Changes in Functional Response of Soil Microbial Community along Chronosequence of Spontaneous Succession on Post Mining Forest Sites Evaluated by Biolog and SIR Methods

Soil formation in post-mining sites is crucial for restoring ecosystem function, and soil formation depend on the accumulation of soil organic matter and the development of an active microbial community. In this study, we used substrate-induced respiration (SIR) and Biolog plates to characterize microbial catabolic profiles in a chronosequence of soil samples from 15 unreclaimed post-mining sites in Sokolov, Czech Republic. The sites had been undergoing spontaneous succession for 3 to 45 years. Biolog ECO plates included 31 substrates. Of substrates used for SIR (glucose, chitin, cellulose, Tween 80, phenylethylamine, N-acetyl-D-glucosamine, L-asparagine, D-mannitol, D-galacturonic acid, α-cyclodextrin, and 4-hydroxy benzoic acid), eight were also used for the Biolog plates. Soil respiration, total bacteria number, and culturable bacteria number were also measured. The total and culturable number of bacteria increased with site age (p < 0.01 and p < 0.05, respectively). The percentage of culturable bacteria decreased with site age (p < 0.01). Biolog analysis indicated that average well-color development (AWCD), evenness, and richness increased with site age. SIR data indicated that only average activities tended to increase with site age (p = 0.06). According to redundancy analysis (RDA), the eight substrates, which were commonly used in both methods (SIR and BIOLOG) explained 74.4% of the variation of data from all Biolog substrates. Among the eight substrates common to both methods, only data for N-acetyl-D-glucosamine were positively correlated (p < 0.01) between Biolog and SIR. Both methods revealed microbial catabolic profile changed along the chronosequence. PCA indicated that site age, soil carbon, and pH were the most important drivers of microbial catabolic profiles.

Application of combined physicochemical and biological processes for enhanced treatment of avermectin fermentation wastewater

This paper aimed at developing the enhanced biological treatment processes for treating avermectin fermentation wastewater (AFW). After UASB treatment and chemical coagulation, the pretreated AFW was subsequently flowed into a rCAA reactor (reactor with repeated coupling of aerobes and anaerobes using macroporous carriers) system for further pollutant degradation and excess sludge reduction. By the treatment with chemical coagulation, COD, total nitrogen and total phosphorus concentration of treated AFW were eliminated to 550–700 mg/L, 130–160 mg/L and 1 mg/L, respectively, and the dark color of the wastewater was greatly bleached. After this decolorized wastewater was treated by the following rCAA bioreactor, the COD could be reduced to around 200–300 mg/L, while the further decrease of COD less than 200 mg/L was difficult. The Biolog analysis and OUR test for the water treated by rCAA bioreactor demonstrated that the effluent from chemical coagulation contained some unknown compounds with low biodegradability and would simplify the microbial community in the subsequent rCAA reactor.

Azospirillum canadense sp. nov., a nitrogen-fixing bacterium isolated from corn rhizosphere

A free-living diazotrophic strain, DS2T, was isolated from corn rhizosphere. Polyphasic taxonomy was performed including morphological characterization, Biolog analysis, and 16S rRNA, cpn60 and nifH gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain DS2T was closely related to the genus Azospirillum (96 % similarity). Chemotaxonomic characteristics (DNA G+C content 67.9 mol%; Q-10 quinone system; major fatty acid 18 : 1ω7c) were also similar to those of the genus Azospirillum. In all the analyses, including phenotypic characterization using Biolog analysis and comparison of cellular fatty acids, this isolate was found to be different from the closely related species Azospirillum lipoferum, Azospirillum oryzae and Azospirillum brasilense. On the basis of these results, a novel species is proposed for this nitrogen-fixing strain. The name Azospirillum canadense sp. nov. is suggested with the type strain DS2T (=NCCB 100108T=LMG 23617T).

First Report of Tomato (Lycopersicon esculentum) Pith Necrosis Caused by Pseudomonas fluorescens and P. corrugata in the Kingdom of Saudi Arabia

