Corn fiber hydrolysis by Thermobifida fusca extracellular enzymes.

Thermobifida fusca was grown on cellulose (Solka-Floc), xylan or corn fiber and the supernatant extracellular enzymes were concentrated. SDS gels showed markedly different protein patterns for the three different carbon sources. Activity assays on a variety of synthetic and natural substrates showed major differences in the concentrated extracellular enzyme activities. These crude enzyme preparations were used to hydrolyze corn fiber, a low-value biomass byproduct of the wet milling of corn. Approximately 180 mg of reducing sugar were produced per gram of untreated corn fiber. When corn fiber was pretreated with alkaline hydrogen peroxide, up to 429 mg of reducing sugars were released per gram of corn fiber. Saccharification was enhanced by the addition of beta-glucosidase or by the addition of a crude xylanase preparation from Aureobasidium sp.

Analysis of ethanol-glucose mixtures by two microbial sensors: application of chemometrics and artificial neural networks for data processing.

Although biosensors based on whole microbial cells have many advantages in terms of convenience, cost and durability, a major limitation of these sensors is often their inability to distinguish between different substrates of interest. This paper demonstrates that it is possible to use sensors entirely based upon whole microbial cells to selectively measure ethanol and glucose in mixtures. Amperometric sensors were constructed using immobilized cells of either Gluconobacter oxydans or Pichia methanolica. The bacterial cells of G. oxydans were sensitive to both substrates, while the yeast cells of P. methanolica oxidized only ethanol. Using chemometric principles of polynomial approximation, data from both of these sensors were processed to provide accurate estimates of glucose and ethanol over a concentration range of 1.0-8.0 mM (coefficients of determination, R(2)=0.99 for ethanol and 0.98 for glucose). When data were processed using an artificial neural network, glucose and ethanol were accurately estimated over a range of 1.0-10.0 mM (R(2)=0.99 for both substrates). The described methodology extends the sphere of utility for microbial sensors.

Transient infrared spectroscopy for detection of toxigenic fungi in corn: potential for on-line evaluation.

An urgent need for rapid sensors to detect contamination of food grains by toxigenic fungi such as Aspergillus flavus prompted research and development of Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) as a highly sensitive probe for fungi growing on the surfaces of individual corn kernels. However, the photoacoustic technique has limited potential for screening bulk corn because currently available photoacoustic detectors can accommodate only a single intact kernel at a time. Transient infrared spectroscopy (TIRS), on the other hand, is a promising new technique that can acquire analytically useful infrared spectra from a moving mass of solid materials. Therefore, the potential of TIRS for on-line, noncontact detection of A. flavus contamination in a moving bed of corn kernels was explored. Early test results based on visual inspection of TIRS spectral differences predict an 85% or 95% success rate in distinguishing healthy corn from grain infected with A. flavus. Four unique infrared spectral features which identified infected corn in FTIR-PAS were also found to be diagnostic in TIRS. Although the technology is still in its infancy, the preliminary results indicate that TIRS is a potentially effective screening method for bulk quantities of corn grain.

Degradation of starch-poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) bioplastic in tropical coastal waters.

Extruded bioplastic was prepared from cornstarch or poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV) or blends of cornstarch and PHBV. The blended formulations contained 30 or 50% starch in the presence or absence of polyethylene oxide (PEO), which enhances adherence of starch granules to PHBV. Degradation of these formulations was monitored for 1 year at four stations in coastal water southwest of Puerto Rico. Two stations were within a mangrove stand. The other two were offshore; one of these stations was on a shallow shoulder of a reef, and the other was at a location in deeper water. Microbial enumeration at the four stations revealed considerable flux in the populations over the course of the year. However, in general, the overall population densities were 1 order of magnitude less at the deeper-water station than at the other stations. Starch degraders were 10- to 50-fold more prevalent than PHBV degraders at all of the stations. Accordingly, degradation of the bioplastic, as determined by weight loss and deterioration of tensile properties, correlated with the amount of starch present (100% starch >50% starch > 30% starch > 100% PHBV). Incorporation of PEO into blends slightly retarded the rate of degradation. The rate of loss of starch from the 100% starch samples was about 2%/day, while the rate of loss of PHBV from the 100% PHBV samples was about 0.1%/day. Biphasic weight loss was observed for the starch-PHBV blends at all of the stations. A predictive mathematical model for loss of individual polymers from a 30% starch-70% PHBV formulation was developed and experimentally validated. The model showed that PHBV degradation was delayed 50 days until more than 80% of the starch was consumed and predicted that starch and PHBV in the blend had half-lives of 19 and 158 days, respectively. Consistent with the relatively low microbial populations, bioplastic degradation at the deeper-water station exhibited an initial lag period, after which degradation rates comparable to the degradation rates at the other stations were observed. Presumably, significant biodegradation occurred only after colonization of the plastic, a parameter that was dependent on the resident microbial populations. Therefore, it can be reasonably inferred that extended degradation lags would occur in open ocean water where microbes are sparse.

