Bacterial polyextremotolerant bioemulsifiers from arid soils improve water retention capacity and humidity uptake in sandy soil

Bacterial polyextremotolerant bioemulsifiers from arid soils improve water retention capacity and... Background: Water stress is a critical issue for plant growth in arid sandy soils. Here, we aimed to select bacteria pro- ducing polyextremotolerant surface-active compounds capable of improving water retention and humidity uptake in sandy soils. Results: From Tunisian desert and saline systems, we selected eleven isolates able to highly emulsify different organic solvents. The bioemulsifying activities were stable with 30% NaCl, at 4 and 120 °C and in a pH range 4–12. Applications to a sandy soil of the partially purified surface-active compounds improved soil water retention up to 314.3% compared to untreated soil. Similarly, after 36 h of incubation, the humidity uptake rate of treated sandy soil was up to 607.7% higher than untreated controls. Conclusions: Overall, results revealed that polyextremotolerant bioemulsifiers of bacteria from arid and desert soils represent potential sources to develop new natural soil-wetting agents for improving water retention in arid soils. Keywords: Polyextremotolerant bioemulsifiers, Desert sandy soil, Water retention, Humidity uptake, Water stress Background BE and BS are used in agriculture for several applica- Biosurfactants (BS) are amphipathic compounds pro- tions ranging from the improvement of the quality of duced by a variety of microorganisms. They can be low- polluted soils, to the control of plant pathogens or for molecular weight, generally glycolipids or lipopeptides, favoring plant–microbe interactions [5]. The potential of or high-molecular weight compounds, which are mainly exopolysaccharides (EPS) with bioemulsifying properties lipopolysaccharides, lipoproteins, or a combination of have been considered for the promotion of plant growth both. The high-molecular weight BS, also called bioemul - by bacterial producers [6, 7] or for their effect on the sifiers (BE), are capable of producing stable emulsions, hydrological behavior of biological soil crusts [8]. Appli- but do not always determine decreases of surface or cation of surfactants to soil has been included among interfacial tensions [1]. There is a growing interest in twelve strategies for the remediation of soil water repel- microbial biosurfactants owing to several advantages lency [9], even though the effect of biosurfactants on soil over conventional surfactants, including biodegradability, water repellency are controversial. Some studies have low toxicity and production from renewable substrates reported that fungi may produce hydrophobins that favor [2–4]. the development of water repellency [10]. One aspect that, to our knowledge, has not been con- sidered in the literature on BS/BE is their potential for *Correspondence: noura.raddadi@unibo.it improving soil water retention [8], especially in arid and Department of Civil, Chemical, Environmental and Materials Engineering desert sandy soils that have limited content of organic (DICAM), Alma Mater Studiorum University of Bologna, via Terracini 28, 40131 Bologna, Italy matter and experience extreme environmental conditions Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Raddadi et al. Microb Cell Fact (2018) 17:83 Page 2 of 12 of limited water supply and nutrient content, high tem- index and the drop collapse activity as well as by measur- peratures, irradiation and evaporation rates and high ing the interfacial surface tension (IFT). salinity [11, 12]. The emulsification activity (EA) of the supernatant was We hypothesize that, by living under extreme con- determined as follows. Two milliliters of culture super- ditions, bacteria in arid and desert soils should have natant and an equal volume of the tested organic solvent evolved capacities and strategies that allow an efficient (n-hexane, n-hexadecane or toluene) were placed in a use of water, including production of BS/BE highly stable test tube that was vortexed at high speed for 2  min and under extreme conditions of temperature and irradiation then allowed to settle for 24  h. The emulsification index and that can facilitate access/retention of low-availa- (EI %) was estimated as the height of the emulsion layer, ble water and nutrients. On the plant leaf surface that divided by the total height, multiplied by 100. experiences analogous extremes such as those in desert The surface qualitative drop collapse activity test was sandy soils [13], epiphytic bacteria have been reported to carried out as follows: 40 µL of the cell free supernatant