From 2002 to 2004, tomato (Lycopersicon esculentum) plants with external stem lesions, adventitious roots, and necrotic pith that was hollowed or chambered were received by the Clinical Lab of the Plant Protection Department from eight greenhouses in the Riyadh, Abha, and El-Kharj regions of Saudi Arabia. Bacteria were isolated on nutrient agar or King's medium B (KMB) from the stems of tomato plants cv. Red Gold, the cultivar most commonly grown in greenhouses. Gram-negative, rod-shaped bacteria were consistently isolated from stems with symptoms of pith necrosis. They were identified as Pseudomonas fluorescens (biotype I) and P. corrugata on the basis of morphological, physiological, and biochemical tests (2). Isolates of P. fluorescens isolated from Abha and El-Kharj were fluorescent on KMB, aerobic, and positive for oxidase, arginine dihydrolase, and gelatin liquefaction. Furthermore, all isolates produced levan-type colonies on sucrose nutrient agar and utilized glucose, 2-ketogluconate, sucrose, and sorbitol. They were negative for tobacco hypersensitivity and nitrate reduction. The strains of P. corrugata isolated from Riyadh were nonfluorescent, aerobic, and positive for oxidase, nitrate reductase, arginine dihydrolase, and utilization of malonate, alanine, trehalose, arginine, mannitol, and m-inositol. They were negative for levan, pectinase, tobacco hypersensitivity, and utilization of cellobiose and sorbitol. The identity of bacterial species was confirmed by Biolog analysis (carbon source utilization at 37°C), with a similarity index of 0.75 for P. corrugata and 0.71 for P. fluorescens. Four-week-old tomato plants (cv. Red Gold) were inoculated by injecting 50 μl of a bacterial suspension into the axils of the first true leaves. The bacterial suspension was prepared from 24-h-old cultures with sterile distilled water. Sterile distilled water was used as the negative control. After inoculation, plants were covered with polyethylene bags for 24 h to maintain high humidity at 25°C (1). Necrotic lesions surrounding injection points were observed 14 days after inoculation. At 4 weeks after inoculation, all inoculated plants showed symptoms of necrotic pith similar to those observed on the samples received. Control plants injected with water remained healthy throughout the experiments. Isolates of P. fluorescens (biotype I) and P. corrugata were reisolated from inoculated plants and were identical to the original strains on the basis of Biolog analysis. To our knowledge, this is the first report of tomato pith necrosis in Saudi Arabia. References: (1) G. Demir. J. Turk. Phytopathol. 19:63, 1990. (2) R. A. Lelliott and D. E. Stead. Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific Publications. Oxford, UK, 1987.

The impact of crop residue amendments and lime on microbial community structure and nitrogen-fixing bacteria in the wheat rhizosphere

Crop management practices can affect the soil microbial community, but it is not clear whether the effect of these practices is measurable at the wheat root–soil interface, where the plant exerts significant influence through root exudation. In this study, wheat plants were grown in soil amended with milled canola, lucerne, lupin, pea, and wheat residues with and without lime, to determine what changes occur to microbial community structure in the rhizosphere. Rhizosphere soil collected from wheat plants at the 5-leaf stage was assessed for overall microbial functional diversity using BIOLOG analysis and the diversity of the functional gene nifH using the polymerase chain reaction (PCR), terminal restriction fragment length polymorphism (T-RFLP), and cloning. Plant development was reduced in all residue amendments except lucerne, and a high positive correlation in the non-limited treatments between plant residue nitrogen (N) content and wheat shoot N suggested microbial competition for available N. Results from BIOLOG analysis indicated significant differences in rhizosphere microbial community structure due to lime, and to a lesser extent, residue type. Diversity, measured by the Shannon Diversity Index, was higher in limed rhizosphere soil, in addition to an increase in soils amended with lucerne, lupin, and pea residues compared with amendment with wheat, canola, and control soil. Each residue amendment promoted unique microbial communities determined by multi-dimensional scaling (MDS) and analysis of similarities (ANOSIM) of the BIOLOG data; the strongest effect was produced by addition of canola residues. N-fixing bacteria were also affected by lime, but residue effects were less apparent, especially between limed samples. The factor that correlated best with both BIOLOG and nifH T-RFLP data in non-limed soil was a combination of residue sodium (Na), copper (Cu), and manganese (Mn). In limed soil, phosphorus (P), calcium (Ca), and pH correlated well with BIOLOG data, and N, potassium (K), and iron (Fe) correlated with nifH T-RFLP data. A clone library of nifH sequences from control and limed, pea-amended soils revealed significant diversity amongst nifH sequences, most clustering with α-proteobacteria, and in some instances with Geobacter sulfurreducens. Clone distribution was significantly different for control soil and pea/lime soil, especially amongst the α-proteobacteria. The results suggest that rhizosphere microorganisms can be influenced by soil amendments, and change, depending on the type of residue applied. The addition of lime, however, produced the most significant changes in microbial community structure and nifH-containing rhizobacteria, highlighting the significant functional changes that occur when soil pH is increased.

