ethanol by batch
simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisiae in the presence of Trichoderma reesei cellulase. SSF of sugarcane bagasse (as received), aspen chips (smallest dimension 3 mm), and mixed hardwood flour (−60 +70
mesh) resulted in 90% conversion to ethanol in 2–5 d at enzyme loadings of 15–30 FPU/g. In most cases, 90% of the final conversion
was achieved within 75 h of inoculation. Comminution of the pretreated substrates did not affect the conversion to ethanol.
The hydrolysate produced from the LHW pretreatment showed slight inhibition of batch growth of S. cerevisiae. Solids pretreated at a concentration of 100 g/L were as reactive as those pretreated at a lower concentration, provided
that the temperature was maintained at 220°C. 相似文献
11.
An aquatic weed biomass, Eicchornia crassipes, present in abundance and leading to a threatening level of water pollution was used as substrate for cellulase and β-glucosidase
production using wild-type strain Aspergillus niger RK3 that was isolated from decomposing substrate. Alkali treatment of the biomass (10%) resulted in a 60–66% increase in
endoglucanase, exoglucanase, and β-glucosidase production by the A. niger RK3 strain in semi-solid-state fermentation. Similarly, the alkali-treated biomass led to a 45–54% increase in endo- and
exoglucanase and a higher (98%) increase in β-glucosidase production by Trichoderma reesei MTCC164 under similar conditions. However, the cocultivation of A. niger RK3 and T. reesei MTCC164 at a ratio of 3:1 showed a 20–24% increase in endo- and exoglucanase activities and about a 13% increase in the β-glucosidase
activity over the maximum enzymatic activities observed under single culture conditions. Multistep physical (ultraviolet)
and chemical ( N-methyl- N′-nitrosoguanidine, sodium azide, colchicine) mutagenesis of the A. niger RK3 strain resulted in a highly cellulolytic mutant, UNSC-442, having an increase of 136, 138, and 96% in endoglucanase,
exoglucanase, and β-glucosidase, activity, respectively. The cocultivation of mutant UNSC-442 along with T. reesei MTCC164 (at a ratio of 3:1) showed a further 10–11% increase in endo- and exoglucanase activities and a 29% increase in β-glucosidase
activity in semi-solid-state fermentation. 相似文献
12.
The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity
by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography
(HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by
sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β- d-glucopyranoside hydrolysis, apparent K
m and V
max values were found to be 0.09 m M and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly
inhibited by Fe 2+ and activated about 35% by Ca 2+. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production
rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation
was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 13C nuclear magnetic resonance spectroscopy. 相似文献
13.
Biochemical conversion of lignocellulosic biomass to ethanol involves size reduction, preprocessing, pretreatment, enzyme
hydrolysis, and fermentation. In recent years, microbial preprocessing has been gaining attention as a means to produce labile
biomass for lessening the requirement of pretreatment severity. However, loss of sugars due to microbial consumption is a
major consequence, suggesting its minimization through optimization of nutrients, temperature, and preprocessing time. In
this work, we emphasized estimation of fungal preprocessing time, at which higher sugar yields can be achieved after preprocessing
and enzyme hydrolysis. The estimation is based on the enzymatic activity profile obtained by treating switchgrass with Phanerochaete chrysosporium for 28 days. Enzyme assays were conducted once in every 7 days for 28 days, for activities of phenol oxidase, peroxidase,
β-glucosidase, β-xylosidase, and cellobiohydrolase. We found no activity for phenol oxidase and peroxidase, but the greatest
activities for cellulases on the seventh day. We then treated switchgrass for 7 days with P. chrysosporium and observed that the preprocessed switchgrass had higher glucan (39%), xylan (17.5%), and total sugar yields (25.5%) than
the unpreprocessed switchgrass (34%, 37.5%, and 20.5%, respectively, p < 0.05). This verifies the utility of using enzyme assays for initial estimation of preprocessing time to enhance sugar yields. 相似文献
14.
A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L
of manure (dry basis [w/w]), 2 g/L KH 2PO 4, 2 mL/L of Tween-80, and 2mg/L of CoCl 2. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL)
and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing
manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced
glucose was significantly ( p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei. 相似文献
15.
The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic
proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and β-glucosidases.
One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of β-glucosidases secreted by this fungus is insufficient for effective cellulose conversion.
