Optimal microbial adaptation routes for the rapid degradation of high concentration of phenol

Pseudomonas fluorescence KNU417 was able to degrade up to 700 mg/L of phenol in 65 h but could not degrade 1,000 mg/L of phenol. Phenol degradation rate was noticeably enhanced by pre-adaptation. In addition, the cell was able to degrade up to 1,300 mg/L of phenol by pre-adapting to 700 mg/L of phenol. Repeated adaptations to the same concentration of phenol showed negligible increase in degradation rate. Also, relatively low concentration of phenol (100–700 mg/L) required only one pre-adaptation while high concentration (1,000 mg/L) did two consecutive stepwise pre-adaptations for rapid degradation. Optimal adaptation routes were suggested for the fast phenol degradation. For example, 1,000 mg/L of phenol was degraded as fast as in 48 h when the cell was pre-adapted to 100 and 300 mg/L of phenol sequentially. The mechanism of adaptation was explained in terms of catechol 1,2-dioxygenase induction, related to aromatic ring cleavage.     

Trinitrotoluene (TNT) as a sole nitrogen source for a sulfate-reducing bacteriumDesulfovibrio sp. (B strain) isolated from an anaerobic digester

A sulfate-reducing bacterium (SRB),Desulfovibrio sp. (B strain), isolated from a continuous anaerobic digester (Boopathy and Daniels, Current Microbiology, 23:327–332, 1991) was found to use 2,4,6-trinitrotoluene (TNT) as sole nitrogen source. This bacterium also used nitrate, nitrite, and ammonium as nitrogen source. A long lag period was noticed when TNT or nitrite was used as nitrogen source. Nitrate, nitrite and TNT also served as electron acceptor in the absence of sulfate for this bacterium. Under nitrogen-limiting condition, 100% removal of TNT was observed within 8 days of incubation. The main intermediate observed was diaminonitrotoluene, which was further converted to toluene via triaminotoluene by reductive deamination process. Under nitrogen-rich conditions (presence of ammonium), TNT was converted to diaminonitrotoluene, and toluene was not produced. This isolate did not degrade TNT all the way to CO₂. This study demonstrated the possibility of using this isolated to decontaminate the soil and water contaiminated with TNT under anaerobic conditions.     

Lactic acid bacteria and yeasts in kefir grains and kefir made from them

In an investigation of the changes in the microflora along the pathway: kefir grains (A)→kefir made from kefir grains (B)→kefir made from kefir as inoculum (C), the following species of lactic acid bacteria (83–90%) of the microbial count in the grains) were identified: Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp. pseudoplantarum and Lactobacillus brevis. Yeasts (10–17%) identified were Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, Candida inconspicua and Candida maris. In the microbial population of kefir grains and kefir made from them the homofermentative lactic streptococci (52–65% and 79–86%, respectively) predominated. Within the group of lactobacilli, the homofermentative thermophilic species L. delbrueckii subsp. bulgaricus and L. helveticus (70–87% of the isolated bacilli) predominated. Along the pathway A→B→C, the streptococcal proportion in the total kefir microflora increased by 26–30% whereas the lactobacilli decreased by 13–23%. K. marxianus var. lactis was permanently present in kefir grains and kefirs, whereas the dominant lactose-negative yeast in the total yeast flora of the kefir grains dramatically decreased in kefir C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 1–6 DOI: 10.1038/sj/jim/7000186 Received 02 August 2000/ Accepted in revised form 15 July 2001     

Decolorization of water and oil-soluble azo dyes by <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>

The capability of Lactobacillus acidophilus and Lactobacillus fermentum to degrade azo dyes was investigated. The bacteria were incubated under anaerobic conditions in the presence of 6 μg/ml Methyl Red, Ponceau BS, Orange G, Amaranth, Orange II, and Direct Blue 15; 5 μg/ml Sudan I and II; or 1.5 μg/ml Sudan III and IV in deMann–Rogosa–Sharpe broth at 37°C for 36 h, and reduction of the dyes was monitored. Both bacteria were capable of degrading all of the water-soluble azo dyes to some extent. They were also able to completely reduce the oil-soluble diazo dyes Sudan III and IV but were unable to reduce the oil-soluble monoazo dyes Sudan I and II to any significant degree in the concentrations studied. Growth of the bacteria was not significantly affected by the presence of the Sudan azo dyes. Metabolites of the bacterial degradation of Sudan III and IV were isolated and identified by liquid chromatography electrospray ionization tandem mass spectrometry analyses and compared with authentic standards. Aniline and o-toluidine (2-methylaniline), both potentially carcinogenic aromatic amines, were metabolites of Sudan III and IV, respectively.     

