Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*.

AIMS: A Lactobacillus buchneri strain NRRL B-30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B-30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B-30929 for fermenting mixed substrates from biomass hydrolysates. METHODS AND RESULTS: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B-30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B-30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1.98-2.35 g l(-1) ethanol from corn stover hydrolysates and 2.92-3.01 g l(-1) ethanol from wheat straw hydrolysates when supplemented with either 0.25x MRS plus 1% corn steep liquor or 0.5x MRS. CONCLUSIONS: The L. buchneri NRRL B-30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are valuable for future research in engineering L. buchneri NRRL B-30929 for fermentative production of ethanol and chemicals from biomass.

Biotechnological production and applications of pullulan.

Pullulan is a unique biopolymer with many useful traits and hundreds of patented applications. However, despite the fact that pullulan has been in commercial production for more than 25 years, few of these potential uses have been widely adopted. In large part this may be due to the relatively high price of pullulan. Nevertheless, the last few years have seen a resurgence in interest in pullulan, particularly for higher-value health and pharmaceutical applications.

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.

Modification of alternan by novel Penicillium spp.

Four strains identified as Penicillium spp. were isolated from soil samples based on their capacity to modify the unique polysaccharide, alternan. Spores from these isolates germinated in medium containing alternan and reduced the apparent molecular weight of alternan as determined by high-performance size exclusion chromatography and viscometry. However, the fungi exhibited limited growth on alternan and did not consume the substrate. The rheological properties of the modified alternan resembled those of commercial gum arabic. Thus, treatment of native alternan with spores from these Penicillium spp. strains constitutes a simple bioconversion method to quantitatively produce novel and potentially useful modified alternan.

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.

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.

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.

Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi.

An apiZYM system, with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium anisopliae. Similar enzyme profiles were obtained for two of the N. rileyi isolates (Mississippi, Ecuador) regardless of whether culture medium (Sabouraud-maltose-yeast) or cuticle (from larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) were used as substrates. Centroid-clustering analysis revealed three distinct enzyme profiles.

Colloidal Gold Cytochemistry of Endo-1,4-beta-Glucanase, 1,4-beta-D-Glucan Cellobiohydrolase, and Endo-1,4-beta-Xylanase: Ultrastructure of Sound and Decayed Birch Wood.

Colloidal gold coupled to endo-1,4-beta-glucanase II (EG II) and 1,4-beta-D-glucan cellobiohydrolase I (CBH I), isolated from Trichoderma reesei (QM9414), and endo-1,4-beta-xylanase from Aureobasium pullulans (NRRLY-2311-1) was used successfully to determine the ultrastructural localization of cellulose and xylan in sound birch wood. In addition, these enzyme-gold complexes demonstrated the distribution of cellulose and xylan after decay by three white rot fungi, Phanerochaete chrysosporium, Phellinus pini, and Trametes versicolor, and one brown rot fungus, Fomitopis pinicola. Transverse sections of sound wood showed that EG II was localized primarily on the S(1) layer of the secondary wall, whereas CBH I labeled all layers of the secondary wall. Oblique sections showed a high concentration of gold labeling, using EG II or CBH I. Preference for the sides of the microfibrillar structure was observed for both EG II and CBH I, whereas only CBH I had a specificity for the cut ends of microfibrils. Labeling with the xylanase-gold complex occurred primarily in the inner regions of the S(2) layer, S(1), and the middle lamella. In contrast, little labeling occurred in the middle lamella with EG II or CBH I. Intercellular regions within the cell corners of the middle lamella were less electron dense and labeled positively when EG II- and xylanase-gold were used. Wood decayed by P. chrysosporium or P. pini was delignified, and extensive degradation of the middle lamella was evident. The remaining secondary walls labeled with EG II and CBH I, but little labeling was found with the xylanase-gold complex. Wood decayed by T. versicolor was nonselective, and erosion of all cell wall layers was apparent. Remaining wall layers near sites of erosion labeled with both EG II and CBH I. Erosion troughs that reached the S(1) layer or the middle lamella had less xylanase-gold labeling in the adjacent cell wall that remained. Brown-rotted wood had very low levels of gold particles present in sections treated with EG II or xylanase. Labeling with CBH I had the lowest concentrations in the S(2) layer near cell lumina and corresponded to sites with the most extensive degradation.