Incomplete Hydrocarbon Biodegradation in Contaminated Soils: Limitations in Bioavailability or Inherent Recalcitrance?

Bioremediation has been used to treat soils contaminated with complex mixtures of organic compounds such as total petroleum hydrocarbons (TPH), oil and grease (O&G), or polycyclic aromatic hydrocarbons (PAHs). Despite the common use and cost-effectiveness of bioremediation for treating hydrocarbon-contaminated soils, it has been observed that a residual fraction remains undegraded in the soil even when optimal biodegradation conditions have been provided. This paper provides a brief review of the two major conceptual models that have been used to explain why a residual hydrocarbon fraction remains in the soil after bioremediation treatment. The contaminant sequestration model is based on the assumption that a certain fraction of hydrocarbons is “locked up” in small soil pores within soil particles or aggregates. These sorbed hydrocarbons are believed to be inaccessible to soil microorganisms. Consequently, limitations in bioavailability are thought to be the major reason for incomplete hydrocarbon biodegradation, particularly in aged or weathered soils. Alternatively, according to the inherent recalcitrance model, incomplete TPH biodegradation may be caused by the presence of certain hydrocarbons that are inherently recalcitrant to biodegradation or are only extremely slowly degradable even under optimal conditions. Each conceptual model provides different explanations regarding the potential risks of the residual hydrocarbon fraction. If the residual TPH is truly sequestered within the soil pore space, it is unlikely that these compounds will pose any significant risk to human or environmental receptors. By contrast, these risks may be considerably greater if the residual TPH fraction consists of inherently recalcitrant compounds that reside mostly on the surface of soil particles and therefore are much more available to potential receptors. Both conceptual models and their implications for the potential risk of the residual TPH fraction are discussed.     

生物降解对黑碳及土壤上苯酚脱附行为的影响

农业土壤和黑碳(BC)两种不同的吸附剂吸附苯酚平衡后分离,每组一部分不做处理,另一部分通过加入无酚灭菌溶液脱附平衡后分离,制备得到在不同吸附位点上吸附有苯酚的两类不同类型的4种吸附苯酚的吸附剂,研究了在不同Pseudomonasputida ATCC 11172菌密度条件下吸附在这4种吸附剂上的苯酚的脱附行为.结果表明,土壤及BC对苯酚的吸附均呈现明显的非线性,可用Freundlich模型描述.吸附态的苯酚能否被微生物利用取决于微生物及吸附剂的性质,BC具有发达的微孔结构,微孔小于假单胞菌细胞尺寸,导致假单胞菌无法直接利用吸附在BC上的苯酚;土壤基本无微孔结构,微生物较易与吸附的苯酚发生表面接触,直接利用吸附态苯酚.BC和土壤上的吸附态苯酚的脱附行为能用三元位点模型很好地描述,模型计算结果表明BC上的苯酚脱附主要受慢速脱附和极慢速脱附控制,微生物降解速率受脱附控制,降解可加速BC上的慢速脱附和极慢速脱附;土壤上的苯酚脱附主要受快速脱附控制,微生物降解不受脱附速率限制,对土壤上的脱附行为基本无影响.     

Degradation of dissolved and sorbed 2,4-dichlorophenol in soil columns by suspended and sorbed bacteria

The influence of sorption of bacteria, as well as 2,4-dichlorophenol (2,4-DCP), on themineralization of 100 g l^-1 of the organic compound was examined in an aquifer material under advective flow conditions (column displacement technique). The study was designed to distinguish the rates and extent of biodegradation of the sorbed and the dissolved trace organic and the contribution of sorbed and suspended bacteria to the degradation. The degradation of dissolved 2,4-DCP was significantly faster thanthe degradation of the same compound sorbed to the solids, and suspended bacteriadegraded the dissolved compound at a higher rate than sorbed bacteria, also on a percell basis. The suspended bacteria degraded 8–12% of the added dissolved 2,4-DCP, while sorbed bacteria made a smaller contribution by degrading about 5% of sorbed 2,4-DCP. No degradation was seen with sorbed 2,4-DCP and suspended bacteria, and a marginal contribution was made by sorbed bacteria on the degradation of dissolved 2,4-DCP (<0.4%).     