Ethene as an auxiliary substrate for the cooxidation of cis-1,2-dichloroethene and vinyl chloride

Cultures able to dechlorinate cis-1,2-dichloroethene (cDCE) were selected with ethene (3–20%, v/v) as the sole source of carbon and energy. One mixed culture (K20) could degrade cDCE (400 μmol l^–1) or vinyl chloride (100 μmol l^–1) in the presence of ethene (≤ 80 μmol l^–1 and ≤ 210 μmol l^–1, respectively). This culture consists of at least five bacterial strains. All five strains were able to degrade cDCE cometabolically in pure culture. The mixed culture K20 was highly tolerant against cDCE (up to 6 mmol l^–1 in the liquid phase). Degradation of cDCE (200 μmol l^–1) was not affected by the presence of trichloroethene (100 μmol l^–1) or tetrachloroethene (100 μmol l^–1). Transformation yields (T_y, defined as unit mass of chloroethene degraded per unit mass of ethene consumed) of the mixed culture K20 were relatively high (0.51 and 0.61 for cDCE and vinyl chloride, respectively). The yield for cDCE with ethene as auxiliary substrate was ninefold higher than any values reported with methane or methane/formate as auxiliary substrate. The viability of the cells of the mixed culture K20 (0.3 mg of cells ml^–1) was unaffected by the transformation of ≤ 200 μmol l^–1 cDCE in 300 min. Received: 9 March 1999 / Accepted: 21 July 1999     

Anaerobic biodegradation of triphenylmethane dyes in a hybrid UASFB reactor for wastewater remediation

Anaerobic digestions have been proved more successful than aerobic systems for the degradation and destruction of dye-containing wastewaters. The performance of a hybrid up flow anaerobic sludge-filter bed (UASFB) reactor was tested with a synthetic wastewater containing Crystal violet (CV) as a carbon source and sodium acetate as a co-substrate. Continuous feeding of the reactor started with an initial OLR of 0.9 g COD/l-d and then it was increased step wise to 4 g COD l⁻¹ d⁻¹, while maintaining constant HRT (24 h). The optimum pH value and temperature for decolorization of crystal violet by this mixed culture species under anaerobic conditions were found to be 8–9 and 30–35°C respectively. N,N-dimethylaminophenol and N,N-bis (dimethylamino) benzophenone (Michler’s Ketone) were detected as the degradative metabolites of Crystal Violet. Subsequently, N,N-dimethylaminophenol was further degraded to aniline in the reactor whereas Michler’s ketone was not degraded under anaerobic conditions. The UASFB bioreactor was able to remove the CV completely up to a loading rate of 100 mg CV l⁻¹d⁻¹.     

Enhanced Degradation of TNT by Genome-Shuffled Stenotrophomonas maltophilia OK-5

In this study, the enhanced degradation of TNT using cultures of genome-shuffled Stenotrophomonas maltophilia OK-5 mt-3 has been examined and the proteome of shuffled strain was compared to the wild-type OK-5 strain. Genome shuffling of S. maltophilia OK-5 was used to achieve a rapid enhancement of TNT degradation. The initial mutant population was generated by NTG treatment and UV irradiation. The wild-type OK-5 strain was able to degrade 0.2 mM TNT within 6 days, yet barely tolerated 0.5 mM TNT while the shuffled OK-5 mt-3 was capable of completely degrading 0.5 mM TNT within 8 days, and 1.2 mM within 24 days. The proteomic analysis of the shuffled OK-5 mt-3 demonstrated the changes in the expression levels of certain proteins compared to wild-type OK-5. These results provide clues for understanding TNT tolerance and improved TNT degradation by shuffled S. maltophilia OK-5 mt-3 and have possible applications in the processing of industrial waste containing relatively high TNT concentrations.     