We investigated the production of cellulases and β-glucosidases in shake-flask cultures by applying three pH-controlling strategies:
( 1) the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment
performed, ( 2) the pH was adjusted to 6.0 daily, and ( 3) the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources—Solka
Floc 200, glucose, lactose, and sorbitol—were added to standard Mandels nutrients. The lowest β-glucosidase activities were
obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of β-glucosidase
were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium
on β-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied. 相似文献
16.
The intergeneric protoplast fusion hybrid (Bs/C 005) between Cellulomonas sp. and Bacillus subtilisproduced extracellular aryl β-glucosidase that is otherwise intracellular in parental Cellulomonassp. This extracellular aryl β-glucosidase was active at relatively higher temperature (60°C) and lower pH (pH 5.0) conditions
than that of Cellulomonas enzyme. It also exhibited increased thermostability and stability over wide range of pH. Cellobiase activity, distinctly
different from aryl β-glucosidase detected in both Cellulomonassp. Bs/C 005, was only intracellular. Cellobiase from Bs/C 005, however, was more thermostable than that of Cellulomonassp. 相似文献
17.
Production of β-cyclodextrin (CD) with high-dextrose equivalent (DE) starch hydrolysates by simultaneous fermentation and
cyclization (SFC) gives higher yields than using only the enzyme CGTase, because fermentation eliminates glucose and maltose
that inhibit CD production, while at the same time, produces ethanol that increases yield. A 10% (w/v) solution of cassava
starch, liquefied with α-amylase, was incubated with CGTase using: only the enzyme, added ethanol (from 1 to 5%), and added
yeast, S. cerevisiae (12% w/v), plus nutrients, the latter being the SFC process. Reaction conditions were: 38αC, pH 6.0, DE from 2 to 25, and
3.3 mL of CGTase/L. The yield of β-CD has decreased with an increase in DE, and maximum reaction yields were found for DE
equal to 3.54, reaching 5.6, 14.7, and 11.5 mM β-CD, respectively. For an increase of DE, of approx 6 times (from 3.54 to
23.79), β-CD yield decreased 6 times for the first, and second reaction media with 3% (v/v) ethanol, and only approx 3 times
for SFC (from 11.5 to 3.73 m M), showing that this process is less sensitive to variations in the DE 相似文献
18.
Validoxylamine A is structurally similar to trehalose and acts a potent competivive inhibitor of trehalase. It has recently
been receiving increased attention as a potential material for the development of new insecticides or drugs. In this study,
β-glucosidase extracted from honeybees ( Apis cerana Fabr.) was used as a catalyst to produce validoxylamine A through enzymatic hydrolysis of validamycin A. β-Glucosidase was
separated and purified from honeybees, and its characteristics were examined. The results showed that β-glucosidase was stable
across a range of temperatures from 30 to 40°C and across a relatively wide range of pH values from 5.0 to 7.5. Investigation
of the biocatalyzed hydrolysis process from validamycin A to validoxylamine A with β-glucosidase revealed that both the substrate
(validamycin A) and the product (validoxylamine A) inhibited β-glucosidase activity. The inhibition constant of the substrate
K value was 5.01 m M, and that of the product K
ip value was 1.32 m M. This product inhibition was competitive. 相似文献
19.
Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting
degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present
study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated
willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration
levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme
activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was
1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained
without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity
even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration.
Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production. 相似文献
20.
Research is needed to allow more efficient processing of lignocellulose from abundant plant biomass resources for production
to fuel ethanol at lower costs. Potential dedicated feedstock species vary in degrees of recalcitrance to ethanol processing.
The standard dilute acid hydrolysis pretreatment followed by simultaneous sacharification and fermentation (SSF) was performed
on leaf and stem material from three grasses: giant reed ( Arundo donax L.), napiergrass ( Pennisetum purpureum Schumach.), and bermudagrass ( Cynodon spp). In a separate study, napiergrass, and bermudagrass whole samples were pretreated with esterase and cellulose before
fermentation. Conversion via SSF was greatest with two bermudagrass cultivars (140 and 122 mg g −1 of biomass) followed by leaves of two napiergrass genotypes (107 and 97 mg g −1) and two giant reed clones (109 and 85 mg g −1). Variability existed among bermudagrass cultivars for conversion to ethanol after esterase and cellulase treatments, with
Tifton 85 (289 mg g) and Coastcross II (284 mg g −1) being superior to Coastal (247 mg g −1) and Tifton 44 (245 mg g −1). Results suggest that ethanol yields vary significantly for feedstocks by species and within species and that genetic breeding
for improved feedstocks should be possible. 相似文献
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