Growth of “Seeded” Cellulolytic Enrichment Cultures on Mesquite Wood

Eleven enrichment cultures were developed by a “seeded” enrichment culture technique, and one was developed by a simple enrichment technique. The seeded enrichment, the pure “seed,” and the simple enrichment cultures were compared during growth on mesquite wood, cotton, carboxymethylcellulose, and cellobiose. All of the enrichment cultures were cellulolytic and exceeded the pure seed cultures in mesquite wood hydrolysis and/or viable cell count. Yeast extract improved, but was not essential for, growth of the seeded enrichment cultures on carboxymethylcellulose. Two of the seeded enrichment cultures, CAD5 and CAD11, grew best at 37°C and pH 7.0 on mesquite wood. A 1.0% (wt/vol) wood concentration was optimum for their growth.     

Single-Cell Protein Production in Alkaline Mesquite Wood Extracts

A nine-member, mixed, cellulolytic, bacterial culture was used to evaluate the effects of sodium hydroxide normality, length and temperature of treatment, and the ratio of volume of alkali to mesquite wood on the suitability of alkali mesquite wood extractives as nutrients for bacterial growth. The presence or absence of air during the extraction process did not significantly affect results. The amount of lignin extracted and the total loss in weight of the wood during extraction were correlated to both alkali concentration and temperature. Neutralized extracts supported bacterial growth; growth was inversely related to the final salt concentration of the neutralized extract. Deionized extracts were superior to acid-neutralized extracts for the support of bacterial growth. The optimum conditions for extraction were 2.5 N NaOH at 30°C for 12 h. The study demonstrated that nutrients as well as growth inhibitory compounds are released from wood by alkali treatment. This study of alkali wood extracts and the previous study of washed alkali treated wood residues provide a data base for the optimization of alkali treatments of hard woods that are to be used as nutrients for the growth of cellulolytic cultures.     

Woody invasion of grasslands: evidence that CO2 enrichment indirectly promotes establishment of Prosopis glandulosa

Grasslands worldwide have been invaded by woody species during the last200 years. Atmospheric CO₂ enrichment may indirectly havefacilitatedinvasion by reducing soil water depletion by grasses. We used a two-stepcorrelative approach to test this hypothesis with the invasive and native shrubhoney mesquite (Prosopis glandulosa Torr. var.glandulosa). 1) Water content to 0.15 m depthwas measured in grassland exposed to a CO₂ gradient from 200 to 550mol/mol to evaluate the prediction that CO₂enrichment lessens soil water depletion by grasses. 2) Soil water content andemergence and survival of mesquite seedlings were measured in adjacentgrasslandplots from which grass roots were excluded to 0.15 m depth toreduce water depletion or that were irrigated to increase soil water levels.With these measurements, we tested the hypothesis that mesquite establishmentislimited by water.Excluding grass roots doubled emergence of mesquite and almost tripledthe fraction of emergent seedlings that survived for 12 weeks following thefirst of two plantings. Seedlings were taller, heavier, and had greater leafarea when grown without grass roots. Root exclusion did not measurably affectsoil water during the 3-week period of seedling emergence, but soilwatercontent over the 12 weeks that seedling survival was studied was higher inplotsfrom which grass roots were excluded and following an April than May planting.Survivorship of mesquite seedlings correlated positively with soil watercontent. Percentage survival of seedlings increased from 1.5% to15% and 28% at the soil water content measured in grasslandexposed to CO₂ concentrations of 270 (preindustrial), 360 (current),and 550 mol/mol (future), respectively. We infer thatrecent and projected increases in atmospheric CO₂ concentration maybe large enough to increase establishment of invading mesquite seedlings ingrasslands that are severely water-limited.     

Inheritance of seed coat color genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) and tagging the genes using SRAP,SCAR and SNP molecular markers