exploit biosurfactants to increase wettability of the leaf was aliquoted as a droplet onto Parafilm (Parafilm M, and to enhance nutrients diffusion through the wax cuti - Germany); the flattening and the spreading of the droplet cle [14]. Similarly, bacteria inhabiting arid environments on the Parafilm surface was followed over 10  min and and especially sandy soils could take advantage from recorded by visual inspection. The assay was scored nega - BS/BE to access limited nutrient supplies and withstand tive or positive if the drop remained beaded or collapsed, fluctuations in moisture availability in a water-deficient respectively. environment. The IFT of the cell-free culture supernatant was meas - In this study, we have screened a collection of bacte- ured with a Drop Shape Analyzer—DSA30 (KRUSS ria from arid Tunisian soils including (i) an inland saline GmbH, Germany) working in the pendant drop mode. system and sand from the Sahara desert and (ii) an arid The drops were produced by a syringe equipped with a vineyard in the North of the country, for BS/BE activities 2.098  mm needle diameter, were left to equilibrate close under different conditions. We have also tested a sub - to the rupture point and IFT values (mN/m) were cal- group of selected BS/BE bacterial isolates for their poten- culated by the instrument software using the Young– tial to improve water retention and humidity uptake in Laplace equation: sandy soils. �p = σ ∗ 1 r1 + 1 r2 ; Methods where ∆p is the differential pressure between the inside Bacterial strains and outside of the drop, σ is the IFT value and r1 and The 24 bacterial isolates used in this study include 23 r2 are the main radii of the drop curvature. Measure- strains obtained from sediment samples recovered from ments were performed at room temperature, at least in the inland saline system Chott El Fejej and desert sand triplicates. A Tween 80 solution at a final concentration from Douz in the South of Tunisia (Additional file  1: of 0.5% (in water), was used as positive control while w/v Table S1); and one (V3E1) isolated from the root system deionized water and non-inoculated growth medium of grapevine plants growing in an arid soil of Northern with 2% glucose were used as negative controls. All w/v Tunisia (Mornag area). The strains were identified based screening assays were performed in duplicate as inde- on partial 16S rRNA gene sequences (database accession pendent experiments. numbers MG594617-MG594638 and MG637030). Stability of bioemulsifiers Screening for biosurfactant/bioemulsifier production Emulsion stability studies were performed using cell-free Cells were inoculated into glucose mineral salts medium culture supernatants. The stability of the bioemulsifiers (GMSM) (g/L: 20.0 glucose; 0.7 K H PO ; 0.9 Na HPO ; activity was determined by investigating the effect of var - 2 4 2 4 2.0 NaNO ; 0.4 MgSO ·7H O; 0.1 C aCl ·2H O; 2  mL ying NaCl concentration, temperature or pH on the EA. 3 4 2 2 2 of trace elements [per liter, 2.0  g F eSO ·7H O, 1.5  g In order to assess the effect of salinity on the bioemul - 4 2 MnSO ·H O, 0.6  g (NH ) Mo O ·4H O]; pH = 6.72). sifier activity, culture supernatants were supplemented 4 2 4 6 7 24 2 The flasks were incubated at 30  °C on a rotary shaker with different NaCl concentrations (8–30% ) and the w/v (150  rpm). The surface activity of cell-free supernatants emulsifying activity was measured as described above. To was tested on culture samples every 24 h for a period of evaluate the stability of the bioemulsifier at different tem - up to 4 days. The cell-free culture supernatants recovered peratures, culture supernatants were subjected to cooling by centrifugation (10,000 rpm, 10 min, 4 °C) followed by (4 °C, 2 h), heating (55 °C, 2 h) or autoclaving (121 °C for filter-sterilization (0.22  µm) were used for evaluation of 20 min) followed by cooling to room temperature before the surface activity by determining the emulsification performing the EI assay. The measured emulsification Raddadi et al. Microb Cell Fact (2018) 17:83 Page 3 of 12 indexes were compared to the corresponding values samples in a sealed desiccator, in which the dishes were obtained using culture supernatants not subjected to the placed on the platform and the space under the platform different treatments. The EA of hexadecane was not eval - was filled with tap water instead of desiccant in order to uated at 4 °C since the melting point of the solvent was of create a high (100%) relative humidity in the environ- 18  °C and hence at lower temperature the emulsion was ment. After 36  h of incubation at constant temperature subjected to