Survival of rhizobia in soil is sensitive to elevated zinc in the absence of the host plant

The survival of free-living rhizobia in soil is sensitive to elevated heavy metals in soil and can explain adverse effects of metals on symbiotic nitrogen fixation in soils. A survival experiment was set-up to derive critical cadmium (Cd) and zinc (Zn) concentrations in a range of field-contaminated soils in the absence of their host plant (Trifolium repens L.). Soils applied with metal salts or sewage sludge >10 years ago were sampled and were inoculated with Rhizobium leguminosarum bv. trifolii (10⁸ cells g⁻¹ soil) and incubated outdoors for up to 6 months. The most probable number (MPN) decreased 1-2 orders of magnitude in uncontaminated soils during the incubation. There was no significant effect of total metal concentrations on rhizobia survival in soils contaminated with Cd salts or with high Ni/Cd sewage sludge with highest Cd concentrations between 18 and 118 mg Cd kg⁻¹. In contrast, survival was strongly affected in soils contaminated by sewage sludge, where Zn was the principal metal contaminant. Neither total Cd nor soil solution Cd was large enough to attribute these effects to Cd when compared with the soil series, where Cd salts had been applied. The MPN decreased at least one order of magnitude above total Zn concentrations of 233 mg Zn kg⁻¹ (soil pH 5.6) and 876 mg Zn kg⁻¹ (soil pH 6.3). The EC50s of log MPN were 204 and 604 mg Zn kg⁻¹, respectively, and were lower than those for the symbiotic nitrogen fixation measured in the pot trial on the same soils (respectively 602 and 737 mg Zn kg⁻¹). This study corroborates the evidence that symbiotic nitrogen fixation is affected by Zn in the field when Zn decreases the free-living population of rhizobia to below a critical threshold.     

Initial results from long-term field studies at three sites on the effects of heavy metal-amended liquid sludges on soil microbial activity

In a long‐term study of the effects on soil fertility and microbial activity of heavy metals contained in sewage sludges, metal‐amended liquid sludges each with elevated Zn, Cu or Cd concentrations were applied over a 3‐year period (1995–1997) to three sites in England. The experiments were sited adjacent to experimental plots receiving metal‐rich sludge cakes enabling comparisons to be made between the effects of heavy metal additions in metal‐amended liquid sludges and sludge cakes. The liquid sludge additions were regarded as ‘worst case’ treatments in terms of likely metal availability, akin to a long‐term situation following sewage sludge additions where organic matter levels had declined and stabilised. The aim was to establish individual Zn (50–425 mg kg^?1), Cu (15–195 mg kg^?1) and Cd (0.3–4.0 mg kg^?1) metal dose–response treatments at each site, but with significantly smaller levels of organic matter addition than the corresponding sludge cake experiments. There were no differences (P > 0.05) in soil respiration rates, biomass carbon concentrations or most probable numbers of clover Rhizobium between the treatments at any of the sites at the end of the liquid sludge application programme. Soil heavy metal extractability differed between the metal‐amended liquid sludge and metal‐rich sludge cake treatments; Zn and Cd extractabilities were higher from the liquid sludge additions, whereas Cu extractability was higher from the sludge cake application. These differences in metal extractability in the treated soil samples reflected the contrasting NH₄NO₃ extractable metal contents of the metal‐amended liquid sludges and sludge cakes that were originally applied.     

A survey of symbiotic nitrogen fixation by white clover grown on metal contaminated soils