Assessing mineralization rates of petroleum hydrocarbons in soils in relation to environmental factors and experimental scale

Mineralization rates of non-volatile petroleum hydrocarbons (HCs) in five different oil-contaminated soils with initial HC contents ranging from 0.1 to 13 g kg^-1 are estimated as a function of environmental factors. The aim of the study is threefold, (i) to study the relevance of environmental factors that may influence the mineralization rate, (ii) to compare mineralization rates estimated in two experiments at different scales, after standardizing them to environmental reference conditions, (iii) to evaluate the CO₂ production rate as a measure for the mineralization rate of HCs. Experiments were performed at laboratory scale (30–50 cm³ soil volume) in closed-jars under constant environmental conditions and in lysimeters (0.81 m³ soil volume) under dynamic climatic and hydrological conditions. A biodegradation model, coupled to transport models for soil heat, water, and gas dynamics is employed for data interpretation. The transport models are used to simulate the environmental conditions that influence the mineralization rate in the non-steady lysimeter experiments. The results show that temperature, O₂ concentration and HC content have an effect on the mineralization rates. Water content could not be identified as a direct governing environmental factor. However, an indirect effect of water content is that it influences the effective gas diffusion coefficient in soils. The CO₂ production rate seems to be a good quantity to express the mineralization rate of HCs for HC contents>1 g kg^-1. Measured CO₂ production rates standardized to reference conditions are similar for the two different experimental scales. This demonstrates that the usage of biodegradation rates obtained in the laboratory to predict the biodegradation rates under field conditions is sound, as long as the differences in environmental conditions have been taken into account.     

Microbial biodegradation of aromatic alkanoic naphthenic acids is affected by the degree of alkyl side chain branching

Naphthenic acids (NAs) occur naturally in oil sands and enter the environment through natural and anthropogenic processes. NAs comprise toxic carboxylic acids that are difficult to degrade. Information on NA biodegradation mechanisms is limited, and there are no studies on alkyl branched aromatic alkanoic acid biodegradation, despite their contribution to NA toxicity and recalcitrance. Increased alkyl side chain branching has been proposed to explain NA recalcitrance. Using soil enrichments, we examined the biodegradation of four aromatic alkanoic acid isomers that differed in alkyl side chain branching: (4′-n-butylphenyl)-4-butanoic acid (n-BPBA, least branched); (4′-iso-butylphenyl)-4-butanoic acid (iso-BPBA); (4′-sec-butylphenyl)-4-butanoic acid (sec-BPBA) and (4′-tert-butylphenyl)-4-butanoic acid (tert-BPBA, most branched). n-BPBA was completely metabolized within 49 days. Mass spectral analysis confirmed that the more branched isomers iso-, sec- and tert-BPBA were transformed to their butylphenylethanoic acid (BPEA) counterparts at 14 days. The BPEA metabolites were generally less toxic than BPBAs as determined by Microtox assay. n-BPEA was further transformed to a diacid, showing that carboxylation of the alkyl side chain occurred. In each case, biodegradation of the carboxyl side chain proceeded through beta-oxidation, which depended on the degree of alkyl side chain branching, and a BPBA degradation pathway is proposed. Comparison of 16S rRNA gene sequences at days 0 and 49 showed an increase and high abundance at day 49 of Pseudomonas (sec-BPBA), Burkholderia (n-, iso-, tert-BPBA) and Sphingomonas (n-, sec-BPBA).     

Accelerated biodegradation of atrazine by a microbial consortium is possible in culture and soil

A mixed enrichment culture of microorganisms capable of accelerated mineralization of atrazine was isolated from soil treated with successive applications of the herbicide. Liquid cultures of this consortium, in the presence of simple carbon sources, mineralized 96% of the applied atrazine (0.56 mM) within 7 days. Atrazine mineralization in culture is initiated with the formation of the metabolite hydroxyatrazine. In soil treated with atrazine at a concentration of 0.14 mM (concentration is based on total soil mass), and then inoculated with the microbial consortium, the parent compound was completely transformed in 25 days. After 30 days of incubation, 60% of the applied atrazine was accounted for as¹⁴CO₂. As was found with the liquid cultures, hydroxyatrazine was the major metabolite. After 145 days, soil extractable hydroxyatrazine declined to zero and 86% of the applied atrazine was accounted for as¹⁴CO₂. No metabolites, other than hydroxyatrazine, were recovered from either the liquid culture or soil inoculated with the consortium. The use of the mixed microbial culture enhanced mineralization more than 20 fold as compared to uninoculated soil.     

