Enhanced biodegradation of diesel fuel through the addition of particulate organic carbon and inorganic nutrients in coastal marine waters

Diesel fuel pollution in coastal waters, resulting from recreational boating and commercial shipping operations, is common and can adversely affect marine biota. The purpose of this study was to examine the effect of additions of particulate organic carbon (POC) in the form of naturally-occurring marsh grass (Spartina alterniflora), inorganic nutrients (nitrogen and phosphorus), inert particles, and dissolved organic carbon (DOC) on diesel fuel biodegradation and to attempt to formulate an effective bioremedial treatment for small diesel fuel spills in marine waters. Various combinations of treatments were added to water samples from a coastal marina to stimulate diesel fuel biodegradation. Diesel fuel was added in concentrations approximating those found in a spill and biodegradation of straight chain aliphatic constituents was estimated by measuring mineralization of ¹⁴C hexadecane added to diesel fuel. All treatments that included POC showed stimulation of biodegradation. However, the addition of inert particles (glass fiber filters and nylon screening) caused no stimulation of biodegradation. The addition of nitrogen and phosphorus alone did not result in stimulation of biodegradation, but nitrogen and Spartina (although not phosphorus and Spartina) did result in stimulation above that of Spartina alone. Maximum biodegradation rates were obtained by the addition of the Spartina POC, ammonium, and phosphate. The addition of mannitol, a labile DOC source with POC and phosphate resulted in a decrease in diesel fuel biodegradation as compared to POC and phosphate alone. The seasonal pattern of diesel fuel biodegradation showed a maximum in the summer and a minimum in the winter. Therefore, of the treatments tested, the most effective for bioremediation of diesel fuel in marine waters is the addition of POC, nitrogen, and phosphorus.     

Enhanced biodegradation of diesel fuel through the addition of particulate organic carbon and inorganic nutrients in coastal marine waters

Diesel fuel pollution in coastal waters, resulting from recreational boating and commercial shipping operations, is common and can adversely affect marine biota. The purpose of this study was to examine the effect of additions of particulate organic carbon (POC) in the form of naturally-occurring marsh grass (Spartina alterniflora), inorganic nutrients (nitrogen and phosphorus), inert particles, and dissolved organic carbon (DOC) on diesel fuel biodegradation and to attempt to formulate an effective bioremedial treatment for small diesel fuel spills in marine waters. Various combinations of treatments were added to water samples from a coastal marina to stimulate diesel fuel biodegradation. Diesel fuel was added in concentrations approximating those found in a spill and biodegradation of straight chain aliphatic constituents was estimated by measuring mineralization of ¹⁴C hexadecane added to diesel fuel. All treatments that included POC showed stimulation of biodegradation. However, the addition of inert particles (glass fiber filters and nylon screening) caused no stimulation of biodegradation. The addition of nitrogen and phosphorus alone did not result in stimulation of biodegradation, but nitrogen and Spartina (although not phosphorus and Spartina) did result in stimulation above that of Spartina alone. Maximum biodegradation rates were obtained by the addition of the Spartina POC, ammonium, and phosphate. The addition of mannitol, a labile DOC source with POC and phosphate resulted in a decrease in diesel fuel biodegradation as compared to POC and phosphate alone. The seasonal pattern of diesel fuel biodegradation showed a maximum in the summer and a minimum in the winter. Therefore, of the treatments tested, the most effective for bioremediation of diesel fuel in marine waters is the addition of POC, nitrogen, and phosphorus.     

Modulation of microbial consortia enriched from different polluted environments during petroleum biodegradation

Environmental microbial communities are key players in the bioremediation of hydrocarbon pollutants. Here we assessed changes in bacterial abundance and diversity during the degradation of Tunisian Zarzatine oil by four indigenous bacterial consortia enriched from a petroleum station soil, a refinery reservoir soil, a harbor sediment and seawater. The four consortia were found to efficiently degrade up to 92.0% of total petroleum hydrocarbons after 2 months of incubation. Illumina 16S rRNA gene sequencing revealed that the consortia enriched from soil and sediments were dominated by species belonging to Pseudomonas and Acinetobacter genera, while in the seawater-derived consortia Dietzia, Fusobacterium and Mycoplana emerged as dominant genera. We identified a number of species whose relative abundances bloomed from small to high percentages: Dietzia daqingensis in the seawater microcosms, and three OTUs classified as Acinetobacter venetianus in all two soils and sediment derived microcosms. Functional analyses on degrading genes were conducted by comparing PCR results of the degrading genes alkB, ndoB, cat23, xylA and nidA1 with inferences obtained by PICRUSt analysis of 16S amplicon data: the two data sets were partly in agreement and suggest a relationship between the catabolic genes detected and the rate of biodegradation obtained. The work provides detailed insights about the modulation of bacterial communities involved in petroleum biodegradation and can provide useful information for in situ bioremediation of oil-related pollution.     