Seed coat color inheritance in Brassica napus was studied in F₁, F₂, F₃ and backcross progenies from crosses of five black seeded varieties/lines to three pure breeding yellow seeded lines. Maternal inheritance was observed for seed coat color in B. napus, but a pollen effect was also found when yellow seeded lines were used as the female parent. Seed coat color segregated from black to dark brown, light brown, dark yellow, light yellow, and yellow. Seed coat color was found to be controlled by three genes, the first two genes were responsible for black/brown seed coat color and the third gene was responsible for dark/light yellow seed coat color in B. napus. All three seed coat color alleles were dominant over yellow color alleles at all three loci. Sequence related amplified polymorphism (SRAP) was used for the development of molecular markers co-segregating with the seed coat color genes. A SRAP marker (SA12BG18388) tightly linked to one of the black/brown seed coat color genes was identified in the F₂ and backcross populations. This marker was found to be anchored on linkage group A9/N9 of the A-genome of B. napus. This SRAP marker was converted into sequence-characterized amplification region (SCAR) markers using chromosome-walking technology. A second SRAP marker (SA7BG29245), very close to another black/brown seed coat color gene, was identified from a high density genetic map developed in our laboratory using primer walking from an anchoring marker. The marker was located on linkage group C3/N13 of the C-genome of B. napus. This marker also co-segregated with the black/brown seed coat color gene in B. rapa. Based on the sequence information of the flanking sequences, 24 single nucleotide polymorphisms (SNPs) were identified between the yellow seeded and black/brown seeded lines. SNP detection and genotyping clearly differentiated the black/brown seeded plants from dark/light/yellow-seeded plants and also differentiated between homozygous (Y2Y2) and heterozygous (Y2y2) black/brown seeded plants. A total of 768 SRAP primer pair combinations were screened in dark/light yellow seed coat color plants and a close marker (DC1GA27197) linked to the dark/light yellow seed coat color gene was developed. These three markers linked to the three different yellow seed coat color genes in B. napus can be used to screen for yellow seeded lines in canola/rapeseed breeding programs.     

Comparison of Yeast Growth in Mesquite Wood Hydrolysate

Hot-water extracts of mesquite (Prosopis glandulosa) wood were assayed for their total carbohydrate, reducing sugar, and glucose content. These hydrolysates were then used as complete media for yeast growth. A total of 10 strains of yeasts were evaluated for their biomass production in the mesquite wood hydrolysates. Levels of utilizable carbohydrate proved to be the limiting factor for yeast growth in the hydrolysates.     

Growth and Cellulase Formation by Cellvibrio fulvus

S ummary : The aerobic cellulolytic bacterium Cellvibrio fulvus grew on several sugars and polysaccharides, but not on highly substituted cellulose derivatives, organic acids and alcohols. Whereas no growth was obtained on long cotton fibres, it occurred on such fibres cut into small pieces, and on filter paper and chromatography powders derived from cotton. Lignin free wood pulp was rapidly degraded. The organism grew best at pH 7–8 and utilized nitrate, ammonium and some amino acids as nitrogen sources. The bacteria have cell-bound cellulase but enzyme was also found in the culture medium. Glucose repressed cellulase formation and the enzyme activity of cultures grown on cellulose was much higher than on sugars. Reducing sugar was not detected in cellulose cultures. The pH optimum for hydrolysis of carboxymethylcellulose (CMC) was 7 and the enzyme was inhibited by mercuric acetate but not by p -chloromercuribenzoate or EDTA. Fractionation of cellulase preparations from cultures grown on partially hydrolysed filter paper gave many components of different molecular weights. The activities of these components against carboxymethylcellulose and microcrystalline cellulose differed.     

Enzyme immunoassay for detection of Salmonellae in foods.

An enzyme immunoassay was developed to detect Salmonella in foods. Indirect test protocols were developed for use with microtitration plates or Gilford microcuvettes. Samples from enrichment cultures were mixed with H-specific immunoglobulin G and allowed to react; unbound antibody was removed by three 5-min centrifugation washes; goat anti-rabbit antibody conjugated to alkaline phosphatase was added and allowed to react; and unbound conjugate was removed by centrifugation washing as before. Salmonella-positive samples were indicated by the production of a chromogenic reaction product after the addition of alkaline phosphatase substrate. The color could be read visually or quantified by absorbance. Ninety-eight food samples were examined to compare the enzyme immunoassay with enrichment serology, immunofluorescence, and the Food and Drug Administration pure culture technique. The enzyme immunoassay was sensitive and specific, and it possessed advantages over methods currently in use. Furthermore, when the enzyme immunoassay was used to screen preenrichment media, the results indicated that it might be decidedly more sensitive than the conventional pure culture technique.     

Isolation of an anaerobic cellulolytic mixed culture

Summary Isolation and enrichment cultures were made for anaerobic cellulose utilizing micro-organisms from non-ruminant sources. Stable mixed cultures were developed which degraded pure cellulose (wet-milled filter paper) in a defined mineral salts medium. Components of the mixed cultures lost viability in monoculture when grown on cellulose. Growth on cellulose was stimulated at low oxygen concentrations, when increased cellulase activity and increased volatile fatty acid production occurred.Low concentrations (0.1–3 mM) of cellobiose, and to a lesser extent, glucose stimulated solubilization of cellulose by the cultures, but higher concentrations had an inhibitory effect.Growth on cellulose was accompanied by production of acetic, propionic and butyric acids. The production and profile of the acids was stable and characteristic of the culture. When an open nonaseptic fermentation was employed, the fatty acid profile was variable and also included valeric acid.     

Chloroform degradation in methanogenic methanol enrichment cultures and by Methanosarcina barkeri 227.