Effects of humic substances on the bioavailability and aerobic biodegradation of polychlorinated biphenyls in a model soil.

The very high hydrophobicity of polychlorinated biphenyls (PCBs) strongly reduces their bioavailability in aged contaminated soils, thus limiting their bioremediation. The biodegradability of PCBs in heavily contaminated soils can be significantly enhanced by soil treatment with surface-active agents. In this work, the effects of naturally occurring surfactants such as humic substances (HS) on the aerobic biodegradation of PCBs in a model soil were studied. The soil was amended with biphenyl (4 g/kg), Fenclor 42 (1,000 mg/kg), the aerobic PCB-biodegrading bacterial co-culture ECO3 (inoculum: 10(8)CFU/mL), and treated in aerobic batch slurry-phase conditions (17.5% w/v) with and without the addition of HS at the rates of 1.5 and 3.0% (w/w). Low PCBs biodegradation and dechlorination yields were observed in the HS-free microcosms, probably as a result of the rapid disappearance of inoculated bacteria. The presence of HS influenced significantly the activity of the specialized biomass and the biodegradation of PCBs in the microcosms. The microcosms that received HS at the 1.5% rate showed a higher persistence of the specialized bacteria and yields of PCB biodegradation and dechlorination about 150 and 100%, respectively, larger than those found for the HS-free microcosms. Lower stimulating effects were observed in the microcosms added with the HS at 3.0% rate. These effects were attributed to an increased solubilization of PCBs in the hydrophobic domains of the humic supramolecular associations and to a different accessibility of PCBs by the specialized bacteria at the different rates of HS addition. Although the slurry-phase treatment generally showed a decrease of the original soil ecotoxicity, the addition of the originally non-toxic HS decreased soil ecotoxicity for the Collembola animal biomarker and increased that towards the Lepidium sativum vegetal biomarker.     

Isolation and characterization of butachlor‐catabolizing bacterial strain Stenotrophomonas acidaminiphila JS‐1 from soil and assessment of its biodegradation potential

Aims: Isolation, characterization and assessment of butachlor‐degrading potential of bacterial strain JS‐1 in soil. Methods and Results: Butachlor‐degrading bacteria were isolated using enrichment culture technique. The morphological, biochemical and genetic characteristics based on 16S rDNA sequence homology and phylogenetic analysis confirmed the isolate as Stenotrophomonas acidaminiphila strain JS‐1. The strain JS‐1 exhibited substantial growth in M9 mineral salt medium supplemented with 3·2 mmol l^?1 butachlor, as a sole source of carbon and energy. The HPLC analysis revealed almost complete disappearance of butachlor within 20 days in soil at a rate constant of 0·17 day^?1 and half‐life (t_½) of 4·0 days, following the first‐order rate kinetics. The strain JS‐1 in stationary phase of culture also produced 21·0 μg ml^?1 of growth hormone indole acetic acid (IAA) in the presence of 500 μg ml^?1 of tryptophan. The IAA production was stimulated at lower concentrations of butachlor, whereas higher concentrations above 0·8 mmol l^?1 were found inhibitory. Conclusions: The isolate JS‐1 characterized as Stenotrophomonas acidaminiphila was capable of utilizing butachlor as sole source of carbon and energy. Besides being an efficient butachlor degrader, it substantially produces IAA. Significance and Impact of the Study: The bacterial strain JS‐1 has a potential for butachlor remediation with a distinctive auxiliary attribute of plant growth stimulation.     

Characterization of cellulosic fibers and fabrics by sorption/desorption

Three cellulosic substrates: lyocell (CLY), viscose (CV), and modal (CMD) in the form of fibers and fabrics were subjected to wet/dry or wash/dry treatments. The accessibility of untreated and treated substrates to water and iodine was investigated using dynamic water-vapor sorption, moisture retention, and iodine sorption methods, to study the influence of treatments on sorption-desorption hysteresis, fraction of moisture sorbed as a monomolecular layer, water retention, and iodine sorption. It was found that the sorption properties of untreated and treated substrates differed with sorbate type as well as substrate type and form.     