There is conflicting evidence about toxic effects of heavy metals in soil on symbiotic nitrogen fixation. This study was set-up to assess the general occurrence of such effects. Soils with metal concentration gradients were sampled from six established field trials, where sewage sludge or metal salts have been applied, or from a transect in a sludge treated soil. Additional contaminated soils were sampled near metal smelters, in floodplains, in sludge amended arable land and in a metalliferous area. Symbiotic nitrogen fixation was measured with ¹⁵N isotope dilution in white clover (Trifolium repens L.) grown in potted soil that was not re-inoculated, and using ryegrass (Lolium perenne L.) as reference crop. The fraction nitrogen in clover derived from fixation (N_dff) varied from 0 to 88% depending on soil. Pronounced metal toxicity on N_dff was only confirmed in a sludge treated soil where nitrogen fixation was halved from the control value at soil total metal concentration of 737 mg Zn kg⁻¹, 428 mg Cu kg⁻¹ and 10 mg Cd kg⁻¹. The N_dff was significantly reduced by increasing metal concentration in soils from two other sites where N_dff was low throughout and where these effects might be attributed to confounding factors. No significant effects of metals on N_dff were identified in all other gradients even up to elevated total metal concentration (e.g. 55 mg Cd kg⁻¹). The variation of N_dff among all soils (n=48), is mainly explained by the number of rhizobia in the soil (log MPN, log (cells g⁻¹ soil)), whereas correlations with total or soil solution metal concentrations were weak (R²<0.25). The is significantly affected by the presence or absence of the host plant at the sampling site. No effects of metals were identified at even at total Zn concentrations of about 2000 mg Zn kg⁻¹, whereas metal toxicity could be identified at lower most probable number (MPN) values. This survey shows that the metal toxicity on symbiotic nitrogen fixation cannot be generalized and that survival of a healthy population of the microsymbiont is probably the critical factor.     

Mudflat soil amendment by sewage sludge: Soil physicochemical properties,perennial ryegrass growth,and metal uptake

Abstract

The fast pace of cropland loss in China is causing alarm over food security and China’s ability to remain self-reliant in crop production. Mudflats after organic amendment can be an important alternative cropland in China. Land application of sewage sludge has become a popular organic amendment to croplands in many countries. Nevertheless, the land application of sludge to mudflats has received little attention. Therefore, the objective of the present work was to investigate the impact of sewage sludge amendment (SSA) at 0, 30, 75, 150 and 300 t ha^?1 rates on soil physicochemical properties, perennial ryegrass (Lolium perenne L.) growth and heavy metal accumulation in mudflat soil. The results showed that the application of sewage sludge increased organic matter (OM) content by 3.5-fold while reducing salinity by 76.3% at the 300 t ha^?1 rate as compared to unamended soil. The SSA reduced pH, electric conductivity (EC) and bulk density in mudflat soil, increased porosity, cation exchange capacity (CEC) and contents of nitrogen (N), phosphorus (P), exchangeable potassium ions (K⁺), sodium ions (Na⁺), calcium ions (Ca²⁺) and magnesium ions (Mg²⁺) in comparison to unamended soil. There were 98.0, 146.6, 291.4 and 429.2% increases in fresh weight and 92.5, 132.4, 258.6 and 418.9% increases in dry weight of perennial ryegrass at 30, 75, 150, and 300 t ha^?1, respectively, relative to unamended soil. The SSA increased metal concentrations of aboveground and root parts of perennial ryegrass (p < 0.05). The metal concentrations in perennial ryegrass were Zn > Cr > Mn > Cu > Cd > Ni, and the metal concentrations in roots were significantly higher than aboveground parts. The metal accumulation in perennial ryegrass correlated positively with sludge application rates and available metal concentrations in mudflat soil. Land application of sewage sludge was proved to be an effective soil amendment that improved soil fertility and promoted perennial ryegrass growth in mudflat soil. However, heavy metal accumulation in plants may cause food safety concern.     

Effect of sewage sludge on nodulation and N2−fixation in alfalfa grown on calcareous loamy soils