solidification. The pH stability was studied (30  °C), the samples were weighed in order to evaluate by adjusting the cell-free culture supernatants to different their weight increase due to humidity uptake. pH values (4–12) using HCl or NaOH solutions, and then the EA was measured as previously described. Further- Statistical analyses more, the stability of the emulsions produced under the Data related to the evaluation of bioemulsifiers stabil - different conditions was monitored for up to 30 months. ity under stressful conditions and to water retention and All the experiments were carried out in duplicate. The moisture uptake assays were statistically assessed using results were reported as residual emulsification activity the analysis of variance (ANOVA) via MATLAB software (REA) percentage (%) expressed as follows: (Version R2017a, The MathWorks Inc, Natick, USA). The statistical analyses aimed to highlight the statisti - REA (%) = EI EI ∗ 100; t 24 cally significant differences among EI % values obtained where EI and EI are the EI (%) values at incubation under standard and stressful conditions, and among the t 24 time t and after 24  h, respectively; and compared with amounts of retained water and uptaken humidity by those obtained with the positive control. sandy soil samples treated with the bioemulsifiers solu - tions, Triton X-100 or tap water. The significance of the Water retention and moisture uptake assays by a sandy soil data was determined by Tukey honestly significant differ - For BS/BE production, selected bacterial strains were ent test. Statistically significant results were depicted by p grown in GMSM and incubated at 30  °C. Cells were values < 0.05. removed by centrifugation (8000 rpm, 10 min, 4 °C) and the collected supernatant was acidified with 6  N hydro - Results and discussion chloric acid solution to pH 2.0 ± 0.3. The precipitate Screening of surface‑active strains and their identification which contained BS/BE was allowed to settle at 4  °C Among the 23 isolates screened (Additional file1: overnight. The precipitated BS/BE were collected by cen - Table  S1, Additional file2: Figure S1), 9 were capable to trifugation (12,000  rpm, 20  min, 4  °C) and resuspended significantly emulsify (EI % > 45%) at least one of the in sterile tap water at a final concentration of 25 g/L. Tri - solvents tested (Table  1). On the basis of the partial 16S ton X-100 applied at a final concentration corresponding rRNA gene sequences, all isolates had between 99 and to its CMC (0.14 g/L) and sterilized tap water were used 100% sequence similarities with their closest relative as controls. Before using in the experiments, the sandy type strain in the databases (Table 1). Bacillus mojavensis soil sample was oven-dried (105 °C, 4 h) and subjected to IFO15718 isolated from Mojave desert [15, 16] was the sterilization by tyndallization followed by drying (55  °C, closest relative species of the R4p, R43 and R39 strains, overnight) and acclimated at 30 °C for 24 h. while Bacillus endophyticus strain 2DT isolated from the The water retention assay was performed as follows: inner tissues of cotton plants [17] was the closest relative 10  g of sandy soil were placed in a glass Petri dish in of L45, L97b and L37 isolates. The partial 16S rRNA gene duplicate and subjected to wetting with 10  ml of a solu- sequence from strain N3 showed 99% homology with tion of BS/BE to be tested or tap water, followed by dry- different B. subtilis including the subsp. spizizenii TU-B- ing at 30  °C until a constant weight was achieved. The 10 isolated from soil collected near Nefta, Tunisia [18]. samples have been thoroughly mixed manually and then Bacillus licheniformis strain DSM 13 was the closest rela- subjected to two cycles of irrigation with 2.5  mL steri- tive of L98 strain, and Bacillus frigoritolerans DSM 8801 lized tap water and drying until constant weight was isolated from arid soil in Morocco was that of R55 and achieved. During the first drying cycle, each sample was R40 strains. The last isolate among those obtained from weighed immediately after irrigation with tap water and chott and that is positive for BS/BE production was pre- every 60  min up to 12.5  h of incubation and then each viously classified as Paenibacillus tarimensis (strain L88) 12  h until constant weight was achieved. Monitoring of [19], while the grapevine rhizosphere isolate V3E1was the samples weight after the second irrigation was per- assigned to Rhizobium sp.. formed at the time of irrigation, after 12.5 h of incubation The eleven desert/chott isolates capable of produc - and then every 60 min until complete