Richard G. Burns

Enzyme activity in soil: Location and a possible role in microbial ecology

The activity of any particular enzyme in soil is a composite of activities associated with various biotic and abiotic components, e.g. proliferating cells, latent cells, cell debris, clay minerals, humic colloids and the soil aqueous phase. The location of the enzyme is at least partially determined by such factors as the size and solubility of its substrate, the species of microorganism, and the physical and chemical nature of the soil colloids. However, enzymes may change location with time, for example, many hydrolases are intracellular sensu stricto but are also found associated with cell debris and clay and organic colloids. There are difficulties in quantifying the various activities, but this may be possible by employing different types of assays, the prudent use of controls and the study of crude enzyme extracts from soil. Enzymes bound to clay and humic colloids (the immobilized or accumulated enzyme fraction) have a residual activity not found in enzymes free in the soil aqueous phase. However, the mere adsorption of enzymes to soil surfaces does not guarantee subsequent activity, and it appears that some mechanism of association with the humic polymer offers the best form of protection, yet permits the retention of enzyme activity. The catalytic activity of extracellular enzymes is discussed and a possible relationship between soil microorganisms, exogenous substrates and immobilized enzymes is suggested.

Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques

Knowledge of microbial numbers and activity in subsoils is essential for understanding the transformation and downward movement of natural and synthetic organics. Soil cores were taken from two soil profiles (surface textures: silty clay loam and loamy sand), and samples extracted from the 0-30 cm (surface), 1.0-1.3 m (mid) and 2.7-3.0 m deep; clay) and 3.9-4.2 m (deep: sand) layers. A variety of soil biotic (microbial numbers, microbial biomass, enzyme activities) and abiotic properties (pH, organic C, texture, CEC) were measured. Bacterial numbers decreased with depth as indicated by viable counts and by calculations based upon biomass carbon and extracted DNA. Direct microscopic counts were the most sensitive method of enumeration and gave bacterial numbers between 37 and 442 X greater than colony formin g units and those calculated from DNA extracted from soil. DNA extracted from soil ranged from 1.23 (sand surface) and 1.34 (clay surface) mug g(-1) d wt soil to 0.02 (sand deep) and 0.01 (clay deep) mug g(-1) d wt soil. Bacterial numbers, estimated from biomass-C measurements, were comparable to direct counts. Large numbers of bacteria were recorded in the subsoils (direct counts: 5.6 X 10(8) sand, 4.5 X 10(8) clay) even though this was equivalent to only 4.7 and 1.7% of those in the surface soils. Fungi were isolated from surface and mid-depth layers of both soils but were absent from the deep soil samples. Enzymatic activities (arylsulphatase, beta-glucosidase, phosphomonoesterases, urease, dehydrogenase, FDA hydrolysis), assayed with or without buffers, also decreased with depth. The exception was urease activity in the clay soil where no difference was seen between mid and deep in non-buffered assays but a 2.9-fold greater activity was exhibited in the mid than in the surface soil when buffered, Strong positive correlations (R > 0.95) were observed between all enzyme activities (except with urease activity in clay soil and non-buffered phosphatase activity in sand soil) and between all methods of estimating bacterial abundance. Strong positive correlations (R > 0.90) were also found between bacterial abundance and enzyme activities and between enzyme activities and organic matter content

Cyanide production by rhizobacteria as a possible mechanism of plant growth inhibition

SummaryVolatile metabolites from a number of rhizosphere pseudomonads prevented lettuce root growth in a seedling bioassay. One of these metabolites was identified as cyanide. Direct contact between rhizobacteria and plant roots produced, with one exception, similar responses. However, not all cyanogenic isolates were plant-growth-inhibitory rhizobacteria. When grown in liquid culture, cyanogenic strains produced an average of 37 nmol HCN ml−1 over a 36-h period and inhibition of root growth occurred at concentrations as low as 20 nmol ml−1. Cyanogenic strains introduced into sand or soil also produced HCN. Two cyanogenic strains ofPseudomonas fluorescens, one (5241) a plant-growth inhibitory rhizobacterium and the other (S97) a plant-growth-promotory rhizobacterium, were used to treat bean and lettuce seedlings prior to planting in soil. Lettuce dry weight was reduced by 49.2% (day 28) and 37.4% (day 49) when inoculated with S241 whereas S97 increased growth initially (+64.5% at day 28, no difference from control at day 49). Equivalent figures for inoculated bean plants were: −52.9% and −65.1% (5241); +40.7% and +23.3% (S97). A more detailed experiment using only bean plants confirmed these contrasting affects. Inhibition by S241 was related to consistently higher levels of rhizosphere cyanide in comparison with S97-treated plants and control soils. S241 also survived in the rhizosphere at higher densities and for a longer period of time than S97. The possible contribution of rhizobacterial cyanogenesis to plant growth inhibition is discussed.

Soil urease: Activity, stability and kinetic properties

Abstract The activity, stability and kinetic characteristics of soil urease are examined and compared with those of a crude enzyme-organic matter extract from soil and of jack bean urease. Methods of organic matter extraction from soil are discussed and the resistance of all three forms of urease to irradiation, proteolysis, temperature, storage and lyophilization is assessed. In addition, measurements are made of Michaelis constants and pH optima. The data confirm previous observations in that at least a proportion of soil urease is remarkably stable in conditions which rapidly inactivate jack bean urease. Stability is maintained with the enzymically-active organic extract.