Uptake of soil cadmium by three field crops and its prediction by a pH-dependent Freundlich sorption model

Crop contamination with cadmium is a function of soil contamination. Here we study the applicability of the soil solution bioavailability hypothesis to Cd: that is, whether uptake of Cd was more directly related to its concentration or activity in the soil solution than in the soil solid phase. Experimental data from past soil-crop surveys for Cd were used to test this hypothesis. It was also investigated whether pH-dependent desorption of cadmium would be an important mechanisms in affecting cadmium activity and thus uptake. To do so we calculated the correlation between the Cd transfer factor (ratio between Cd level in plant dry material and Cd level in the topsoil) and either the soil pH, or the calculated soil solution Cd concentrations. There was no correlation between the Cd contents of the soil and of the edible parts of leafy plants (endive, spinach and lettuce). There was a strong negative correlation between soil pH and the log transfer factor for Cd at pH 4.5–7.2 and thus plant content. There also was a negative correlation between soil pH and calculated cadmium concentrations in the soil solution. For spinach grown on soils with pH > 7.2 the transfer factor increased, which is tentatively ascribed to cadmium mobilization by dissolved organic matter. The soil solution hypothesis should be further tested by pot and field trials. Special attention should be paid to the role of pH and dissolved organic matter. A C Borstlap Section editor     

Comparison of Pesticide Sorption by Physicochemically Modified Soils with Natural Soils as a Function of Soil Properties and Pesticide Hydrophobicity

The objectives of this paper were to determine the efficiency of physicochemically modified soils with a surfactant in the sorption of pesticides, the stability against washing of the pesticides sorbed, and the effective sorption capacity of surfactant adsorbed by soils as a function of pesticide hydrophobicity and soil characteristics. Five soils of different characteristics and five pesticides (penconazole, linuron, alachlor, atrazine and metalaxyl) with different Kow values were selected and octadecyltrimethylammonium bromide (ODTMA) was chosen as model of cationic surfactants. Sorption-desorption isotherms were obtained and constants Kf and Kfd for natural soils (from Freundlich equation) and K and Kd for ODTMA-soils (from linear equation) were determined. Sorption on ODTMA-soils was higher than on natural soils. K increased 27–165 times for penconazole, 22–77 times for linuron, 7–14 times for alachlor, 9–23 times for atrazine, and 21–333 times for metalaxyl in relation to Kf. Sorption coefficients normalized to 100% of total organic matter (TOM) from organo soils K_OM (K 100/%TOM), were always higher than those from natural soils Kf_OM (Kf 100/%OM), indicating that the organic matter (OM) derived from the ODTMA (OM_ODTMA) had a greater sorption capacity than the OM of the natural soil. K_OM values were also higher than the Kow (octanol/water distribution coefficient) value for each pesticide. The similarity of the high K_OM values for the sorption of each pesticide by the five soils and the linearity of isotherms point to a partitioning of the pesticides between surfactant and water. The use in this work of different soils and various pesticides, unusual in this type of investigation, allowed us to obtain equations to know the sorbed amount of a given pesticide by the surfactant-modified soils as a function of the OM content derived from the cation and the Kow of the pesticide. The results obtained are of interest when it becomes necessary to increase the sorption capacity of soils with low OM contents with a view to delaying pesticide mobility in soils from pollution point sources (high concentration in small area), and preventing the pollution of waters.     

Remediation of PCBs in soil by surfactant washing and biodegradation in the wash by Pseudomonas sp. LB400

Solutions from the washing of polychlorinated biphenyl (PCB)-contaminated soil with a variety of commercial nonionic or anionic surfactants were incubated with Pseudomonas sp. LB400 in an attempt to remediate the soil and destroy the PCBs. Nonionic surfactants washed more PCBs from the soil (up to 89%) but inhibited their biodegradation. Anionic surfactants washed less PCBs from the soil but were more effective in biodegradation tests, removing up to 67% of total PCBs.     

Mechanisms for release of sediment-bound phosphate to water and the effects of agricultural land management on fluvial transport of particulate and dissolved phosphate

Sediment-bound phosphate includes organic and inorganic forms, but the inorganic fraction contains most of the P that can be released into water. The non-apatite inorganic P (NAIP) fraction of sediment-bound phosphate varies considerably with geochemistry and anthropogenic additions (fertilizer, livestock wastes, P adsorbed from municipal wastewater discharges). A small fraction of the NAIP is sufficiently labile to desorb into water, and this release can be described by dissolution or adsorption/ desorption mechanisms. Agricultural practices, such as phosphate fertility management and conservation tillage, which affect the levels of phosphate and sediment leaving the land, will determine the partition of sediment-bound P and dissolved P in water draining into lakes, with implications for the availability of that phosphate to phytoplankton.     