Edible oil degradation by using yeast coculture of <Emphasis Type="Italic">Rhodotorula pacifica</Emphasis> ST3411 and <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> ST3412

To develop a microbial treatment of edible oil-contaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH₄–N] = 240 mg/L) and phosphorus sources ([PO₄–P] = 90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20°C was 39.8% ± 9.9% (means ± standard deviations of eight replicates). The highest degradation rate was observed at 20°C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671 ± 52.0 to 143 ± 96.7 ppm in 24 h, and the degradation rate was 79.4% ± 13.8% (means ± standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells.     

Transformation of 2,4,6-trinitrotoluene (TNT) by <Emphasis Type="Italic">Raoultella</Emphasis> <Emphasis Type="Italic">terrigena</Emphasis>

Manufacture of nitroorganic explosives generates toxic wastes leading to contamination of soils and waters, especially groundwater. For that reason bacteria living in environments highly contaminated with 2,4,6-trinitrotoluene (TNT) and other nitroorganic compounds were investigated for their capacity for TNT degradation. One isolate, Raoultella terrigena strain HB, removed TNT at concentrations between 10 and 100 mg l⁻¹ completely from culture supernatants under optimum aerobic conditions within several hours. Only low concentrations of nutrient supplements were needed for the cometabolic transformation process. Radioactivity measurements with ring-labelled ¹⁴C–TNT detected about 10–20% of the initial radioactivity in the culture supernatant and the residual 80–90% as water-insoluble organic compounds in the cellular pellet. HPLC analysis identified aminodinitrotoluenes (2-ADNT, 4-ADNT) and diaminonitrotoluenes (2,4-DANT) as the metabolites which remained soluble in the culture medium and azoxy-dimers as the main products in the cell extracts. Hence, the new isolate could be useful for the removal of TNT from contaminated waters.     

Production of flavour compounds by yogurt starter cultures

The present work studied the production of carbonyl compounds and saturated volatile free fatty acids by pure cultures of Streptococcus thermophilus and Lactobacillus bulgaricus, and by starter cultures for Bulgarian yogurt during cultivation and cooling. The mixed cultures formed volatile aromatic compounds more actively than the pure cultures. A guiding factor in the preparation of the starter cultures was the biochemical activity of Lactobacillus bulgaricus in synthesizing the major carbonyl compounds, acetaldehyde, diacetyl and the volatile fatty acids C₂–C₁₀. The activity of the yogurt cultures in synthesizing carbonyl compounds was at its highest during milk coagulation and cooling, up to 7 h. However, maximum concentration was reached by 22–31 h. In the cooled 22–h starter cultures, acetaldehyde predominated (1415.0–1734.2 μg per 100 g) followed by diacetyl (165.0–202.0 μg per 100 g), acetoin (170.0–221.0 μg per 100 g), acetone (66.0–75.5 μg per 100 g), ethanol (58.0 μg per 100 g), and butanone-2 (3.6–3.8 μg per 100 g). The thermophilic streptococcus and lactobacillus cultures, and the starter cultures contained predominantly acetic, butyric and caproic acids. Received 19 June 1997/ Accepted in revised form 10 January 1998     

Degradation of chlorobenzene by strain <Emphasis Type="Italic">Ralstonia pickettii</Emphasis> L2 isolated from a biotrickling filter treating a chlorobenzene-contaminated gas stream

A Ralstonia pickettii species able to degrade chlorobenzene (CB) as the sole source of carbon and energy was isolated from a biotrickling filter used for the removal of CB from waste gases. This organism, strain L2, could degrade CB as high as 220 mg/L completely. Following CB consumption, stoichiometric amounts of chloride were released, and CO₂ production rate up to 80.2% proved that the loss of CB was mainly via mineralization and incorporation into cell material. The Haldane modification of the Monod equation adequately described the relationship between the specific growth rate and substrate concentration. The maximum specific growth rate and yield coefficient were 0.26 h⁻¹ and 0.26 mg of biomass produced/mg of CB consumed, respectively. The pathways for CB degradation were proposed by the identification of metabolites and assay of ring cleavage enzymes in cell extracts. CB was degraded predominantly via 2-chlorophenol to 3-chlorocatechol and also partially via phenol to catechol with subsequent ortho ring cleavage, suggesting partially new pathways for CB-utilizing bacteria.     