Biodegradation of diesel fuel by a microbial consortium in the presence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues

Fast development of ionic liquids as gaining more and more attention valuable chemicals will undoubtedly lead to environmental pollution. New formulations and application of ionic liquids may result in contamination in the presence of hydrophobic compounds, such as petroleum mixtures. We hypothesize that in the presence of diesel fuel low-water-soluble ionic liquids may become more toxic to hydrocarbon-degrading microorganisms. In this study the influence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues (side-chain length from C₃ to C₁₈) on biodegradation of diesel fuel by a bacterial consortium was investigated. Whereas test performed for the consortium cultivated on disodium succinate showed that toxicity of the investigated ionic liquids decreased with increase in side-chain length, only higher homologues (C₈–C₁₈) caused a decrease in diesel fuel biodegradation. As a result of exposure to toxic compounds also modification in cell surface hydrophobicity was observed (MATH). Disulphine blue active substances method was employed to determine partitioning index of ionic liquids between water and diesel fuel phase, which varied from 1.1 to 51% for C₃ and C₁₈ homologues, respectively. We conclude that in the presence of hydrocarbons acting as a solvent, the increased bioavailability of hydrophobic homologues is responsible for the decrease in biodegradation efficiency of diesel fuel.     

Bioremediation strategies for diesel and biodiesel in oxisol from southern Brazil

Leaks and spillages during the extraction, transport and storage of petroleum and its derivatives may result in environmental contamination. Biodiesel is an alternative energy source that can contribute to a reduction in environmental pollution. The aim of the present work was to evaluate biodegradation of diesel, biodiesel, and a 20% biodiesel-diesel mixture in oxisols from southern Brazil, using two bioremediation strategies: natural attenuation and bioaugmentation/biostimulation. Fuel biodegradation was monitored over 60 days by dehydrogenase activity, CO₂ evolution and gas chromatography. The bacterial inoculum employed for bioaugmentation/biostimulation consisted of Bacillus megaterium, Bacillus pumilus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia and PCR-DGGE using 16S RNAr primers showed that some members of this consortium survived in the soil after 60 days. The biodegradation of pure biodiesel was higher for bioaugmentation/biostimulation than for natural attenuation, suggesting that the addition of the microbial consortium, together with adjustment of the macronutrient ratio, increased biodiesel degradation. The results of dehydrogenase and respiratory activity, together with GC analysis, suggested that the presence of biodiesel may, by stimulating general microbial degradative metabolism, increase the biodegradation of petroleum diesel. The microbial community was altered by both treatments, with natural attenuation producing a lower diversity index than the amended soil. The bioaugmentation/biostimulation strategy was showed to have a high potential for cleaning up soils contaminated with diesel and biodiesel blends.     

Interactions between rhamnolipid biosurfactants and toxic chlorinated phenols enhance biodegradation of a model hydrocarbon-rich effluent

Surfactant-mediated treatment increases hydrocarbon solubilization and potentially facilitates biodegradation, unless toxic co-contaminants inhibiting microbial activity are present in the hydrocarbon mixture. We assessed the effect of rhamnolipids on the performance of a bacterial consortium degrading diesel fuel employed as a model hydrocarbon-rich effluent, co-contaminated with toxic phenol, 4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP). This approach led to the unexpected finding that rhamnolipids reduced toxicity of 4-CP and 2,4-DCP to the hydrocarbon-degrading cells. The facts that rhamnolipids decreased diesel fuel - water partition coefficient (K_FW) of 4-CP and 2,4-DCP and modified aggregate size distribution profiles of the dispersed diesel fuel - chlorinated phenols solutions, suggest the existence of specific interactions between rhamnolipids and the co-contaminants. Due to the polar nature of 4-CP and 2,4-DCP, possible explanations involve adsorption of 4-CP and 2,4-DCP on the surface of biosurfactant aggregates. This property of rhamnolipids is of interest to those using biosurfactants for microbial treatment of hydrocarbon-rich wastewaters co-contaminated with toxic compounds.     