Application of Carbon Source Utilization Patterns To Measure the Metabolic Similarity of Complex Dental Plaque Biofilm Microcosms

ABSTRACT Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H2-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (Streptococcus mutans plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.

Characterization of a Sinorhizobium Isolate and Its Extracellular Polymer Implicated in Pollutant Transport in Soil

ABSTRACT A bacterium isolated from soil (designated 9702-M4) synthesizes an extracellular polymer that facilitates the transport of such hydrophobic pollutants as polynuclear aromatic hydrocarbons, as well as the toxic metals lead and cadmium in soil. Biolog analysis, growth rate determinations, and percent G+C content identify 9702-M4 as a strain of Sinorhizobium meliloti. Sequence analysis of a 16S rDNA fragment gives 9702-M4 a phylogenetic designation most closely related to Sinorhizobium fredii. The extracellular polymer of isolate 9702-M4 is composed of both an extracellular polysaccharide (EPS) and a rough lipopolysaccharide. The EPS component is composed mainly of 4-glucose linkages with monomers of galactose, mannose, and glucuronic acid and has pyruval and acetyl constituents. The lipid fraction and the negative charge associated with carbonyl groups of the exopolymer are thought to account for the binding of polynuclear aromatic hydrocarbons and cationic metals.

Effect of solids retention time on the performance and biological characteristics of a membrane bioreactor

The purpose of this study was to compare the performance of a pilot-scale membrane bioreactor (MBR) in the treatment of municipal strength wastewater at solid retention times (SRTs) ranging from 30 days to two days. Cumulative nitrogen and phosphorus mass balances resulted in closures exceeding 90% at each steady state period. Biomass production rate and biomass viability generally increased with decreasing SRT, whereas overall enzymatic activity did not change significantly at most SRTs, but was highest at the two day SRT. Nitrification decreased at two day SRT but did not fail completely. At higher SRTs, nitrification was not noticeably affected by the sludge age. Phospholipid fatty acid (PLFA) analysis showed substantially diverse biomass in the sludge at different SRTs. Different ratios of gram positive bacteria, eukaryotic organisms, and yeast cells were observed in the mixed liquor at varying SRTs. On the other hand, BIOLOG analysis indicated that the overall capacity of the biomass to degrade different carbon substrates did not change significantly at different SRTs. The concentration of metals in the MBR mixed liquor declined steadily with decreasing SRT. The MBR effluent contained negligible amounts of Fe, Zn, Mn, and Co at each condition, indicating the retention of these metals regardless of the SRT.

First Report of a Bulb Decay of Onion by Enterobacter cloacae in Colorado

Sweet Spanish onion cultivars (Allium cepa L.) in northern Colorado displayed symptoms of postharvest bulb rot during September to November of 1999. This disease appears identical to that reported from infected onions in California in 1988 and is presumably associated with high temperature stress (1). Mature, firm bulbs harvested from scattered fields in Weld County exhibited a brownish discoloration and breakdown of inner scales. Gram negative, rod-shaped, cream-colored bacteria were consistently recovered from infected bulb tissue on nutrient agar. Physiological tests showed that the bacteria utilized glucose in an oxidative and fermentative manner and were catalase positive and oxidase negative. A representative strain was identified by Microbe Inotech Laboratories (St. Louis, MO) as Enterobacter cloacae (Jordan) Hormaeche & Edwards (2) using Biolog analysis, with a similarity index of 0.81. To confirm pathogenicity, a 0.5- to 1.0-ml suspension of bacteria (108 CFU/ml sdw) was injected into firm onion bulbs (7.5 to 10.0 cm diameter). After incubation for 14 days at 22°C in closed plastic bags in the dark, bulbs were cut in half and evaluated. Tan to brown discoloration and initial dry rot, similar to that observed postharvest, was observed in inoculated bulbs. The pathogen was reisolated from six of eight bulbs inoculated with the representative strain. No discoloration or disease developed on eight control bulbs injected with water. To our knowledge, this is the first report of E. cloacae from onion grown in Colorado. References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) Hormaeche and Edwards. Int. J. System. Bacteriol. 30:293, 1960.

Detection and Characterization of Plasmid pJP4 Transfer to Indigenous Soil Bacteria

ABSTRACT Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism,Escherichia coli ATCC 15224, by plate mating withRalstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 107 and 108 transconjugants g of dry soil−1 for samples supplemented with 500 and 1,000 μg of 2,4-D g of dry soil−1, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genusBurkholderia or the genus Pseudomonas. No mercury-resistant, 2,4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.