Reductive transformation of TNT by Escherichia coli resting cells: kinetic analysis

Microbial 2,4,6-trinitrotoluene (TNT) biotransformation via sequential nitro-reduction appears a ubiquitous process, but the kinetics of these transformations have been poorly understood or described. TNT transformation by Escherichia coli was monitored and a kinetic model for reductive TNT depletion was developed and experimentally calibrated in this report. Using resting cells of aerobically pregrown E. coli, TNT was quickly reduced to hydroxylaminodinitrotoluenes. The standard Michaelis–Menten model was modified to include three additional parameters: product toxicity (T _c), substrate inhibition (K _i), and intracellular reducing power (RH) limitation. Experimentally measured product toxicity (5.2 μmol TNT/mg cellular protein) closely matched the best-fit model value (2.84 μmol TNT/mg cellular protein). Parameter identifiability and reliability (k _m, K _s, T _c, and K _i) was evaluated and confirmed through sensitivity analyses and via Monte Carlo simulations. The resulting kinetic model adequately described TNT reduction kinetics by E. coli resting cells in the absence or presence of reducing power limitation.     

Synergistic Effect Between Two Bacteriocin-like Inhibitory Substances Produced by Lactobacilli Strains with Inhibitory Activity for <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis>

Group B streptococci (GBS) are bacterial species that colonize the vagina in pregnant women and as such may cause serious infections in neonates that passed through the birth channel. The objective of this work was to study the inhibitory activities produced by each bacteriocin-like inhibitory substance (BLIS) of Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23, and the effects of the combined BLIS-es of these lactobacilli on GBS. The interactions between the BLIS-es were assessed by qualitative and quantitative methods on agar plates. The minimum inhibitory concentrations (MICs) and fractional inhibitory concentrations (FICs) were determined by a modification of the broth microdilution and checkerboard methods, respectively. Antibiotic susceptibilities of all S. agalactiae strains were assayed and the results of these tests were evaluated for statistical significance. A 7.5% of GBS isolates were recovered from 760 pregnant women and 91% of those strains were susceptible to each BLIS produced by L. fermentum, L. rhamnosus, and also to a mixture of them. The comparisons among the BLIS-es showed statistically significant differences, with a combination of the BLIS-es from the two Lactobacillus species being better than the BLIS of each one alone (P < 0.05) as GBS growth inhibitors. Synergistic activities between the BLIS-es were found on 100% of susceptible GBS strains, MICs ranges of BLIS of L23 and L60 were 80–160 and 160–320 UA ml⁻¹, respectively. By the checkerboard method, the BLIS-es combination showed synergistic effect on all sensitive strains tested, with values of FICs ranging from 0.131 to 0.218. The BLIS-es produced by these lactobacilli of vaginal origin were able to inhibit S. agalactiae isolates. The results indicate that these strains may have probiotic potential for the control of GBS in women and may consequently prevent GBS infections in newborns.     

Degradation of chloroform by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in calcium alginate beads

A Bacillus sp., capable of degrading chloroform, was immobilized in calcium alginate. The beads in 20 g alginate l⁻¹ (about 2 × 10⁸ cells/bead) could be re-used nine times for degradation of chloroform at 40 μM. The immobilized cells had a higher range of tolerance (pH 6.5–9 and 20–41°C) than free cells (pH 7–8.5 and 28–32°C). At 5 g alginate l⁻¹, leakage of the cells from the beads was 0.51 mg dry wt ml⁻¹. This species is the first reported Bacillus that can degrade chloroform as the sole carbon source.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation of <Emphasis Type="Italic">cry</Emphasis>1Ac Gene into Shoot-Tip Meristem of Diploid Cotton <Emphasis Type="Italic">Gossypium arboreum</Emphasis> cv. RG8 and Regeneration of Transgenic Plants