The influence of different rates of sludge applications to calcareous loamy soils of Saudi Arabia, on nodulation and symbiotic N₂?fixation in alfalfa plants (Medicago sativa L.) was studied in a pot experiment. The effect of heavy metals accumulation in soil due to continuous irrigation of the test soil with sewage water was also investigated. Application of up to 80 g sludge per pot enhanced nodulation, nitrogenase activity, dry matter yield and N-contents of alfalfa plants growing in loamy soils either previously irrigated with sewage water or well water. However, sludge applied at the rate of 160–200 g pot^?1 inhibited the nodulation, N accumulation and dry matter yield of alfalfa. The response of alfalfa to sludge was dependent on the rhizobial strain used. Our results also showed that accumulation of heavy metals due to continuous irrigation of a calcareous sandy loam soil with sewage water, for more than 10 years, didn't inhibit N₂?fixation in alfalfa plants, but enhanced it. Microelements in alfalfa plants increased with increase in the rate of sludge application. Although high rates of sludge application affected nodulation and N₂?fixation of alfalfa, dry matter and the nitrogen contents of the plants were not highly affected. Therefore, the inhibitory effect of high rates of sludge was most probably due to the toxic effect of heavy metals on the microsymbiont rather than on the plants.     

Initial results from a long-term, multi-site field study of the effects on soil fertility and microbial activity of sludge cakes containing heavy metals

In a long‐term study of the effects on soil fertility and microbial activity of heavy metals contained in sewage sludges, metal‐rich sludge cakes each with high Zn, Cu or Cd concentrations were applied annually for 4 years (1994–1997) to nine sites throughout Britain. These sites were selected to represent agricultural soils with a range of physical and chemical properties, typical of those likely to be amended with sewage sludge. The aim was to establish individual total Zn (approx. 60–450 mg kg^?1), total Cu (approx. 15–200 mg kg^?1) and total Cd (approx. 0.2–4 mg kg^?1) metal dose–response treatments at each site. Sludges with low metal concentrations were added to all treatments to achieve as constant an addition of organic matter as possible. Across the nine sites, soil pH was the single most important factor controlling Zn (P < 0.001; r² = 92%) and Cd extracted with 1 m NH₄NO₃ (P < 0.001; r² = 72%), and total iron content the most important factor controlling Cu extracted with 1 m NH₄NO₃ (P < 0.001; r² = 64%). There were also positive relationships (P < 0.001) between soil organic carbon (C) concentrations and soil biomass C and respiration rates across the nine sites. Oxidation of sludge C following land application resulted in approximately 45% of the digested sludge cake C and approximately 64% of the ‘raw’ sludge cake C being lost by the end of the 4‐year application period. The sludge cake applications generally increased soil microbial biomass C and soil respiration rates, whilst most probable numbers of clover Rhizobium were generally unchanged. Overall, there was no evidence that the metal applications were damaging soil microbial activity in the short term after the cessation of sludge cake addition.     

Survival and plasmid stability of rhizobia introduced into a contaminated soil

The behaviour of Rhizobium strains introduced separately into soil from a contaminated site with high concentrations of heavy metals (mainly Zn and Hg), and the role of plasmids in the ecology of these rhizobia strains were studied. Six Rhizobium leguminosarum biovar trifolii strains, from different sources and with different plasmid contents, were selected. Two of them were isolated from nodules of subterranean clover plants (Trifolium subterraneum) grown in the contaminated soil and four were from an uncontaminated soil. After inoculation with approximately 10⁷ cells g⁻¹ soil, of each strain, survival and plasmid stability were assessed over a period of 12-18 months. Differences in survival of Rhizobium strains were only detected more than 12 months after inoculation. After 18 months it was clear that survival in contaminated soil was greatest in the two strains originally isolated from that contaminated soil, and also by two of the strains originally isolated from uncontaminated soil. The latter two strains were also the only ones that showed changes in their plasmid profiles. The remaining isolates had the lowest populations, and their plasmid profiles were unchanged and similar to the parent strains.     

