Enumeration of wax-degrading microorganisms in water repellent soils using a miniaturised Most-Probable-Number method

Soil Research ◽  
2005 ◽  
Vol 43 (2) ◽  
pp. 171 ◽  
Author(s):  
Margaret M. Roper ◽  
V. V. S. R. Gupta
Keyword(s):  
Agricultural Soils ◽  
Carbon Sources ◽  
Coconut Oil ◽  
Water Repellency ◽  
Most Probable Number ◽  
Water Repellent ◽  
Natural Habitats ◽  
Probable Number ◽  
Degrading Bacteria ◽  
Pure Cultures

The ability of a soil to reduce waxes that cause water repellency depends, in part, on the presence of populations of wax-degrading microorganisms. Therefore, estimating the size of populations of wax-degrading bacteria in water repellent soils is likely to be a useful indicator of the potential for bioremediation of water repellency. A Most Probable Number (MPN) method that selects specifically for the function of wax degradation in bacteria was developed and tested using both pure cultures and natural populations of wax-degrading bacteria. Two carbon sources (coconut oil and hexadecane) that contain fatty acids or hydrocarbons implicated as causative agents of water repellency were compared in terms of growth and emulsification by a range of known wax-degrading bacteria. Emulsification resulting from surfactant production, a direct measure of wax degradation, was the criterion for a positive test. All the bacteria tested emulsified coconut oil, which was chosen as the carbon source in subsequent testing of the MPN assay. Population estimates of pure cultures of wax-degrading bacteria (liquid suspensions and inoculated into sterile soils) made using the MPN and a plating technique showed good agreement between the 2 methods, indicating that the MPN method gives an accurate measure of size of the wax-degrading population. Extensive replication of MPN counts of known wax-degrading bacteria inoculated into sterile soils or populations in soils collected from a range of natural habitats showed little variation within samples, indicating that the new method was reproducible. Based on its accuracy and reproducibility the MPN method was deemed suitable for estimating populations in a range of soils. Monitoring wax-degrading populations under different agricultural managements and at different times of the year, i.e. seasonal dynamics, could provide clues for the development of new managements that minimise the risk of water repellency in agricultural soils.

Managing soils to enhance the potential for bioremediation of water repellency

Soil Research ◽  
2005 ◽  
Vol 43 (7) ◽  
pp. 803 ◽  
Author(s):  
Margaret M. Roper
Keyword(s):  
Management Practices ◽  
Crop Yields ◽  
Water Repellency ◽  
Water Infiltration ◽  
Most Probable Number ◽  
Water Repellent ◽  
Gradual Decline ◽  
Kaolinite Clay ◽  
Probable Number ◽  
Degrading Bacteria

Water repellency can significantly reduce crop and pasture establishment and production in sandy soils. Management practices that increase the rate of water infiltration into dry soils following the first rains at the end of the dry season were investigated. In the laboratory, addition of water to water repellent soil and maintenance of warm moist conditions produced a gradual decline in water repellency. This was supported by results in the field which showed that under daily irrigation there was a gradual decline in water repellency over time. However, under dryland conditions, other mechanisms to increase water infiltration had to be found. In the laboratory, after the addition of lime and kaolinite clay, there was an initial rapid decline in repellency, indicative of a physical mechanism, followed by a more gradual decline suggesting a biological response. In the field, under dryland conditions, the addition of lime and kaolinite clay resulted in a reduction in water repellency, and in the case of lime, this effect increased with the size of application. Estimates of the numbers of wax-degrading bacteria in the treated soils, using a most-probable-number assay, showed at least a 10-fold increase in lime-treated sands, but not in the clay-treated sands. The results suggest that lime may provide a viable alternative for increasing the wettability of soils by physical mechanisms and by promoting microbial activity by bacteria responsible for wax degradation, resulting in more consistent plant germination and establishment, and increased crop yields.

Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure

1996 ◽  
Vol 42 (3) ◽  
pp. 252-258 ◽  
Author(s):  
Brian A. Wrenn ◽  
Albert D. Venosa
Keyword(s):  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Oxidation Products ◽  
Most Probable Number ◽  
Probable Number ◽  
Microtiter Plates ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic ◽  
Pure Cultures ◽  
Plate Counts

A most-probable-number (MPN) procedure was developed to separately enumerate aliphatic and aromatic hydrocarbon degrading bacteria, because most of the currently available methods are unable to distinguish between these two groups. Separate 96-well microtiter plates are used to estimate the sizes of these two populations. The alkane-degrader MPN method uses hexadecane as the selective growth substrate and positive wells are detected by reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20 °C. Polycyclic aromatic hydrocarbon degraders are grown on a mixture of phenanthrene, anthracene, fluorene, and dibenzothiophene in a second plate. Positive wells turn yellow to greenish-brown from accumulation of the partial oxidation products of the aromatic substrates and they can be scored after a 3-week incubation period. These MPN procedures are accurate and selective. For pure cultures, heterotrophic plate counts on a nonselective medium and the appropriate MPN procedure provide similar estimates of the population density. Bacteria that cannot grow on the selective substrates do not produce false positive responses even when the inoculum density is very high. Thus, this method, which is simple enough for use in the field, provides reliable estimates for the density and composition of hydrocarbon-degrading microbial populations.Key words: most probable number, polycyclic aromatic hydrocarbon, alkane, hydrocarbon, bacteria.

A Method of Profiling Microbial Communities Based on a Most-Probable-Number Assay That Uses BIOLOG Plates and Multiple Sole Carbon Sources

1999 ◽  
Vol 65 (10) ◽  
pp. 4419-4424 ◽  
Author(s):  
Masashi Gamo ◽  
Tadashi Shoji
Keyword(s):  
Bacterial Communities ◽  
Carbon Sources ◽  
High Rate ◽  
Most Probable Number ◽  
Probable Number ◽  
New Approach ◽  
Mixing Ratios ◽  
Color Development ◽  
Assay Procedure ◽  
Pure Cultures

ABSTRACT A new approach to the community-level BIOLOG assay was proposed. This assay, which we call the BIOLOG-MPN assay, is a most-probable-number (MPN) assay that uses BIOLOG plates and multiple sole carbon sources, and the profiles obtained by this assay consist of MPNs estimated for the substrates in the BIOLOG plates. In order to demonstrate the performance of the BIOLOG-MPN assay, it was applied to pure cultures, model bacterial communities that contain two strains in different ratios, and microbial community samples. MPN estimation using BIOLOG plates worked well for the substrates on which utilizers can grow at a sufficiently high rate for color development under the conditions of the assay procedure. Furthermore, the results obtained using model communities showed that the MPNs obtained reflected the mixing ratios of pure cultures in the model communities. The profiles obtained using model communities and community samples were differentiated properly by statistical analyses. The results suggest that the BIOLOG-MPN assay is a promising procedure for obtaining a quantitative picture of the community structure.

scholarly journals Effects of Monoculture, Crop Rotation, and Soil Moisture Content on Selected Soil Physicochemical and Microbial Parameters in Wheat Fields

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
A. Marais ◽  
M. Hardy ◽  
M. Booyse ◽  
A. Botha
Keyword(s):  
Soil Moisture ◽  
Microbial Communities ◽  
Crop Rotation ◽  
Management Practices ◽  
Agricultural Soils ◽  
Soil Microbial Communities ◽  
Soil Microbial Activity ◽  
Most Probable Number ◽  
Probable Number ◽  
Soil Microbial

Different plants are known to have different soil microbial communities associated with them. Agricultural management practices such as fertiliser and pesticide addition, crop rotation, and grazing animals can lead to different microbial communities in the associated agricultural soils. Soil dilution plates, most-probable-number (MPN), community level physiological profiling (CLPP), and buried slide technique as well as some measured soil physicochemical parameters were used to determine changes during the growing season in the ecosystem profile in wheat fields subjected to wheat monoculture or wheat in annual rotation with medic/clover pasture. Statistical analyses showed that soil moisture had an over-riding effect on seasonal fluctuations in soil physicochemical and microbial populations. While within season soil microbial activity could be differentiated between wheat fields under rotational and monoculture management, these differences were not significant.