Identification and Kinetic Characteristics of an Indigenous Diesel-degrading Gordonia alkanivorans Strain

Summary An indigenous strain Gordonia alkanivorans CC-JG39 was isolated from oil-contaminated sludge of a local gas station located in central Taiwan. The bacterial isolate was able to grow on diesel-containing Bushnell–Haas medium and also tolerate various chemical additives frequently used in petroleum products (e.g. BETX, methyl-tert-butyl ether, and naphthalene). Kinetics of diesel-limited cell growth and biodegradation of diesel followed a Monod-type model. The kinetic constants for cell growth (μ_max and K_S,G) were 0.158 h⁻¹ and 3196 mg/l, respectively, while those for biodegradation of diesel (v_{max, diesel} and K_S,D) were 3.59 mg/h/mg cell and 2874 mg/l, respectively. G. alkanivorans CC-JG39 produced extracellular surface-active material, leading to a low surface tension of nearly 33 mN/m. The CC-JG39 strain also possessed the ability to float towards the oil/water interface. These features might play some roles in enhancing the mass transfer efficiency between oil substrate and the bacterial cells. Therefore, G. alkanivorans CC-JG39 may have potential applications in bioremediation of oil pollution sites.     

Use of Rice Husk as Bulking Agent in Bioremediation of Automobile Gas Oil Impinged Agricultural Soil

Evaluation of rice husk (RH) as bulking agent in bioremediation of automobile gas oil (AGO) hydrocarbon polluted agricultural soil using renewal by enhanced natural attenuation (RENA) as control was the subject of the present investigation. The effect of different parameters such as total petroleum hydrocarbon (TPH), dehydrogenase activity (DHA), optical density and pH on bioremediation performance were evaluated. The studied parameters such as microbial dynamics, percentage degradation and DHA were found to be higher in RH-amended system and differed significantly with control at P < 0.05. RH resulted in high removal efficiency of 97.85 ± 0.93% under a two-month incubation period, while RENA had lesser removal efficiency of 53.15 ± 3.81%. Overall hydrocarbon biodegradation proceeded very slowly in the RENA particularly from week 0 to 4. Experimental data perfectly fitted into the first-order kinetic and generated high r² values (0.945), first-order degradation constant (0.47 day^?1), and shorter degradation half-life (1.50 d)—t_1/2 = Ln2/K and Ln2 numerically equals to 0.693 and hence written as 0.693/K. Micrococcus luteus and Rhizopus arrhizus were isolated in the present study, which displayed extreme AGO hydrocarbon biodegradative abilities. The use of RH in hydrocarbon-polluted soil significantly increased biodegradation rate and resulted in effective AGO cleanup within 2 months period. Therefore, RH provides an alternative source of bioremediation material in field application for abundant petroleum hydrocarbon soil pollution.     

Empirical Models Estimating Carbon Dioxide Accumulation in Two Petroleum Hydrocarbon–Contaminated Soils

Bioremediation is a popular method in degrading diesel fuel contaminants from soil. Bioremediation can be enhanced by estimating the effect of important environmental parameters on microbial activity. Respirometry was used to develop empirical models describing the effects of temperature, moisture, nitrogen, and phosphorus concentration on microbial activity in a diesel-contaminated soil from Wyoming. Carbon dioxide (CO₂) data were analyzed using a base equation where its coefficient values were functions of each parameter. Two physiologically different groups of microorganisms were identified from the results under different operating temperatures. The empirical correlations were combined into one model and this model was tested against a hydrocarbon-contaminated soil collected from a site in Egypt with similar history of contamination. The predicted CO₂ evolution agreed well with the actual data obtained from the Egyptian soil samples, showing a sound predicting power of the empirical model for petroleum hydrocarbon biodegradation. Overall, the empirical correlations developed from the respirometric data provide a method to describe microbial activity in diesel-contaminated soils.     

Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.     