施肥对灌漠土作物产量、土壤肥力与重金属含量的影响

有机物还田是提升土壤肥力的主要措施,但也存在造成土壤金属污染的潜在风险。为查明不同有机物还田对土壤质量及作物产量的影响,本文通过长期定位试验,研究了无肥对照、常规施化肥(氮磷配施)以及70%常规化肥与牛粪、沼渣、污泥、鸡粪、菌渣和猪粪配施对土壤理化性状、有机碳和氮的固存率、氮磷钾活化系数、作物产量及重金属含量的影响。结果表明:牛粪、沼渣、污泥、菌渣、鸡粪和猪粪与70%化肥配施虽作物产量与常规施化肥相似,但6种有机物处理土壤有机质、全氮和碱解氮含量都较常规施化肥处理显著增加,污泥、鸡粪和猪粪处理土壤全磷与速效磷含量较常规施化肥处理显著增加,而且牛粪、沼渣、鸡粪和猪粪处理的速效钾、土壤磷活化系数和土壤钾活化系数较常规施化肥处理也显著提升。牛粪、沼渣、污泥、菌渣、鸡粪和猪粪处理土壤有机碳固存率为36.42%～71.61%,较常规施化肥处理都显著提高;而其氮固存率为6.47%～49.44%,仅有菌渣处理与常规施化肥处理差异不显著,而其他处理较常规施化肥处理显著增加。长期施鸡粪和菌渣处理的土壤铜含量较常规施化肥处理显著增加,增加量分别为4.17mg·kg~(-1)和14.2mg·kg~(-1);而污泥、鸡粪和菌渣处理的土壤锌含量较常规施化肥处理显著增加,增加量分别为13.53 mg·kg~(-1)、22.60 mg·kg~(-1)和49.73mg·kg~(-1)。综上,等有机质(4 500kg×hm~(-2))的牛粪、沼渣、污泥、菌渣、鸡粪和猪粪可替代30%氮磷肥,作物产量不受影响;不同有机物培肥土壤效果为污泥、鸡粪和猪粪优于牛粪和沼渣,而沼渣的培肥效果略差。为保证土壤环境质量稳定不恶化,种植小麦时有机物铜和锌的年携入量应分别低于53.01g×hm~(-2)和221.30 g×hm~(-2),而种植玉米时应分别低于153.40 g×hm~(-2)和347.04 g×hm~(-2)。     

Possibilities of using sewage sludge as nitrogen fertilizer for maize

Abstract

The objective of this study was to determine the effects of nitrogen fertilizer sources of ammonium sulphate and municipal sewage sludge on yield, N content and uptake of the maize (Zea mays L.). Nutrient and heavy metals were determined in soil and plant. The experiment with three sludge rates (256, 513 and 1026 kg total N ha^?1 or 9.5, 18.0 and 38.1 t ha^?1 sludge), two nitrogen rates (80 and 160 kg N ha^?1) and zero-N control were conducted on a clay loam soils under irrigated conditions in Eastern Anatolia region in Turkey. Treatments were arranged in a randomized complete block design with four replications. Yield, N content and total N uptake of maize increased significantly with sludge application. 9.5 t and 19.0 t ha^?1 sewage sludge applications did not significantly affect heavy metal content of leaf and grain. However, 38.1 t ha^?1 sludge applications increased leaf Pb and Zn. DTPA-extractable Cd, Cu, Fe, Pb and Zn concentrations of the soil increased at applications of 38.1 t ha^?1 sewage sludge, whereas applications of 9.5 t and 19.0 t ha^?1 sludge only resulted in elevated levels of Cu and Zn, We conclude that if sewage sludge is to be used in production of maize, applications rate up to 19 t ha^?1 could be accepted. However, this means also that the N requirement of maize crop is not covered by the sludge; therefore, the rest of nitrogen could be supplied as inorganic N.     

Impact of heavy metal amended sewage sludge on forest soils as assessed by bacterial and fungal biosensors