An Agrobacterium-mediated gene transfer protocol was developed for the diploid cotton Gossypium arboreum using meristematic cells of shoot tips, followed by direct shoot organogenesis or multiple shoot induction of putative transformants. Seven-day- old shoot tips of in vitro-germinated seedlings of G. arboreum cv. RG8 were excised by removing cotyledonary leaves and providing “V”-shaped oblique cuts on either side of explants. Excised explants were inoculated with an overnight-grown culture of Agrobacterium tumefaciens carrying a plant cloning vector harboring the cry1Ac gene. The explants were co-cultivated in Murashige and Skoog (MS) medium supplemented with 30 mg/L acetosyringone, 100 mg/L myoinositol, 10 mg/L thiamine, and 30 g/L glucose for three days in the dark. Following co-cultivation, explants were incubated on the same medium supplemented with 20 mg/L kanamycin, for first three passages of 10–12 days each and subsequently on 50 mg/L kanamycin to facilitate stable expression of transgene. Explants were then transferred to a fresh MS medium supplemented with either kinetin (0.1 mg/L), myoinositol (100 mg/L), thiamine (10 mg/L) and glucose (30 g/L) or benzyl adenine, BA (2 mg/L), kinetin (1 mg/L), myoinositol (100 mg/L), thiamine (10 mg/L), and glucose (30 g/L) to induce either single or multiple putative transformant shoots, respectively. Following 6 weeks, shoots were transferred to a rooting medium consisting of liquid MS supplemented with 0.05–0.1 mg/L NAA and glucose (15 g/L). Rooted plantlets were first acclimatized in liquid MS with 0.05 mg/L NAA and 15 g/L glucose, transferred to plastic pots containing soilrite Mix-TC (a mixture of Irish peat moss and horticultural grade expanded perlite, 75:25), and grown under controlled temperature and humidity conditions in a growth chamber. Acclimatized plants were then transferred to clay pots and grown in the greenhouse. These plants were confirmed as transgenic for cry1Ac gene using polymerase chain reaction, enzyme linked imunosorbent assay, and Southern blot analyses.     

Dominance of <Emphasis Type="Italic">Prevotella</Emphasis> and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.     

Copper-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater

Six copper-resistant bacterial strains were isolated from wastewater of tanneries of Kasur and Rohi Nala. Two strains tolerated copper at 380 mg/L, four up to 400 mg/L. Three strains were identified as members of the genusSalmonella; one strain was identified asStreptococcus pyrogenes, one asVagococcus fluvialis and the last was identified asEscherichia coli. The pH and temperature optimum for two of them were 7.0 and 30 °C, respectively; four strains had corresponding optima at 7.5 and 37 °C, respectively. All bacterial isola-tes showed resistance against Ag⁺ (280–350 mg/L), Co²⁺ (200–420), Cr^VI (280–400), Cd²⁺ (250–350), Hg²⁺ (110–200), Mn²⁺ (300–380), Pb²⁺ (300–400), Sn²⁺ (480–520) and Zn²⁺ (300–450). Largesized plasmids (>20 kb), were detected in all of the strains. After the isolates were cured of plasmids with ethidium bromide, the efficiency of curing was estimated in the range of 60–90%. Reference strain ofE. coli was transformed with the plasmids of the bacterial isolates which grew in Luria-Bertani medium containing 100 mg/L Cu²⁺. The capability to adsorb and afterwards accumulate Cu²⁺ inside their cells was assayed by atomic absorption spectrophotometer; all bacterial cells had the ability to adsorb 50–80 % of the Cu²⁺ and accumulate 30–45 % Cu²⁺ inside them after 1 d of incubation.     

Diversity of lactic acid bacteria and yeasts in Airag and Tarag,traditional fermented milk products of Mongolia