Kinetic Modeling and Error Analysis for Decontamination of Different Petroleum Hydrocarbon Components in Biostimulation of Oily Soil Microcosm

A microcosm study was constructed to investigate the effect of complex co-substrate (corn steep liquor, CSL) addition on indigenous bacterial community, rate and extent of petroleum hydrocarbons (PH) degradation in an oily soil with total petroleum hydrocarbons (TPH) content of 63353 mg kg^?1. TPH degradation was found to be characterized by a rapid phase of degradation during the first three weeks where 76% removal of TPH occurred, followed by a slower degradation phase, where further 7% of the initial TPH was removed by the end of incubation period, 35 d. Branched alkanes are more resistant to microbial degradation than n-alkanes. Furthermore, the unresolved complex mixtures (UCM) of hydrocarbons are less degradable than n- and iso-alkanes. Pristane (Pr) was the most recalcitrant aliphatic compound studied in this work. These results in addition to the extensive bacterial growth observed (from 10⁷ to 10¹⁰ CFU g^?1 soil) give strong support that the addition of CSL resulted in increased degradation rates. The indigenous bacteria grew exponentially during the incubation period of 35 d with a growth rate of 0.26 d^?1. Kinetic modeling was performed to estimate the rates of biodegradation of each hydrocarbon type component in the studied system. Five different error functions were used in this study to evaluate the fitness of the model equation to the obtained experimental data. This showed that the degradation of ∑nC₂₀-nC_24, ∑nC₃₅-nC₄₂ and nC₁₈ can be better represented by a second order model, whereas the TPH, total resolvable peaks (TRP), nC₁₇, UCM, ∑nC₁₀-nC₁₄, ∑nC₁₅-nC_19, ∑nC₂₅-nC₂₉, ∑nC₃₀-nC_34, ∑nC_n, and ∑isoC_n and isoprenoids Pr and phytane (Ph) were similarly following the first order model.     

Diesel Biodegradation Capacities and Biosurfactant Production in Saline-Alkaline Conditions by Delftia sp NL1, Isolated from an Algerian Oilfield

Abstract

In this study, a diesel oil-degrading bacterium was isolated from an oilfield water injection (water-bearing formations, 1,205?m depth) in Algeria. The bacterial strain, designated NL1, was cultivated on diesel oil as sole carbon and energy sources. Molecular analyses of the 16S rRNA gene sequence (KY397882) placed NL1 strain closely related to distinct cultivated species of the Delftia genus. Optimal diesel oil biodegradation by Delftia sp NL1 strain occurred at pH 11, 40?°C, 2?M NaCl and initial hydrocarbon concentration of 5% (v/v) as sole carbon source. GC-MS analyses evidenced that strain Delftia sp NL1 was able to degrade more than 66.76% of diesel oil within only 7?days. On the other hand, and in the same conditions, biosurfactant production by Delftia sp NL1 was also evaluated evidencing high emulsifying capacity (E₂₄ = 81%), ability to lower the surface tension of growing media (with the value of 25.7?mN m^?1), and production of glycolipids (8.7?g L^?1) as biosurfactants. This research presents indigenous strain Delftia sp NL1 for diesel degradation and synthesis of biosurfactant in extreme conditions. In this sense, strain NL1 is a good candidate for possible in situ oil recovery and in wastewater treatment in refineries and oil terminals in petroleum industry.     

Metagenomic analysis of microbial communities across a transect from low to highly hydrocarbon-contaminated soils in King George Island,Maritime Antarctica

Soil samples from a transect from low to highly hydrocarbon-contaminated soils were collected around the Brazilian Antarctic Station Comandante Ferraz (EACF), located at King George Island, Antarctica. Quantitative PCR (qPCR) analysis of bacterial 16S rRNA genes, 16S rRNA gene (iTag), and shotgun metagenomic sequencing were used to characterize microbial community structure and the potential for petroleum degradation by indigenous microbes. Hydrocarbon contamination did not affect bacterial abundance in EACF soils (bacterial 16S rRNA gene qPCR). However, analysis of 16S rRNA gene sequences revealed a successive change in the microbial community along the pollution gradient. Microbial richness and diversity decreased with the increase of hydrocarbon concentration in EACF soils. The abundance of Cytophaga, Methyloversatilis, Polaromonas, and Williamsia was positively correlated (p-value = <.05) with the concentration of total petroleum hydrocarbons (TPH) and/or polycyclic aromatic hydrocarbons (PAH). Annotation of metagenomic data revealed that the most abundant hydrocarbon degradation pathway in EACF soils was related to alkyl derivative-PAH degradation (mainly methylnaphthalenes) via the CYP450 enzyme family. The abundance of genes related to nitrogen fixation increased in EACF soils as the concentration of hydrocarbons increased. The results obtained here are valuable for the future of bioremediation of petroleum hydrocarbon-contaminated soils in polar environments.     