The effects of methanol addition and consumption on chloroform degradation rate and product distribution in methanogenic methanol enrichment cultures and in cultures of Methanosarcina barkeri 227 were investigated. Degradation of chloroform with initial concentrations up to 27.3 microM in enrichment cultures and 4.8 microM in pure cultures was stimulated by the addition of methanol. However, methanol consumption was inhibited by as little as 2.5 microM chloroform in enrichment cultures and 0.8 microM chloroform in pure cultures, suggesting that the presence of methanol, not its exact concentration or consumption rate, was the most significant variable affecting chloroform degradation rate. Methanol addition also significantly increased the number of moles of dichloromethane produced per mole of chloroform consumed. In enrichment cultures, the number of moles of dichloromethane produced per mole of chloroform consumed ranged from 0.7 (methanol consumption essentially uninhibited) to 0.35 (methanol consumption significantly inhibited) to less than 0.2 (methanol not added to the culture). In pure cultures, the number of moles of dichloromethane produced per mole of chloroform consumed was 0.47 when methanol was added and 0.24 when no methanol was added. Studies with [14C]chloroform in both enrichment and pure cultures confirmed that methanol metabolism stimulated dichloromethane production compared with CO2 production. The results indicate that while the addition of methanol significantly stimulated chloroform degradation in both methanogenic methanol enrichment cultures and cultures of M. barkeri 227, the prospects for use of methanol as a growth substrate for anaerobic chloroform-degrading systems may be limited unless the increased production of undesirable chloroform degradation products and the inhibition of methanol consumption can be mitigated.     

Carboxymethylcellulase produced by facultative bacteria from the hind-gut of the termite Reticulitermes hesperus.

Bacillus cereus RW1 and Serratia marcescens RW3, isolated from the hind-gut of the termite Reticulitermes hesperus, both grew well on mesquite wood and produced moderate amounts of carboxymethylcellulase. Carboxymethylcellulose (CMC) gels were depolymerized rapidly by B. cereus RW1 and slowly by S. marcescens RW3. The depolymerization of CMC was pH and temperature sensitive. Depolymerization of gels by growing cultures of B. cereus RW1 and the action of cell-free extracts of B. cereus RW1 on CMC sols were optimum at pH 6.0 and 5.5, respectively. Glucose and cellobiose increased the rate of CMC gel depolymerization. Enzyme synthesis rather than growth was stimulated by the addition of glucose to a culture of RW1 growing on a non-cellulosic substrate. Bacillus cereus RW1 produced both cell-free and cell-bound carboxymethylcellulase.     

Mathematical modelling of a mixed culture cultivation process for the production of polyhydroxybutyrate

Mixed cultures submitted to acetate "feast" and "famine" cycles are able to store intracellularly high quantities of polyhydroxybutyrate (PHB). It was demonstrated in a previous study that the intracellular PHB content can be increased up to 78.5% (g HB/gVSS) of cell dry weight in a sequencing batch reactor (SBR) with optimised operating conditions. The specific PHB formation rate was also shown to be higher for mixed cultures than for pure cultures. Such high intracellular PHB contents and specific productivity open new perspectives for the industrial production of polyhydroxyalkanoates (PHA) using mixed cultures instead of pure cultures. The main goal in this work was to develop a mathematical model of mixed cultures envisaging the optimisation of PHB production. A relatively simple two-compartments cell model was developed based on experimental observations and other models proposed in the literature. A convenient experimental planing allowed to identify the kinetic parameters and yield coefficients. Experiments were performed with and without ammonia limitation enabling the analysis of PHB formation independently of the cell growth process. The experimental true yields partially confirm the theoretical values proposed in the literature. The final model exhibited high accuracy in describing the process state of most experiments performed, thus opening good perspectives for future model-based optimisation studies.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.     

Isolation of<Emphasis Type="Italic">Arcobacter butzleri</Emphasis> and<Emphasis Type="Italic">A. cryaerophilus</Emphasis> in samples of meats and from meat-processing plants by a culture technique and detection by PCR

A pilot survey of sources of contamination with arcobacters (representing a potential risk for humans) was done in a wide range of samples involved various kinds of meat (beef, pork, meat products, chilled chickens, etc.) from a retail level and domestic farming. Sanitary practices in slaughterhouses and production lines were checked in two different plants (a beef and pork production and a chicken processing plant). The method is based on a selective enrichment to isolate suspect strains, in combination with a PCR technique specific for arcobacters. The choice of a suitable enrichment broth and a plating agar was made with the use of pure bacterial strains and by means of real meat samples seeded with Arcobacter butzleri. The PCR technique was optimized to allow differentiation of a 1223 bp product, typical of the genus Arcobacter, and a product of 686 bp, specific for A. butzleri a total number of 198 samples were tested, of that 33 (17%) were found to be positive for the genus Arcobacter but only 22 (11%) for A. butzleri.     