Bacterial and fungal bioluminescence-based biosensors were used as indicators of potential heavy metal toxicity to microorganisms in the needle litter of a mature Pinus radiata forest under heavy metal contaminated sewage sludge. Sewage sludge was amended with increasing concentrations of Cu, Ni and Zn and applied to the surface of a mature P. radiata forest. The response of the bacterial and fungal biosensors to soluble Cu, Ni and Zn in needle litter extracts was investigated. The bioluminescence response of the bacterial biosensor Escherichia coli HB101 pUCD607 declined as water-soluble Zn concentrations increased. The effective concentrations that gave a 50% reduction in bioluminescence (EC₅₀ values) for water-soluble Zn and total litter Zn were 1.3 mg l⁻¹ and 3700 mg kg⁻¹, respectively. The bioluminescence response of the fungal biosensor Armillaria mellea declined as soluble Cu concentrations increased. The EC₅₀ values for water-soluble Cu and total litter Cu were 0.12 mg l⁻¹ and 540 mg kg⁻¹, respectively. No decline in bioluminescence was noted for either the bacterial or fungal biosensor on exposure to increasing concentrations of water-soluble Ni. The use of a combination of bacterial and fungal biosensors offers a rapid and sensitive tool for assessing toxicity of heavy metals to microorganisms and, thus, elucidating the environmental impact of contaminants in sewage sludge on litter dwelling microorganisms.     

Heavy metal toxicity in Rhizobium leguminosarum biovar viciae isolated from soils subjected to different sources of heavy-metal contamination: Effects on protein expression

Heavy metals adversely influence microorganisms, affecting their growth, morphology and activities. Metals also can exert a selective pressure on the organisms, resulting in microbial populations with higher tolerance to metals. Given the importance of legumes in animal and human consumption and their use in maintaining soil fertility, some attention has been given to the effects that heavy metals exert on Rhizobium isolates. In this context, Rhizobium leguminosarum biovar viciae was isolated from areas with different heavy metal contents and their tolerances were compared. Alterations in the protein pool of Rhizobium populations were also evaluated. Physicochemical parameters were determined and heavy metal concentrations in soils were analysed by ICP-AES. Isolates were screened for their tolerance in YEM media supplemented with different heavy metals (Zn, Pb, Co, Cd, Ni, Cr). Proteins were extracted and separated by SDS-PAGE. EI₁ and EI₂ (engineering industries) soils presented the highest metal concentration, and were therefore the most polluted soils. Isolates showed different growth responses to heavy metals. C (control soil) and M (mines) isolates were less tolerant than EI₁, EI₂ and CI (chemical industries) isolates. Metals influenced their protein profiles, most of the alterations corresponding to decreases in polypeptide expression. However, in tolerant isolates these alterations corresponding basically to increases, as occurred in CI isolates.This work suggests that there is a relationship between Rhizobium's tolerance, heavy metal soil contamination and alterations in protein pool. As a result, the analysis of protein alterations seems to be a good indicator to estimate the level of stress imposed on Rhizobium populations submitted to heavy-metal contamination.     

Population size of indigenous Rhizobium leguminosarum biovar trifolii in long-term field experiments with sewage sludge cake,metal-amended liquid sludge or metal salts: Effects of zinc,copper and cadmium

There is conflicting evidence, and therefore continuing concern, as to whether metals in sewage sludge are deleterious to soil microbial processes and long-term agricultural productivity. Nine field experiments with sewage sludge cakes, three with metal-amended liquid sludges and three with inorganic metal salts were set up across Britain in 1994 to give individual metal dose–response treatments to try to answer this question. This study reports on the effects of Zn, Cu and Cd on the population size of Rhizobium leguminosarum biovar trifolii, a nitrogen fixing symbiont of white clover (Trifolium repens), in soils from these experiments over 11 years. Significant (P < 0.05) reductions in indigenous rhizobial numbers occurred on the Zn metal dose–response treatments at eight of the sludge cake sites in 2005, but few consistent effects were evident on the Cu or Cd metal dose–response treatments during the 11-year monitoring period. The soil total Zn concentrations where effects occurred were near to the UK statutory limit of 300 mg kg^?1 for soils receiving sewage sludge. No significant reductions occurred in any treatments on the metal-amended liquid sludge or inorganic metal salt experiments in which the metals would be expected to be in a more bioavailable form, even after 11 years. The effects in the sludge cake experiments were related consistently with soil total Zn, with no recovery to date. The reductions in clover rhizobial numbers in the sludge cake experiments were due to Zn effects on free-living rhizobia in the soil, with gradual die-off over a long time with increasing soil total Zn concentrations. Currently, no consistent adverse effects on rhizobia have been seen at the UK limits for Cu and Cd of 135 and 3 mg kg^?1, respectively.     