Changes in aggregate stability, nutrient status, indigenous microbial populations, and seedling emergence, following inoculation of soil withNostoc muscorum

The potential of the N2-fixing cyanophyteNostoc muscorum for improving the aggregate stability of a poorly structured silt loam soil was studied in a greenhouse experiment. Inoculum rates were 1.61×105 cells g-1 soil dry weight (low rate) and 4.04×105 cell g-1 soil dry weight (high rate), approximately equivalent to a field application of 2 and 5 kg ha-1 cells dry weight, respectively.N. muscorum numbers had increased 8-fold (low rate) and 10-fold (high rate) by 300 days after inoculation, indicating not only survival but proliferation. Increases in soil polysaccharides, determined as soil carbohydrate C, were 2.96–3.49 time the values in the non-inoculated soils and aggregate stability had incrased by an average of 18% on day 300. Inoculation withN. muscorum also had a pronounced effect on soil chemical and biological properties, with total C increasing by 50–63% and total N increasing by 111–120%. Increases in the soil indigenous microbial population were recorded, with numbers of bacteria 500, fungi 16, and actinomycetes 48 times the non-inoculated values on day 300 in the high-rate soil. The emergence of lettuce seedlings (Lactuca sativa var. Saladin) in undisturbed inoculated 300-day soils was 56% (low rate) and 52% (high rate) higher than in non-inoculated soils. However, homogenising soils and irrigating (to smulate ploughing and surface crusting) significantly reduced this increase in both treatments, although emergence in inoculated soils was still greater by 45% (low) and 24% (high). It is recommended that inoculated soils be left undisturbed prior to planting. The effects ofN. muscorum on soil physical, chemical, and biological properties indicate the possible benefits of cyanobacteria as soil inoculants, not only for the improvement of soil aggregate stability but also as a means of improving seedling emergence.

Covalent immobilization of laccase on activated carbon for phenolic effluent treatment

SummaryLaccase was covalently immobilised to activated carbon using four derivatisation methods. The highest bound activity was obtained using diimide coupling of laccase to carboxyl groups on the carbon. The maximum bound activity was reached at 11.5 mg laccase/g carbon. The carbon-immobilised laccase (CIL) was stable at pH values from 4.0 to 9.0. CIL stored at 4°C lost 38± 5% activity in the first 4 days, then a further 22±5% in 126 days. CIL showed increased stability to low pH although the pH optimum was unchanged. The activation energy of CIL was lower than soluble laccase. Oxidation of 2,6-dimethoxyphenol (DMP) by CIL in a packed-bed system was only 30±10% of that in a fluidised bed system. Of the initial activity 10–30% was retained after oxidation of seven batches of DMP. CIL removed colour from two industrial effluents. Colour was removed from pulp mill bleach plant effluent at 115 colour units per enzyme unit per hour and the removal rate increased with increasing effluent concentration.

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases.

SummaryColour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml−1) precipitated 1.2 g l−1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.

Interactions between Proteins and Soil Mineral Surfaces: Environmental and Health Consequences

Proteins have long been recognized as important compounds in the biogeochemical cycles of terrestrial ecosystems. They can, for example, provide a source of nitrogen for plants and soil microorganisms following proteolysis and ammonification. Extracellular enzymes liberated in soil are essential catalysts in the mobilization of carbon, nitrogen, phosphorus and sulphur from macromolecular organic matter. Proteins are also implicated in new environmental topics, such as soil carbon storage, horizontal transmission of spongiform encephalopathies and potential negative effects of insecticidal toxins released from transgenic plants.

Enhanced Mineralization of [U-14C]2,4-Dichlorophenoxyacetic Acid in Soil from the Rhizosphere of Trifolium pratense

ABSTRACT Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-14C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/α-ketoglutarate dioxygenase (tfdA) were investigated. The rhizosphere effect on [14C]2,4-D mineralization (50 μg g−1) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T. pratense soil but not for 116-day T. pratense rhizosphere soil or for L. perenne rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g−1) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of α-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in T. pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.

Activity, origins and location of cellulases in a silt loam soil

Summary“Cellulase” activity in a silt loam soil was assayed and characterised using a microcrystalline cellulose substrate (Avicel). Activity was maximal between pH 5.3 and pH 6.0. A 64% loss in activity was observed on air-drying the soil. However, the residual activity was stable to storage at 40°C for 7 days and was resistant to the action of added protease. The component endoglucanase and β-D-glucosidase activities in field-moist and air-dried soil were also assayed. The proportion of the soil microbial population able to utilise cellulose was investigated and the persistence of two free (soluble) cellulase preparations of microbial origin was determined following their addition to soil. A rapid decline in the endoglucanase activity of a Streptomyces sp. cellulase preparation was observed while 30% of the original activity of a Trichoderma viride cellulase preparation could still be detected after 20 days. From the data obtained in this study it appears that the major portion of the β-D-glucosidase activity is bound to and protected by the soil colloids. By contrast, the major portion of the exo- and endoglucanase activity appears to be “free” in the soil solution, attached to the outer surfaces of cellulolytic microorganisms or associated in enzyme substrate complexes. The low residual activity measured in air-dried soil may owe its stability to an association with soil colloids or with recalcitrant cellulosic material present in soil.