Colony-Forming Unit Enumeration by a Plate-MPN Method

1983 ◽  
Vol 46 (10) ◽  
pp. 836-841 ◽  
Author(s):  
S-T. TAN ◽  
R. B. MAXCY ◽  
W. W. STROUP
Keyword(s):  
Standard Technique ◽  
Most Probable Number ◽  
Plate Method ◽  
Probable Number ◽  
Microbial Counts ◽  
Colony Forming Unit ◽  
Pure Cultures ◽  
Petri Plate ◽  
Mpn Test ◽  
Surface Plate

Concepts of the standard surface plate method and the most probable number method (MPN) were combined to provide a new enumeration technique (plate-MPN). Three discrete 0.01-ml samples of an appropriate decimal dilution were inoculated onto each quadrant of a pre-dried petri plate. The discrete spots from the inoculum were then observed for growth after incubation. Results were interpreted analogous to a 3-tube MPN test using presently available tables. Application of the test to pure cultures and mixed flora provided no evidence to indicate the plate-MPN technique to be any less accurate than the standard technique for microbial counts. The plate-MPN technique was less precise than the standard technique. However, the plate-MPN technique has many advantages over traditional methods.

Prevalence, Concentration, and Antimicrobial Resistance Profiles of Salmonella Isolated from Florida Poultry Litter

2020 ◽  
Vol 83 (12) ◽  
pp. 2179-2186
Author(s):  
ALAN GUTIERREZ ◽  
JAYSANKAR DE ◽  
KEITH R. SCHNEIDER
Keyword(s):  
Antimicrobial Resistance ◽  
Antimicrobial Agents ◽  
Agricultural Soils ◽  
Geometric Mean ◽  
Poultry Litter ◽  
Ammonia Nitrogen ◽  
Animal Origin ◽  
Plate Count ◽  
Most Probable Number ◽  
Probable Number

ABSTRACT For over a decade, Salmonella contamination has increasingly led to outbreaks of foodborne illness associated with fresh produce. The use of untreated animal manures, or biological soil amendments of animal origin, to amend agricultural soils holds a risk of contamination from foodborne pathogens, such as Salmonella. This study was conducted to determine the prevalence, concentration, serotypes, and antimicrobial resistance profiles of Salmonella in poultry litter from Florida farms. Litter pH, total Kjeldahl nitrogen, total ammonia nitrogen, total phosphorus (P2O5), total potassium (K2O), moisture content, total solids, total ash, organic matter, and aerobic plate count (APC) were also measured. Litter samples (n = 54) were collected from 18 broiler farms across three seasons (spring, summer, and winter). Salmonella concentrations were enumerated using a most-probable-number (MPN) method, and antimicrobial susceptibility testing was performed. The prevalence of Salmonella in litter samples was 61.1%, with a geometric mean of 0.21 ± 20.7 MPN/g. Across all seasons, Salmonella concentrations were not influenced by the chemical, physical, or microbial properties measured. Recovered Salmonella isolates (n = 290) were grouped into serogroups O:4 (43.1%), O:7 (26.9%), O:8 (11.0%), O:1,3,10,19 (7.9%), and O:9,46 (7.2%). Serotyping Salmonella isolates (n = 47) resulted in 12 serotypes, with the most common being Typhimurium (27.7%), Kentucky (17.0%), Enteritidis (14.9%), and Mbandaka (14.9%). Antimicrobial resistance to tetracycline (29.8%), sulfisoxazole (23.4%), and streptomycin (14.9%) was observed. No isolates were resistant to more than two antimicrobial agents. This study provides valuable information for future risk assessments for the use of poultry litter as an untreated biological soil amendment of animal origin. HIGHLIGHTS

scholarly journals Composition and Function of Sulfate-Reducing Prokaryotes in Eutrophic and Pristine Areas of the Florida Everglades

2002 ◽  
Vol 68 (12) ◽  
pp. 6129-6137 ◽  
Author(s):  
Hector Castro ◽  
K. R. Reddy ◽  
Andrew Ogram
Keyword(s):  
Carbon Sources ◽  
Original System ◽  
Agricultural Runoff ◽  
Florida Everglades ◽  
Most Probable Number ◽  
Probable Number ◽  
Sulfate Reducing ◽  
Limited Degree ◽  
Sulfate Reduction Rates ◽  
And Function