Detection of ethanol in a two-component glucose/ethanol mixture using a nonselective microbial sensor and a glucose enzyme electrode.

Chemometric theory was applied to a microbial sensor for determinations of ethanol in the presence of glucose. Microbial sensors, consisting of Gluconobacter oxydans cells immobilized on Clark-type amperometric oxygen electrodes, exhibited good sensitivity but low selectivity toward ethanol and glucose. An Eksan-G commercial glucose analyzer was used as a second sensor for multivariate calibration and analyses. Microbial sensors exhibited nearly complete additivity for total glucose plus ethanol concentrations from 0.0 to 0.6 mM. Within this linear range, chemometric analyses provided estimates of ethanol concentration with measurement errors of less than 8%. Multivariate calibration thus is a promising approach to enhance the usefulness of microbial sensors.

Effects of high oxygen concentrations on microbial biosensor signals. Hyperoxygenation by means of perfluorodecalin.

Amperometric biosensors register oxygen depletion in response to analyte catabolism, and thus are limited by the availability of dissolved oxygen. Microbial sensors containing immobilized cells of Gluconobacter oxydans were hyperoxygenated to 400% of control levels and the effects on sensor responses to glucose were determined. Oxygenated perfluorodecalin (a completely fluorinated organic substance) was as effective in hyperoxygenation as direct sparging with O2, increasing sensor base medium oxygen concentrations from 9.3 to 37 mg/l. Hyperoxygenation enhanced maximal biosensor response amplitudes, particularly at high cell loading densities. Maximal response rates were also improved, although less dramatically. Results suggest that hyperoxygenation may be a new general approach for modulating biosensor responses.

Neural network pattern recognition of photoacoustic FTIR spectra and knowledge-based techniques for detection of mycotoxigenic fungi in food grains.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS), a highly sensitive probe of the surfaces of solid substrates, is used to detect toxigenic fungal contamination in corn. Kernels of corn infected with mycotoxigenic fungi, such as Aspergillus flavus, display FTIR-PAS spectra that differ significantly form spectra of uninfected kernels. Photoacoustic infrared spectral features were identified, and an artificial neural network was trained to distinguish contaminated form uncontaminated corn by pattern recognition. Work is in progress to integrate epidemiological information about cereal crop fungal disease into the pattern recognition program to produce a more knowledge-based, and hence more reliable and specific, technique. A model of a hierarchically organized expert system is proposed, using epidemiological factors such as corn variety, plant stress and susceptibility to infection, geographic location, weather, insect vectors, and handling and storage conditions, in addition to the analytical data, to predict Al. flavus and other kinds of toxigenic fungal contamination that might be present in food grains.

Identification of Fourier transform infrared photoacoustic spectral features for detection of Aspergillus flavus infection in corn.

Aspergillus flavus and other pathogenic fungi display typical infrared spectra which differ significantly from spectra of substrate materials such as corn. On this basis, specific spectral features have been identified which permit detection of fungal infection on the surface of corn kernels by photoacoustic infrared spectroscopy. In a blind study, ten corn kernels showing bright greenish yellow fluorescence (BGYF) in the germ or endosperm and ten BGYF-negative kernels were correctly classified as infected or not infected by Fourier transform infrared photoacoustic spectroscopy. Earlier studies have shown that BGYF-positive kernels contain the bulk of the aflatoxin contaminating grain at harvest. Ten major spectral features, identified by visual inspection of the photoacoustic spectra of A. flavus mycelium grown in culture versus uninfected corn, were interpreted and assigned by theoretical comparisons of the relative chemical compositions of fungi and corn. The spectral features can be built into either empirical or knowledge-based computer models (expert systems) for automatic infrared detection and segregation of grains or kernels containing aflatoxin from the food and feed supply.

Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides.

Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4-O-acetyl-beta-D-xylopyranoside in almost 90% yield. Methyl 2,3-di-O-acetyl beta-D-xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4- and 3,4-), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated beta-1,4-linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di-O-acetylated residues. The T. reesei AcXE attacked acetylated methyl beta-D-glucopyranosides and beta-D-mannopyranosides in a manner similar to the xylopyranosides.

Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates.