We used culture- and molecular-biology-based methods to investigate microbial diversity in the traditional Mongolian fermented milks “Airag” (fermented mare’s milk) and “Tarag” (fermented milk of cows, yaks, goats, or camels). By rRNA or functional gene sequencing, we identified 367 lactic acid bacteria (LAB) strains and 152 yeast strains isolated from 22 Airag and 31 Tarag samples. The total concentration of LAB in Airag (10^{7.78 ± 0.50} c.f.u. ml^–1; mean ± SD) was significantly lower (P < 0.01) than in Tarag (10^{8.35 ± 0.62} c.f.u. ml⁻¹), whereas the total concentration of yeasts in Airag (10^{7.41 ± 0.61} c.f.u. ml^-1) was significantly higher (P < 0.01) than in Tarag (10^{5.86 ± 1.29} c.f.u. ml^-1). Lactobacillus helveticus and Lactobacillus kefiranofaciens were isolated from Airag as the predominant LAB strains at levels of about 10⁷ c.f.u. ml⁻¹, whereas Lactobacillus delbrueckii subsp. bulgaricus, L. helveticus, and Streptococcus thermophilus were the predominant isolates from Tarag at about 10⁷ c.f.u. ml⁻¹. The lactose-fermenting Kluyveromyces marxianus was isolated predominantly from Airag as its major alcoholic fermentation component. Non-lactose-fermenting yeasts such as Saccharomyces cerevisiae, Issatchenkia orientalis, and Kazachstania unispora were the predominant isolates from Tarag, at about 10⁵ c.f.u. ml⁻¹. The apparent geographic differences in the L. kefiranofaciens and S. thermophilus contents of Tarag strongly suggested that differences among the animal species from which the milk was sourced, rather than geographic distances, were the most important factors influencing the diversity of the microbial composition of traditional fermented milks in Mongolia.     

Molecular diversity of bacteria in Yunnan yellow cattle (Bos taurs) from Nujiang region, China

The rumen content of four Yunnan Yellow Cattle (Bos taurs) were collected to determine the bacteria diversity by using 16S rRNA gene sequence analysis. A total of 129 sequences were examined and the sequences were referred as 107 OTU (Operational Taxonomy Unit) according to the similarity level of 97% in gene sequence. Similarity analysis revealed that Yunnan Yellow Cattle had 12 sequences (10 OTU) shared 97% or greater similarity with cultured rumen bacteria Butyrivibrio fibrisolvens, Succiniclasticum ruminis, Ruminococcus bromii, Clostridium proteoclasticum, Ruminococcus flavefaciens, Pseudobutyrivibrio ruminis, Jeotgalicoccus psychrophilus, and Prevotella ruminicola, which accounting for 9.3% of the total clones (9.2% of the total OTU). The further 12 sequences (9 OTU) shared 90–97% similarity with cultured bacteria Clostridium aminobutyricum, butyrate-producing bacterium, Schwartzia succinivorans, Prevotella ruminicola, Eubacterium ruminantium, Ruminococcus albus, and Clostridium termitidis, also accounting for 9.3% of the total sequences (8.3% of the total OTU). The remaining 105 sequences (90 OTU) shared less than 90% similarity with cultured bacteria, accounting for 81.4% of the total sequences (82.5% of the total OTU). According to the phylogenetic analysis, all sequences were phylogenetically placed within phyla of low G+C subdivision (accounting for 72.1 and 72.5% of the total clones and OTU, respectively) and CFB subdivision (Cytophaga-Flexibacter-Bacteroides; accounting for 27.9 and 27.5% of the total clones and OTU, respectively). Among the examined clones, rare bacteria Jeotgalicoccus psychrophilus was detected in the rumen of cattle.     

Species of Agave with antimicrobial activity against selected pathogenic bacteria and fungi

The in vitro antimicrobial activity against pathogenic bacteria, yeast, and molds were examined in extracts of the Agave species A. lecheguilla, A. picta, A. scabra and A. lophanta using an agar diffusion technique. The extracts of A. picta produced zones of inhibition of 9–13 mm for E. coli, L. monocytogenes, S. aureus, and V. cholerae, while B. cereus and Y. enterocolitica were not inhibited. The other Agave species did not show any detectable inhibitory activity against the bacteria tested; however, all four Agave sp. were inhibitory against all yeast and molds analyzed as evident by 9–20 mm zones of inhibition. The minimum microbicidal concentration (MMC) of the active extract ranged from 1.8 to 7.0 mg/ml for the sensitive bacteria, and 2.0–3.0 mg/ml for yeast. In the case of molds, the minimum inhibitory concentration (MIC) of the active extracts ranged from 3.0 to 6.0 mg/ml. Together, these data suggest that the Agave sp. analyzed are potential antimicrobial candidates with a broad range of activity.