Depolymerisation and biodegradation of a synthetic tanning agent by activated sludges, the bacteria Arthrobacter globiformis and Comamonas testosteroni, and the fungus Cunninghamella polymorpha

Degradation of a synthetic tanning agent CNSF (a condensation product of 2-naphthalenesulfonic acid (2-NSA) and formaldehyde) by four activated sludges, two previously characterised bacterial strains, Arthrobacter sp. 2AC and Comamonas sp. 4BC, and the fungus Cunninghamella polymorpha, was studied in batch culture at 25°C by determining the changes in the concentrations of CNSF and its component monomers and oligomers (n2–n11). The loss of individual oligomers was correlated with the length of the NSA-CH₂ chain. Approximately 25% of the total CNSF was degraded (i.e. mineralised) by the microbes contained in the four activated sludges and by the two bacterial isolates but with different lag phases and at different overall rates. The decline in CNSF concentration was due almost entirely to the biodegradation of the monomers (34.3% of CNSF) and, in particular, 2-NSA (27% of CNSF). There was no change in the n2–n11 components. The growth of C. polymorpha, on the other hand, arose from extracellular depolymerisation of CNSF oligomers and the biodegradation of the lower molecular mass products. Between 38% and 42% of total CNSF was degraded by C. polymorpha at 25°C. The order of oligomer degradation was inversely related to degree of polymerisation. Eighty percent and 90% of the n4 and n5 and 100% oligomers n6–n11 were degraded after 120h. At a higher temperature (37°C) oligomers n4–n11 were degraded completely after 120h. A combination of biodegradation (75%) and sorption to fungal biomass (25%) accounted for the measured loss of all oligomers from the solution phase. The CNSF degradation rates and the volume of fungal biomass produced (and therefore the extent of biosorption) were dependent on the presence of a second carbon source (both optimum at glucose 5g/l). This is the first report that identifies and distinguishes between depolymerisation, sorption and biodegradation processes in the removal of CNSF and its component oligomers. The use of combinations of the depolymerising fungus C.polymorpha, and the monomer-degrading bacteria, Arthrobacter sp. 2AC and Comamonas sp. 4BC, have potential for wastewater treatment.     

Methyl-beta-cyclodextrin-enhanced solubilization and aerobic biodegradation of polychlorinated biphenyls in two aged-contaminated soils

The bioremediation of aged polychlorinated biphenyl (PCB)-contaminated soils is adversely affected by the low bioavailability of the pollutants. Randomly methylated-beta-cyclodextrins (RAMEB) were tested as a potential PCB-bioavailability-enhancing agent in the aerobic treatment of two aged-contaminated soils. The soils, contaminated by about 890 and 8500 mg/kg of Aroclor 1260 PCBs, were amended with biphenyl (4 g/kg), inorganic nutrients (to adjust their C:N ratio to 20:1), and variable amounts of RAMEB (0%, 0.5%, or 1.0% [w/w]) and treated in both aerobic 3-L solid-phase reactors and 1.5-L packed-bed loop reactors for 6 months. Notably, significant enhancement of the PCB biodegradation and dechlorination, along with a detectable depletion of the initial soil ecotoxicity, were generally observed in the RAMEB-treated reactors of both soils. RAMEB effects were different in the two soils, depending upon the treatment conditions employed, and generally increased proportionally with the concentration at which RAMEB was applied. RAMEB, which was slowly metabolized by the soil's aerobic microorganisms, was found to markedly enhance the occurrence of the indigenous aerobic, cultivable biphenyl-growing bacteria harboring genes homologous to those of two highly specialized PCB degraders (i.e., bphABC genes of Pseudomonas pseudoalcaligenes KF707 and bphA1A2A3A4BC1 genes of Rhodococcus globerulus P6) and chlorobenzoic acid-degrading bacteria as well as the occurrence of PCBs in the water phase of the soil reactors. These findings indicate that RAMEB enhanced the aerobic bioremediation of the two soils by increasing the bioavailability of PCBs and the occurrence of specialized bacteria in the soil reactors.     