Availability of Heavy Metals for Brassica Chinensis Grown in an Acidic Loamy Soil Amended with a Domestic and an Industrial Sewage Sludge

The use of sewage sludge on agriculture provides an alternativefor sewage sludge disposal. Therefore, it was the aim of thepresent study to evaluate the feasibility of using a domestic(Tai Po sludge) and an industrial (Yuen Long sludge) sewagesludge produced in Hong Kong for the growth of vegetable crops.The acidic loamy soil with or without lime treatment was amendedseparately with each sludge at application rates of 0, 5, 10, 25and 50% (v/v) for the growth of a common local vegetable crop,Brassica chinensis. The plant available metal contents, asindicated by the DTPA extraction, increased with an increase insludge amendment, but decreased with lime amendment at eachsludge application rate due to the reduced metal availabilityat a higher pH. Sludge amendment enhanced the dry weight yieldof B. chinensis and the increase was more obvious for thesoil with lime treatment. The industrial sludge caused a loweryield than that of the domestic sludge amendment and asignificant reduction in yield at high application rates of YuenLong sludge was also noted. Tissue heavy metal contents, exceptfor Fe, increased as the sludge amendment rate increased whileplant grown in Yuen Long sludge amended soil contained higher Crand Zn contents at each sludge application rate. Liming the soilreduced the heavy metal contents in the plant tissues, exceptfor Fe, which were all below the allowable levels for vegetablecrops. The present experiment demonstrates that liming wasimportant in facilitating the growth of B. chinensis in sludge amended soil. The optimal sludge amendment rate for thesoil with lime amendment was 25% Tai Po sludge and 10% YuenLong sludge, while for the soil without lime amendment was 10% and5%, respectively.     

Sequential fractionation and plant availability of heavy metals as affected by sewage sludge applications to soil

Abstract

The effect of sewage sludge applications on extractability and uptake by chard and lettuce of soil cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), leaf (Pb), and zinc (Zn) was investigated. Ten different treatments (0, 150, 300, and 500 kg N ha^‐1) as mineral fertilizer, and 400, 800, and 1,200 kg N ha^‐1 of aerobically and anaerobically‐digested sewage sludges were applied annually to a sandy‐loam soil since 1984. Seven years after the start of the treatments, higher levels of heavy metals were detected in the soil, depending on the type of metal, depth of sampling, type of sludge used, and, especially, rate of application. Following a sequential extraction procedure incorporating 0.1M CaCl₂, 0.5M NaOH, and 0.05M Na₂EDTA, most of the heavy metals in soil were detected in the Na₂EDTA solution and the residual fractions. Large amounts of Cd appeared to be extracted by CaCl₂, whereas substantial amounts of Cu and Ni were isolated by NaOH. The effect of treatments on the percentages of the metals found in each fraction depended on the type of metal, sampling depth, sludge used, and application rate. No significant increases were found in the heavy metal contents of chard and lettuce leaves, but some of the treatments resulted in a significant decrease of Cd and Cr levels in lettuce leaves.     

Comparison of routine soil tests and EPA method 3050 as extractants for heavy metals in Delaware soils

Abstract

Agricultural use of sewage sludges can be limited by heavy metal accumulations in soils and crops. Information on background levels of total heavy metals in soils and changes in soil metal content due to sludge application are; therefore, critical aspects of long‐term sludge monitoring programs. As soil testing laboratories routinely, and rapidly, determine, in a wide variety of agricultural soils, the levels of some heavy metals and soil properties related to plant availability of these metals (e.g. Cu, Fe, Mn, Zn, pH, organic matter, texture), these labs could participate actively in the development and monitoring of environmentally sound sludge application programs. Consequently, the objective of this study was to compare three soil tests (Mehlich 1, Mehlich 3, and DTP A) and an USEPA approved method for measuring heavy metals in soils (EPA Method 3050), as extractants for Cd, Cu, Ni, Pb and Zn in representative agricultural soils of Delaware and in soils from five sites involved in a state‐monitored sludge application program.