ABSTRACT As a result of agricultural activities in regions adjacent to the northern boundary of the Florida Everglades, a nutrient gradient developed that resulted in physicochemical and ecological changes from the original system. Sulfate input from agricultural runoff and groundwater is present in soils of the Northern Everglades, and sulfate-reducing prokaryotes (SRP) may play an important role in biogeochemical processes such as carbon cycling. The goal of this project was to utilize culture-based and non-culture-based approaches to study differences between the composition of assemblages of SRP in eutrophic and pristine areas of the Everglades. Sulfate reduction rates and most-probable-number enumerations revealed SRP populations and activities to be greater in eutrophic zones than in more pristine soils. In eutrophic regions, methanogenesis rates were higher, the addition of acetate stimulated methanogenesis, and SRP able to utilize acetate competed to a limited degree with acetoclastic methanogens. A surprising amount of diversity within clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) genes was observed, and the majority of DSR sequences were associated with gram-positive spore-forming Desulfotomaculum and uncultured microorganisms. Sequences associated with Desulfotomaculum fall into two categories: in the eutrophic regions, 94.7% of the sequences related to Desulfotomaculum were associated with those able to completely oxidize substrates, and in samples from pristine regions, all Desulfotomaculum-like sequences were related to incomplete oxidizers. This metabolic selection may be linked to the types of substrates that Desulfotomaculum spp. utilize; it may be that complete oxidizers are more versatile and likelier to proliferate in nutrient-rich zones of the Everglades. Desulfotomaculum incomplete oxidizers may outcompete complete oxidizers for substrates such as hydrogen in pristine zones where diverse carbon sources are less available.

scholarly journals Distribution and Physiological Characteristics of Hyperthermophiles in the Kubiki Oil Reservoir in Niigata, Japan

2000 ◽  
Vol 66 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Yoh Takahata ◽  
Miyuki Nishijima ◽  
Toshihiro Hoaki ◽  
Tadashi Maruyama
Keyword(s):  
Cell Density ◽  
Carbon Sources ◽  
Most Probable Number ◽  
Oil Reservoir ◽  
Formation Water ◽  
Probable Number ◽  
Initial Cell ◽  
Initial Cell Density ◽  
Inorganic Substances ◽  
Subterranean Environment

ABSTRACT The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58°C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 × 103 to 4.6 × 104cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. DominantThermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50°C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0.01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.

Dynamics of Escherichia coli in agricultural soils receiving swine manure slurry or liquid municipal biosolids

2006 ◽  
Vol 86 (5) ◽  
pp. 841-849 ◽  
Author(s):  
Andrew Scott ◽  
Ken Conn ◽  
George Lazarovits ◽  
Edward Topp
Keyword(s):  
Escherichia Coli ◽  
Field Experiments ◽  
Agricultural Soils ◽  
Swine Manure ◽  
Most Probable Number ◽  
Probable Number ◽  
E Coli ◽  
Loam Soil ◽  
Municipal Biosolids ◽  
Manure Slurry

The fate of two E. coli stains, one that grows in soil receiving swine manure slurry (SMS) (Strain C279) and one that does not (Strain C278) were evaluated in laboratory and field experiments. A sensitive (detection limit 5 cells g soil-1) microplate-format most probable number (MPN) method for enumerating E. coli was developed and validated. In laboratory incubations, there was a general relationship between manure volatile fatty acid (VFA) content and the ability of the SMS to support growth in soil. Strain C279 grew in SMS-amended loam or sandy soil, but not in silt loam soil. Swine manure slurry inoculated with both Strains C278 and C279 was surface or subsurface applied in field microplots. The population of E. coli increased 50-fold within 3 d, and the ratio of Strain C279 to Strain C278 increased as much as 20-fold during the experiment, consistent with the growth of Strain C279 under these circumstances. Liquid municipal biosolids (LMB) obtained from four cities did not support the growth of Strain C279. The dynamics of E. coli populations in soils receiving surface and subsurface (10 cm) applications of SMS were comparable. Key words: Escherichia coli, water quality, liquid municipal biosolids, swine manure slurry

scholarly journals Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria

2001 ◽  
Vol 67 (7) ◽  
pp. 3168-3173 ◽  
Author(s):  
Linping Kuai ◽  
Arjun A. Nair ◽  
Martin F. Polz
Keyword(s):  
Dry Weight ◽  
Environmental Water ◽  
Microtiter Plate ◽  
Most Probable Number ◽  
Precipitation Reaction ◽  
Simple Method ◽  
Probable Number ◽  
Cell Counts ◽  
Pure Cultures ◽  
Reducing Bacteria

ABSTRACT A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 101 and 105 cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.

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