Protocol for determining bioavailability and biokinetics of organic pollutants in dispersed, compacted and intact soil systems to enhance in situ bioremediation

The development of effective in situ and on-site bioremediation technologies can facilitate the cleanup of chemically-contaminated soil sites. Knowledge of biodegradation kinetics and the bioavailability of organic pollutants can facilitate decisions on the efficacy of in situ and on-site bioremediation of contaminated soils and determine the attainable treatment end-points. Two kinds of compounds have been studied: (1) phenol and alkyl phenols, which represent hydrophilic compounds, exhibiting high water solubility and moderate to low soil partitioning; and (2) polycyclic aromatic hydrocarbons which are hydrophobic compounds with low water solubility and exhibit significant partitioning in soil organic carbon. Representative data are given for phenol and naphthalene. The results provide support for a systematic multi-level protocol using soil slurry, wafer and porous tube or column reactors to determine the biokinetic parameters for toxic organic pollutants. Insights into bioremediation rates of soil contaminants in compact soil systems can be attained using the protocol. Received 04 December 1995/ Accepted in revised form 31 January 1997     

Degradation of polycyclic aromatic hydrocarbons with three to seven aromatic rings by higher fungi in sterile and unsterile soils

Seven commercial 3- to 7-ring (R) polycyclic aromatic hydrocarbons (PAH) as well as PAH derived from lignite tar were spiked into 3 soils (0.8 to 9.7% of organic carbon). The disappearance of the original PAH was determined for the freshly spiked soils, for soils incubated for up to 287 d with their indigenous microflora, and for autoclaved, unsterile and pasteurized soils inoculated with basidiomycetous and ascomycetous fungi. Three to 12 d after spiking, 22 to 38% of the PAH could no longer be recovered from the soils. At 287 d, 88.5 to 92.7%, 83.4 to 87.4%, and 22.0 to 42.1% of the 3-, 4-, and 5- to 7-R PAH, respectively, had disappeared from the unsterile, uninoculated soils. In 2 organic-rich sterile soils, the groups of wood- and straw-degrading, terricolous, and ectomycorrhizal fungi reduced the concentration of 5 PAH by 12.6, 37.9, and 9.4% in 287 d. Five- to 7-R PAH were degraded as efficiently as most of the 3- to 4-R PAH. In organic-rich unsterile soils inoculated with wood- and straw-degrading fungi, the degradation of 3- to 4-R PAH was not accelerated by the presence of fungi.The 5- to 7-R PAH, which were not attacked by bacteria, were degraded by fungi to 29 to 42% in optimum combinations of fungal species and soil type. In organic-poor unsterile soil, these same fungi delayed the net degradation of PAH possibly for 2 reasons. Mycelia of Pleurotus killed most of the indigenous soil bacteria expected to take part in the degradation of PAH, whereas those of Hypholoma and Stropharia promoted the development of opportunistic bacteria in the soil, which must not necessarily be PAH degraders. Contemporarily, the contribution of the fungi themselves to PAH degradation may be negligible in the absence of soil organic matter due to the lower production of ligninolytic enzymes. It is concluded that fungi degrade PAH irrespective of their molecular size in organic-rich and wood chip-amended soils which promote fungal oxidative enzyme production.     

Identification of Putative Biomarkers for Toluene-Degrading Burkholderia and Pseudomonads by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Peptide Mass Fingerprinting

The use of peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to identify and phenotypically characterize toluene-degrading bacteria via biomarkers of degradation and taxonomical classification. Pseudomonas putida F1, P. mendocina KR1, and Burkholderia sp. JS150 were grown on toluene, extracted, electrophoretically separated, and analyzed by MALDI-TOF MS. Catabolic enzymes were identified and results substantiated using tandem MS.     

The protective effect against Leishmania infection conferred by sand fly bites is limited to short-term exposure

Under laboratory conditions, hosts exposed twice to sand fly saliva are protected against severe leishmaniasis. However, people in endemic areas are exposed to the vector over a long term and may experience sand fly-free periods. Therefore, we exposed mice long- or short-term to Phlebotomus duboscqi bites, followed by Leishmania major infection either immediately or after a sand fly-free period. We showed that protection against leishmaniasis is limited to short-term exposure to sand flies immediately before infection. Our results may explain the persistence of leishmaniasis in endemic areas and should be taken into account when designing anti-Leishmania vaccines based on sand fly saliva.