Substrate specificity of a purified acetylxylan esterase from Schizophyllum commune was investigated on a variety of methyl per-O-acetyl glycopyranosides, methyl di-O-acetyl-beta-D-xylopyranosides and acetylated polysaccharides. The enzyme preferentially deacetylated the 3-position of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside. Removal of the 3-acetyl group from the xylopyranoside was accompanied by a slower deacetylation at positions 2 and 4. A similarly slower, accompanying deacetylation occurred primarily at position 2 with the glucopyranoside. Such specificity corresponds well to the expected function of the esterase in acetylxylan degradation. Of the three possible diacetates of methyl beta-D-xylopyranoside, the 3,4-diacetate was found to be the most rapidly deacetylated. Unexpectedly, products of its deacetylation were a mixture of 2- and 4-monoacetate. The formation of the methyl 2-O-acetyl-beta-D-xylopyranoside involved an enzyme-mediated acetyl group transfer because the rate of the enzyme-catalyzed reaction exceeded the rate of spontaneous migration of acetyl groups. This is the likely mechanism for acetyl removal from position 2 in the native substrate. The enzyme exhibited the highest regioselectivity with methyl 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranoside. An 80% conversion of this substrate to methyl 4,6-di-O-acetyl-beta-D-mannopyranoside, a new mannose derivative, was achieved. In contrast to the majority of lipases and esterases exploited for regioselective deacetylation, the S. commune acetylxylan esterase did not attack the C-6 acetyl linkages in methyl hexopyranosides when other acetyl groups were available.

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans.

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE.

FET-microbial sensor for xylose detection based on Gluconobacter oxydans cells.

A potentiometric biosensor for xylose was devised utilizing Gluconobacter oxydans whole cells. Immobilization methods based on physical adsorption were used for G. oxydans cells and extracellular pH changes resulting from xylose dehydrogenation were monitored by a field effect transistor (FET). The G. oxydans, FET-based sensor detected xylose at a lower limit of 0.5 mM. From 5.0 to 30 mM xylose, the response of the sensor was linear. Expectedly, output signals were significantly suppressed by buffer (Tris-HCl). Responses were essentially stable for at least four weeks of storage and showed only a slight loss of initial xylose sensitivity. Xylitol exerted an insignificant influence on the sensor's response to xylose. However, the response to glucose was 5 times higher in relation to that of xylose at the same concentration (1 mM). For xylose determinations in the presence of glucose, a two-step assay is discussed.

Binding of extracellular carboxymethylcellulase activity from the marine shipworm bacterium to insoluble cellulosic substrates.

The binding of extracellular endoglucanase, a carboxymethylcellulase (CMCase), produced by the marine shipworm bacterium to insoluble cellulose substrates was investigated. Up to 70% of CMCase activity bound to cellulosic substrates, and less than 10% bound to noncellulosic substrates. CMCase binding to cellulose was enhanced in basal salt medium or sodium phosphate buffer containing 0.5 M NaCl. Increased cellulose particle size correlated with decreased CMCase binding. Also, cellulose treated with either 5 N NaOH or commercial cellulase reduced the CMCase binding to these surfaces. Pretreatment of CMCase preparations with 0.01% sodium dodecyl sulfate, 5% beta-mercaptoethanol, and 5 mM EDTA or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) had little effect on binding to cellulose. While pretreatment of CMCase with trypsin, chymotrypsin, and pronase had little effect on CMCase enzymatic activity, the ability to bind to cellulose was greatly diminished by these treatments.

Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae.

The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated. The beta-glucosidase from Cl. lusitaniae appeared to be a soluble cytoplasmic enzyme. This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source. Glucose, but not cellobiose, uptake was observed for cells grown on glucose. The Ks and Vmax values for cellobiose transport were different when Cl. lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1). The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions. The beta-glucosidase from C. wickerhamii was extracytoplasmically located. This yeast transported glucose, but not cellobiose, under all growth conditions tested. The Ks for glucose uptake was 0.13-0.28 mM when C. wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose. The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1). Cellobiose did not inhibit glucose uptake in either yeast. Glucose partially inhibited cellobiose transport in C. lusitaniae, but only if the yeast was grown aerobically. In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin.

Adhesive properties of a symbiotic bacterium from a wood-boring marine shipworm.

Adhesive properties of a cellulolytic, nitrogen-fixing bacterium isolated from a marine shipworm by Waterbury et al. (J. B. Waterbury, C. B. Calloway, and R. D. Turner, Science 221:1401-1403, 1983) are described. S-labeled cells of the shipworm bacterium bound preferentially to Whatman no. 1 cellulose filter paper, compared with its binding to other cellulose substrata or substrata lacking cellulose. The ability of the bacteria to bind to Whatman no. 1 filter paper was significantly reduced by glutaraldehyde or heat treatment of cells. Pretreatment of cells with azide, valinomycin, gramicidin-D, bis-hexafluoroacetylacetone (1799), or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone inhibited adhesion activity. Cells pretreated with pronase or trypsin also exhibited reduced binding activity, but chymotrypsin and peptidase had no effect on adhesion activity. Cellodextrins and methyl cellulose 15 inhibited the adhesion of shipworm bacteria to filter paper, whereas glucose, cellobiose, and soluble carboxymethyl cellulose had no significant effect. The divalent cation chelators EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid] had little or no effect on adhesive properties of shipworm bacteria. Also, preabsorbing the substratum with extracellular endoglucanase isolated from the shipworm bacterium or 1% bovine serum albumin had no apparent effect on bacterial binding. Low concentrations (0.01%) of sodium dodecyl sulfate solubilized a fraction from whole cells, which appeared to be involved in cellular binding activity. After removal of sodium dodecyl sulfate, several proteins in this fraction associated with intact cells. These cells exhibited up to 50% enhanced binding to filter paper in comparison to cells which had not been exposed to the sodium dodecyl sulfate-solubilized fraction.