微生物强化修复石油污染土壤的研究进展

微生物修复被认为是去除石油污染物和修复石油污染土壤的一种经济、高效且无二次污染的绿色清洁技术。受土壤环境条件和石油污染物性质等因素制约,土壤中土著石油降解微生物常存在数量不足、活性偏低、生长缓慢等问题,导致修复效果不佳、修复周期偏长。微生物强化修复技术可有效提高微生物降解效能,通过投加具有降解效能的功能菌株或菌剂、营养物质、表面活性剂、生长基质及固定化微生物等手段,可改善提升土著微生物对石油污染土壤的修复效果。文中梳理了已报道的石油降解微生物的种类,总结了微生物修复石油污染土壤的主要影响因素,阐述了微生物强化修复石油土壤的多种有效策略,提出了微生物强化修复石油污染的未来发展方向。     

Impacts of diesel fuel exposure on the microphytobenthic community of an intertidal sand flat

This study employed simulated spills of weathered diesel fuel and measured the initial effects on the intertidal sand flat microphytobenthic (MPB) communities. The goals were to examine the impacts of short-term (hours) and longer-term (days) exposure to petroleum on the native sand flat MPB in coastal North Carolina and to assess recovery of the community following the exposure. We assessed changes in biomass (chlorophyll a), primary productivity (¹⁴C bicarbonate incorporation), photophysiology (P vs. I curves) and species composition (microscopy) and compared diesel exposed samples to unamended controls. We found that short-term impacts of diesel fuel pollution were confined to primary productivity and photophysiology of sand flat MPB. Short-term effects were only detected at relatively high concentrations that are not common outside of a major spill event. In the longer term, diesel fuel was again found to have effects on primary productivity, but at higher concentrations than would be likely to occur in industrialized coastal areas. However, negative impacts on photophysiology were detected at diesel fuel concentrations slightly above typical ambient conditions in coastal waters in industrialized areas. Biomass as measured by chlorophyll a was not affected by any concentration in the longer-term exposure to diesel fuel. Cell counts in the longer-term experiments found cyanobacteria had larger negative impacts from diesel fuel exposure than did diatoms. The recovery portion of this study showed the sand flat MPB communities were fairly resilient following both additions of diesel fuel. However, photophysiology and cell counts did not return to conditions equivalent to the control. Data from this study indicate that the effects of petroleum pollution on the MPB community of tidal sand flats should be considered alongside effects on other coastal microalgae in ecological and damage assessments.     

Coupling hydrocarbon degradation to anaerobic respiration and mineral diagenesis: theoretical constraints

The diagenetic mineral assemblages in petroleum reservoirs control the formation fluid pH and pCO₂. Anaerobic biodegradation of petroleum is controlled by the transfer of electrons from reduced organic species to inorganic, redox sensitive, aqueous and mineral species in many cases through intermediates such as H₂ and CH₃COO^?. The terminal electron accepting reactions induce the dissolution or precipitation of the same minerals that control the ambient pH and pCO₂ in petroleum reservoirs. In this study, we develop a model for anaerobic biodegradation of petroleum that couples the production of acetate and H₂ to ‘late stage’ diagenetic reactions. The model reveals that the principal terminal electron accepting process and electron donor control the type of diagenetic reaction, and that the petroleum biodegradation rate is controlled through thermodynamic restriction by the minimum ΔG required to support a specific microbial metabolism, the fluid flux and the mineral assemblage. These relationships are illustrated by modeling coupled microbial diagenesis and biodegradation of the Gullfaks oil reservoir. The results indicate that the complete dissolution of albite by acids generated during oil biodegradation and the corresponding elevated pCO₂ seen in the Gullfaks field are best explained by methanogenic respiration coupled to hydrocarbon degradation and that the biodegradation rate is likely controlled by the pCH₄. Biodegradation of Gullfaks oil by a consortium that includes either Fe³⁺‐reducing or ‐reducing bacteria cannot explain the observed diagenetic mineral assemblage or pCO₂. For octane, biodegradation, not water washing, was the principal agent for removal at fluid velocities <20 m Myr^?1.     