Media carbon induction of extracellular cellulase activities inNeocallimastix frontalis isolate EB188

Extracellular cellulase induction in the ruminal fungusNeocallimastix frontalis isolate EB188 was followed. Glucose media-established cultures produced cellulase when switched to a variety of cellulose-containing media. High levels of cellulase and xylanase activities were present in cultures switched to sigma cell 100, solka floc, avicel, sisal fiber, and wheat straw, but not those switched to glucose, carboxymethylcellulose, or wood chips. Several assay substrates were used to show differential cellulase induction as well as-glucosidase activity. Cellulases hydrolyzed short oligosaccharides and released glucose from insoluble cellulose. Cellobiase activity was also indicated. Cellulase activity tolerated brief exposure to high temperature, was insensitive to certain metal ions, and possessed pH optima between 5.0 and 6.5.     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

A growth-inhibitory substance exuded from freeze-dried mesquite (Prosopis juliflora (Sw.) DC.) leaves

When lettuce seeds were incubated in a concentric circle aroundfreeze-dried mesquite leaves in a dish containing agar culture medium, thegrowth, especially radicle growth, of the seedlings was inhibited, the morestrongly the closer they were to the leaves. This result indicates thatallelopathic substance(s) inhibiting the lettuce growth are exuded from themesquite leaves. A potent substance was isolated from the exudates of thefreeze-dried mesquite leaves and identified as L-tryptophan by spectralanalyses. L-Tryptophan inhibited the radicle growth of lettuce and barnyardgrass at concentrations greater than1.5×10^–3 . The content ofL-tryptophan in the exudates of freeze-dried mesquite leaves (1 eq.) was 4.8×10^–3 . Theseresults suggest that L-tryptophan may play an important role in the allelopathyof mesquite leaves.     

Simple and rapid method for the isolation of Aspergillus niger mutants with enhanced cellulase and xylanase activity

Summary An agar plate isolation technique was developed for screening the mutants of Aspergillus niger. Fungal growth in solid media containing carboxymethylcellulose(CMC) was selected within 2–3 days. The mutants selected by the new technique showed a remarkable enhancement of CMCase, FPase, -glucosidase, xylanase, and -xylosidase activities. These results clearly show that the new isolation technique is a practical method for the selection of better strains.     

Establishment and development of Tabebuia rosea (Bignoniaceae) seedlings in a semideciduous tropical forest under management, Pacific coast of Mexico

We evaluated the effect of soil "scarification" and vegetation clearing treatments on the natural regeneration and initial development of Tabebuia rosea (Bertold) DC. seedlings in a moderate sized semideciduous tropical forest subjected to wood harvesting on the coast of Jalisco, Mexico. The treatments were applied under "seed" trees, and the number of germinated seedlings and their development were evaluated for nine months. Soil "scarification" promoted seed germination and initial seedling development, while the control of the competing vegetation increased the seedling growth and reduced their mortality. These results should be taken into account for the natural regeneration of this species, after clearing, to improve wood production, and should be incorporated into the silvicultural techniques currently developed in the region.     

Effects of radial growth, tree age, climate, and seed origin on wood density of diverse jack pine populations

Several models of the effects of silviculture, radial growth, and tree age on wood density have been developed, but they have rarely considered the roles of diverse seed origins and climate. We developed a model to test the effects of radial growth, tree age, climate, and seed-source origins on wood density in 21 diverse populations of jack pine in a common garden in Petawawa, Ontario, Canada over the last 24 years using a linear mixed-effects model. Although we found significant differences in wood density among diverse seed origins, there were no differences between seed origins having the same ring age and ring width, indicating an indirect effect on wood density of seed-source origin via radial growth. High variation in wood density among trees within the same population and between populations indicated high genetic control of wood density. The climate effect was significant on wood density in all populations, but smaller when radial growth was controlled. Climate effect did not differ significantly among populations. Precipitation in July negatively affected latewood density, whereas precipitation in May in the current year and September of the previous year negatively affected earlywood density. We concluded that a single model of jack pine wood density and radial growth could be used, either controlling for climate effects or not, as the relationship between wood density and radial growth is preserved among the diverse populations, and the climate effect controlling for radial growth in the model was only slight.     

Direct extraction of bacterial plasmids from food for polymerase chain reaction amplification.

In this report we describe a simple and rapid technique using DNA affinity columns that permits direct extraction of bacterial plasmids from a variety of foods for polymerase chain reaction amplification. The procedure was used to detect virulent enteroinvasive Escherichia coli in several artificially seeded matrices, including seafoods, greens, dairy products, enrichment media, and water. Polymerase inhibitors present in both foods and enrichment media were removed efficiently.