Measurement of protein biomass by Fourier transform infrared-photoacoustic spectroscopy.

A relatively new analytical technique, Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS), provides spectra of bacteria, fungi, and other microorganisms in solid states not suitable for conventional absorption spectroscopy. In this paper the feasibility of quantitative measurement of protein biomass on solid substrates by FTIR-PAS is examined and discussed. By measuring photoacoustic absorption bands from amide groups in the protein of microorganisms, the increase in biomass that occurs during growth was monitored directly and accurately. Incorporation of polyacrylonitrile into the sample as an internal standard was shown to be a convenient method for improving both the reliability and the range of detection by photoacoustic spectroscopy. Results of FTIR-PAS measurements of known quantities of microbial mass in simulated growth experiments suggest that the technique may be especially suitable for assays of microorganisms used in solid-state biosyntheses of drugs, hormones, and other biological agents.

Purification and characterization of an extracellular endoglucanase from the marine shipworm bacterium.

Bacterial cultures isolated from the gland of Deshayes of marine shipworm (Psiloteredo healdi) produced extracellular endoglucanase activity when cultured with 1% cellulose. An endoglucanase of subunit relative molecular mass 58,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to homogeneity from cell-free culture medium. Similarly, the relative molecular mass of the native enzyme was 60,100 as determined by gel permeation chromatography. No carbohydrate appeared to be associated with the purified protein. The action of the purified enzyme on various cellodextrins was also studied. Only interior glucosyl linkages of cellodextrin chains larger than cellotriose were cleaved by the enzyme and the centermost bond of cellohexaose was preferentially cleaved. The Km values of the purified endoglucanase were 0.12 mM for cellotetraose, 0.05 mM for cellopentaose, and 0.11 mM for cellohexaose. Glucose, cellobiose, and cellotriose did not inhibit enzymatic activity.

Extracellular endoglucanase activity by a novel bacterium isolated from marine shipworm.

An extracellular enzyme preparation from shipworm bacterium cultures dramatically increased reducing sugar content of carboxymethylcellulose (CMC3), but did not solubilize sugar from particulate cellulose. The preparation degraded cellodextrins larger than cellotriose (G3). Only interior cellodextrin chain linkages were cleaved and the center-most bond of cellohexaose (G6) was preferentially cleaved. Activity maxima were observed at 60 degrees C and between pH 5.0 and 7.0. The activity was resistant to protease treatment and little loss of activity was observed after 14 d at 25 degrees C.

Growth characteristics of a novel nitrogen-fixing cellulolytic bacterium.

Growth characteristics of a cellulolytic nitrogen-fixing bacterium isolated from a marine shipworm by Waterbury et al. (J. B. Waterbury, C. B. Calloway, and R. D. Turner, Science 221:1401-1403, 1983) are described. When grown microaerobically, the bacterium exhibited doubling times of about 2 days in cellulose-supplemented synthetic medium devoid of combined nitrogen. Maximum growth was reached 12 to 16 days after inoculation. Growth optima for pH, temperature, and NaCl concentration were 8.5, 30 to 35 degrees C, and 0.3 M, respectively. During growth the bacterium produced succinic acid (0.026%) and acetic acid (0.010%). Formic acid (0.010%) was produced during the stationary growth phase. No growth was observed when glucose was the sole carbon source. Cellobiose supported weak growth, while longer-chain-length cellodextrins supported extensive growth. Analysis of residual carbohydrates in the medium during growth indicated that the bacterium catabolized a terminal glucose moiety from the cellodextrin chain.

H2O2-dependent decolorization of Poly R-481 by particulate fractions from Phanerochaete chrysosporium.

A cell-free preparation from Phanerochaete chrysosporium culture medium decolorized the polymeric dye Poly R-481. The majority of this decolorization activity sedimented when centrifuged at 150,000 X g, indicating that it was associated with a particulate body. The activity was sensitive to heat, azide and cyanide, was stimulated by exogenously added H2O2, and was optimal around pH 4. Electron micrographs of the sedimented culture medium fraction showed the presence of numerous particulate structures. A similar dye decolorization activity from sonicated mycelium also sedimented at 150,000 X g.