Soil tests extracted less than 30% of total (EPA 3050) metals from most soils, with average percentages of total metal extracted (across all soils and metals) of 15%, 32%, and 11% for the Mehlich 1, Mehlich 3, and DTPA, respectively. Statistically significant correlations between total and soil test extractable metal content were obtained with all extractants for Cu, Pb, and Zn, but not Cd and Ni. The Mehlich 1 soil test was best correlated with total Cu and Zn (r=0.78***, 0.60***, respectively), while the chelate‐based extractants (DTPA and Mehlich 3) were better correlated with total Pb (r=0.85***, 0.63***). Multiple regression equations for the prediction of total Cu, Ni, Pb, and Zn, from soil test extractable metal in combination with easily measured soil properties (pH, organic matter by loss on ignition, soil volume weight) had R² values ranging from 0.41*** to 0.85***, suggesting that it may be possible to monitor, with reasonable success, heavy metal accumulations in soils using the results of a routine soil test.     

Assessment of Arsenic and Selenium Concentration with Chlorophyll Contents of Sugar Beet (Beta vulgaris var.saccharifera) and Wheat (Triticum aestivum) Exposed to Municipal Sewage Sludge Doses

The present study was conducted to assess the suitability of sewage sludge amendment in soil for Beta vulgaris var. saccharifera (sugar beet) and Triticum aestivum (wheat) by evaluating the arsenic and selenium accumulation and physiological responses of plants grown at 10%, 25%, and 50% sewage sludge amendment rate. Sewage sludge amendment was modified by the physicochemical properties of soil, thus increasing the availability of heavy metals in the soil and consequently with higher accumulation in plant parts. The chlorophyll contents increased after the sewage sludge treatments except for 50%. The sewage sludge amendment led to a significant increase in arsenic and selenium concentrations of the soil. The heavy metal accumulation in the soil after the treatments did not exceed the limits for the land application of sewage sludge recommended by the US Environmental Protection Agency. The increased concentration of heavy metals in the soil due to the sewage sludge amendment led to increases in heavy metal uptake in the leaves and root concentrations of arsenic and selenium in plants as compared to those grown on unamended soil. Accumulation was more in roots than shoots and leaves for most of the heavy metals. Concentrations of arsenic and selenium were more than the permissible limits of national standards in the edible portion of sugar beet and wheat grown on different sewage sludge amendments ratios. The study concludes that the sewage sludge amendment in the soil for growing sugar beet and wheat may not be a good option due to risk of contamination of arsenic and selenium.     

PLANT GROWTH PROMOTING RHIZOBACTERIA INCREASE GROWTH,YIELD AND NITROGEN FIXATION IN PHASEOLUS VULGARIS

Nitrogen (N) fixation by legume-Rhizobium symbiosis is important to agricultural productivity and is therefore of great economic interest. Growing evidence indicates that soil beneficial bacteria can positively affect symbiotic performance of rhizobia. The effect of co-inoculation with plant growth-promoting rhizobacteria (PGPR) and Rhizobium, on nodulation, nitrogen fixation, and yield of common bean (Phaseolus vulgaris L.) cultivars was investigated in two consecutive years under field conditions. The PGPR strains Pseudomonas fluorescens P-93 and Azospirillum lipoferum S-21 as well as two highly effective Rhizobium strains were used in this study. Common bean seeds of three cultivars were inoculated with Rhizobium singly or in a combination with PGPR to evaluate their effect on nodulation and nitrogen fixation. A significant variation of plant growth in response to inoculation with Rhizobium strains was observed. Treatment with PGPR significantly increased nodule number and dry weight, shoot dry weight, amount of nitrogen fixed as well as seed yield and protein content. Co-inoculation with Rhizobium and PGPR demonstrated a significant increase in the proportion of nitrogen derived from atmosphere. These results indicate that PGPR strains have potential to enhance the symbiotic potential of rhizobia.     