Effects of nutrient and temperature on degradation of petroleum hydrocarbons in sub-Antarctic coastal seawater

In an attempt to evaluate the potential of petroleum bioremediation at high latitudes environments, microcosm studies using Antarctic coastal seawater contaminated with diesel or crude oil were conducted in Kerguelen Archipelago (49°22′S, 70°12′E). Microcosms were incubated at three different temperatures (4, 10 and 20°C). During experiments, changes observed in microbial assemblages (total direct count, heterotrophic cultivable microorganisms and hydrocarbon-degrading microorganisms) were generally similar for all incubation temperatures, but chemical data showed only some slight changes in biodegradation indices [Σ(C12–C20)/Σ(C21–C32) and C17/pristane]. The complete data set provided strong evidence of the presence of indigenous hydrocarbon-degrading bacteria in Antarctic seawater and their high potential for hydrocarbon bioremediation. The rate of oil degradation could be increased by the addition of a commercial fertilizer, but water temperature had little effects on biodegradation efficiency which is in conflict with the typical temperature-related assumption predicting 50% rate reduction when temperature is reduced by 10°C. Global warming of Antarctic seawater should not increase significantly the rate of oil biodegradation in these remote regions.     

Symbiotic performance of grain and wild herbaceous legumes in the Okavango Delta and Tswapong region of Botswana

The low inherent soil fertility, especially nitrogen (N) constrains arable agriculture in Botswana. Nitrogen is usually added to soil through inorganic fertilizer application. In this study, biological nitrogen fixation by legumes is explored as an alternative source of N. The objectives of this study were to measure levels of N₂ fixation by grain legumes such as cowpea, Bambara groundnut and groundnut in farmers’ fields as well as to estimated N₂ fixation by indigenous herbaceous legumes growing in the Okavango Delta. Four flowering plants per species were sampled from the panhandle part of the Okavango Delta and Tswapong area. Nitrogen fixation was measured using the ¹⁵N stable isotope natural abundance technique. The δ¹⁵N values of indigenous herbaceous legumes indicated that they fixed N₂ (?1.88 to +1.35 ‰) with the lowest value measured in Chamaecrista absus growing in Ngarange (Okavango Delta). The δ¹⁵N values of grain legumes growing on farmers’ fields ranging from ?1.2 ‰ to +3.3 ‰ indicated that they were fixing N₂. For grain legumes growing at most farms, %Ndfa were above 50% indicating that they largely depended on symbiotic fixation for their N nutrition. With optimal planting density, Bambara groundnuts on farmers’ fields could potentially fix over 90 kg N/ha in some parts of Tswapong area and about 60 kg N/ha in areas around the Okavango Delta. Results from this study have shown that herbaceous indigenous legumes and cultivated legumes play an important role in the cycling of N in the soil. It has also been shown that biological N₂ on farmer’s field could potentially supply the much needed N for the legumes and the subsequent cereal crops if plant densities are optimized with the potential to increase food security and mitigate climate change.     

Influence of soil pollution on the composition of a microbial community

The abundance dynamics and composition of indigenous soil microbial communities were studied in soils polluted with naphthalene, dioctyl phthalate, diesel fuel, and crude oil. DGGE analysis of the 16S rRNA genes amplified from the total soil DNA revealed that the bacterial community of uncontaminated soil was more diverse and included no dominant species. In the soil samples polluted with the crude oil, diesel fuel, or dioctyl phthalate, Pseudomonas became the dominant bacteria since the third day of the experiment. In the soil polluted with naphthalene, two genera of bacteria (Pseudomonas and Paenibacillus) were dominant in population on the third day of the experiment, while on the 21th day of the experiment Arthrobacter became dominant. During the experiment, the average number of indigenous bacterial degraders increased approximately by two orders of magnitude. While the key genes of naphthalene catabolism, nahAc and nahH, were not detected in the pristine soil, they were found in a significant amount on the third day after naphthalene addition. Three degrader strains harboring the plasmids of naphthalene biodegradation (IncP-9 group) were isolated on the third day from the soil polluted with naphthalene. Two of these plasmids, although isolated from various degraders, were shown to be identical.