Transport of zinc,copper, and lead in a sewage sludge amended calcareous soil

The application of sewage sludge on farmland is practised in many countries since sludge is rich in macro- and micro- nutrients. However, increasing use of sewage sludge on farmland has raised concerns about the potential transport of heavy metals into food chains and groundwater. This study determined for a calcareous soil the effects of sludge application on soil physical properties and transport of zinc (Zn), copper (Cu), and lead (Pb). Secondary anaerobic digested sewage sludge was applied at rates of 0, 25, 50, and 100 t/ha (on a dried weight basis) for four consecutive years and mixed in the top 20-cm of soil. Corn (Zea mays L.) was planted as a spring crop, followed by wheat (Triticum aestivum) as a winter crop. Sludge application increased the dissolved organic matter content and modified the soil structure, increased the soil infiltration rate, saturated hydraulic conductivity, and aggregate stability, and decreased the bulk density. Sludge application greatly increased DTPA (diethylenetriamine pentaacetic acid)-extractable soil metal concentrations to 50 cm depth and significantly to 1 m. In the plots that received four application of 100 t/ha sewage sludge, the mean concentrations of Zn, Cu, and Pb in subsoil increased by 1600, 7, and 4.5 times, respectively, compared with the control. The results indicate that a combination of enhanced soil physical properties, heavy and inefficient irrigation and high organic matter content with heavy metals cause significant metal mobility. High sludge applications pose risks of groundwater and food chain contamination and rates are best restricted to those reflecting the nutrient demand of crops (20 t/ha every 4 to 5 yr or an average of 4 to 5 t/ha/yr).     

Fate of gram-negative bacterial biomass in soil—mineralization and contribution to SOM

Soil microorganisms contribute to the formation of non-living soil organic matter (SOM) by metabolic transformation of plant-derived material. After cell death, their biomass components with a specific molecular character become incorporated into SOM imprinting its chemical properties, although this process has not yet been quantified. In order to elucidate the contribution to SOM formation, we investigated the fate of gram-negative bacterial model biomass (Escherichia coli usually introduced into soil with manure or feces) during incubation of soil with isotopically (¹³C) and genetically (lux gene) labeled cells. The decline of living cells was monitored by the loss of bioluminescence. The carbon turnover and mineralization was balanced by bulk soil stable isotope analysis, and the persistence of nucleic acids was investigated by PCR amplification of the lux gene. During incubation, the number of viable E. coli cells decreased rapidly (99.9% within the first 42 d) serving as substrate for other microorganisms or for the formation of SOM, and bioluminescent cells could only be detected during the first 56 d. However, the lux gene was still detected after 224 d, which indicates stabilization of DNA in SOM. Although the survival of E. coli in soil is limited, only about 65% of the added labeled biomass carbon was mineralized to ¹³CO₂ and 51% remained in soil after 224 d with an average ¹³C recovery of 117%. The amount of ¹³C found in the PLFA representative of living cells had decreased to 25% of the initial value, suggesting a proportional decrease of the ¹³C in the soil microbial biomass. The extent of this decrease is higher than the mineralization of the bulk E. coli C and thus the difference of around 25% has to be stabilized as metabolites, or in non-living SOM. The data provide evidence that the genetic information and a considerable part of the carbon from dying bacterial biomass were retained in both the soil microbial food web and in non-living SOM.     

Quantification of nitrogen fixation by the peanut/Rhizobium symbiotic system in a virgin sandy soil

Nitrogen balance studies were conducted to quantify the nitrogen fixed by peanut/Rhizobium symbiotic system under field conditions in a sandy soil. Large scale inoculation with three NifTAL strains of cowpea rhizobia, 1000, 169, 1371 was done using two inoculation techniques: peat-based inoculant or injection of inoculant with irrigation water through an injection tank attached to the central pivot system. The results show nitrogen fixation amounting up to 186 kg N ha^?1 in peat-based inoculant and 171 kg N ha^?1 in liquid inoculant injected through the irrigation system. However, no significant differences in yield response were recorded between both inoculation techniques.