drying. ing BS/BE were spore-formers, a group of bacteria well The moisture uptake assay was performed by incubat - adapted to the arid conditions of the desert primarily ing the Petri dishes containing 10  g of dried sandy soil for their capacity to produce spores that are resistant Raddadi et al. Microb Cell Fact (2018) 17:83 Page 4 of 12 Table 1 List of  BS/BE producers obtained from  the  screening of  23 desert bacterial isolates grown on  MSM with  2% w/v glucose as  carbon source. Emulsification index (EI %) and  interfacial surface tension (IFT) values are expressed and mean value ± SD of two and three replicates, respectively, while drop collapse results as positive (+) or negative (−) Isolate ID 16SrDNA Closest type strains 16S rDNA Drop collapse IFT (mN/m) EI24 (%) Accession (GenBank Accession identity Toluene Hexane Hexadecane No No) (%) Bacillus sp. L37 MG594627 Bacillus endophyticus 99 + 69.28 ± 0.31 57.63 ± 4.24 56.07 ± 2.18 61.11 ± 1.92 (NR_025122) Bacillus sp. L45 MG594628 Bacillus endophyticus 99 − 70.00 ± 0.47 52.53 ± 1.14 59.67 ± 1.48 53.33 ± 9.43 (NR_025122) Paenibacillus tarimensis KF111690 Paenibacillus tarimensis 99 − 74.45 ± 0.16 44.64 ± 2.06 50.93 ± 1.07 48.32 ± 6.27 L88 (NR_044102) Bacillus sp. L97b MG594634 Bacillus endophyticus 99 − 72.48 ± 0.36 57.64 ± 3.67 53.11 ± 5.13 47.36 ± 8.45 (NR_025122) Bacillus sp. L98 MG594631 Bacillus licheniformis 99 + 55.07 ± 0.26 48.82 ± 6.06 50.83 ± 2.18 46.43 ± 5.05 (NR_118996) Bacillus sp. N3 MG594629 Bacillus subtilis 99 + 36.86 ± 1.11 50.81 ± 2.17 34.84 ± 3.21 51.67 ± 7.07 subsp. spizizenii (NR_112686) Bacillus sp. R4p MG594635 Bacillus mojavensis 99 + 28.99 ± 0.35 55.67 ± 6.95 44.91 ± 4.96 28.57 ± 0.00 (NR_112725) Bacillus sp. R39 MG594626 Bacillus mojavensis 99 + 28.36 ± 0.63 11.15 ± 0.00 5.30 ± 2.26 0.00 ± 0.00 (NR_112725) Bacillus sp. R40 MG594633 Bacillus frigoritolerans 99 − 70.25 ± 0.46 52.12 ± 7.54 44.90 ± 4.52 58.33 ± 2.36 (NR_115064) Bacillus sp. R43 MG594630 Bacillus mojavensis 99 + 28.57 ± 0.76 26.67 ± 1.33 32.69 ± 6.78 11.51 ± 2.59 (NR_112725) Bacillus sp. R55 MG594632 Bacillus frigoritolerans 99 − 71.10 ± 0.85 59.98 ± 2.54 49.20 ± 2.92 53.77 ± 8.08 (NR_115064) to heating, desiccation and irradiation [20]. Several of culture supernatants did not exhibit a remarkable sur- the strains were affiliated to species previously isolated face tension reduction but were able to highly emulsify from soil and sand from other desert ecosystems, such the tested organic solvents. Indeed, the formation of as Bacillus mojavensis [15] and Paenibacillus tarimensis emulsions is typical of BE while BS are the compounds [21]. that are able to significantly reduce the surface tension Even though it should be confirmed by a larger range of of aqueous media to around 40 mN/m [14, 22]. isolates and ecosystems, it can be noted that comparing Bacillus spp. are well known BS/BE producers and the bacterial BS/BE producers with the initial collection their applications were related to hydrocarbon (crude of isolates (Additional file1: Table S1), almost all of them oil) degradation and microbial enhanced oil recovery (10 out of 11) belong to the genus Bacillus. This obser - [23–25]. Among others, B. endophyticus isolate TSH42 vation suggests that that BS/BE activities are important was reported to produce surfactin, fengycin and iturin features in the Bacillus genus for the adaptation to arid lipopeptides that exhibit fungal biocontrol activity [26]; conditions. however their ability to reduce the growth medium The maximum emulsifying activity was recorded surface tension or to emulsify organic solvents has between 24 and 96  h of incubation. The significant not been evaluated previously. B. mojavensis has been highest emulsification index was observed using hexa - previously studied for lipopeptide biosurfactant pro- decane as solvent (61.11 ± 1.92% by Bacillus endophyti- duction [27] but not for bioemulsification activities. B. cus L37; Table 1). Cell-free culture supernatants of four licheniformis was the only one previously characterized isolates (Bacillus sp. isolate N3, Bacillus sp. isolates for its production of lipopeptide biosurfactants capa- R4p, R39 and R43) significantly decreased the medium ble to reduce the medium surface tension to 28 mN/m surface tension from 74.66 ± 0.21  mN/m, with a low- [28] and to determine bioemulsification activity [24, est value of 28.36 ± 0.63  mN/m (Table  1). These results 25]. Interestingly, while Paenibacillus sp. isolates have suggest that under these experimental conditions, most been previously studied for their BS/BE production and of the isolates (8 out of 11) produced BE since their activity [29–32], no data regarding B. frigoritolerans were available in literature. Raddadi et al. Microb Cell Fact (2018) 17:83 Page 5 of 12 Stability of bioemulsifiers the maximum salt stress tested (30%), a total absence of Based on BS/BE activities, ten bioemulsifying isolates activity was recorded for all the culture supernatants as were selected and the stability of their EA was evaluated well as for Tween 80 (Fig. 2a). In the presence of a lower under different conditions. The activity of the crude BEs NaCl concentration (8%) the hexane E A was main- was evaluated from cell-free culture supernatants after tained statistically unchanged compared to the standard exposure to low water activity conditions as well as to conditions for Tween 80 and strains L37, L45, L97b, R4p extremes of pH or temperature (Figs. 1, 2, 3). In presence and R55; while it was completely lost for R43 and L98 of toluene under standard conditions, six of the isolates and significantly reduced for the remaining isolates (L88, (L37, L45, L97b, R4p, R40 and R55) did not exhibit a sta- N3, R40). Considering pH as stressful condition, under tistically significant decrease of their BE activity com - acidic conditions EA was significantly reduced for iso - pared to the positive control (Tween 80, Fig.  1a), while lates R40 and R55 with a complete loss for strain L98 at EI (%) of the remaining isolates (L88, L98, N3 and pH 4 (Fig. 2b). In general, alkaline pH did not affect EA 24 24 R43) were significantly reduced. Different trends were with the exception of the strain R40 (Fig.  2b). Looking observed when salt stresses were applied to the solution. to the temperature, also in this case for some strains the With the increase of NaCl concentration (up to 30%), autoclaving procedure resulted in a reduction (N3, R4p) only five isolates (L37, L45, L88, N3 and R55) showed EI or complete loss (L88 and R40) of the EA . The incuba - 24 24 values statistically comparable to those obtained under tion at 55  °C resulted in a significant reduction of EA standard conditions, suggesting how stress can strongly for the isolates L37, L88, R40 and R55 and a complete affect the activity of bacterial BEs. Remarkably, while loss for N3 and R43, while the activity remained statisti- the EA was significantly reduced for the strains R40 cally stable for the other strains. The reduction of EA at 24 24 and L97b, it was completely lost for R4p, L98 and R43 55  °C (although it was stable at higher temperature, i.e., (Fig. 1a). to autoclaving), for example in the case of isolates L37, When the stress applied was determined by extreme R43 and R55, could be ascribable to partial degradation pH (4 and 12), a different scenario was observed (Fig.  1b). of the bioemulsifiers as a result of the activation of extra - Under these conditions, no significant changes in EA cellular hydrolytic enzymes (for example proteases and/ values were reported for L45, L97b, R55, N3, R4p and or lipases) present in the culture supernatant of these iso- L37, as well as for the positive control (Tween 80), com- lates. Indeed, the cell free culture supernatants were pre- pared to standard conditions. The toluene EA by culture heated and further incubated at 55  °C once the organic supernatant of isolate P. tarimensis L88 was significantly solvents to be tested were added, which could has led the enhanced at pH 4, while remained statistically equiva- bioemulsifier polymeric compounds degradation. The lent to standard conditions at pH 12. On the contrary, activity of extracellular hydrolytic enzymes at relatively a significant EA reduction/loss was recorded for iso- high temperature (55 °C) can be expected considering the lates R40 (pH 4 and pH 12), R43 (pH 12) and L98 (pH 4) origin of the isolates from a hot desert. On the contrary, a (Fig. 1b). With the exception of isolates N3, R43 and R40, low temperature (4 °C) reduced EA of R55, R40 and N3 all the remaining cell-free culture supernatants retained but did not affect that of the Tween 80 or the remaining EA at low temperature (4  °C), which were statistically strains (Fig. 2c). identical to that of Tween 80 (Fig.  1c). When the tem- The stability of the emulsification activity in hexade - perature was increased to 55 °C, isolates L97b, L88, L98, cane under stressful conditions was evaluated only for R4p, R55 and L45 showed E A comparable to the cor- isolates exhibiting a significant stability in hexane and responding standard conditions and to Tween 80, while toluene solvents (L37, L45, L88, L97b, R4p and R55) as a reduction/loss of activity was recorded for strains L37, reported in Fig.  3a–c. The presence of different salt con - R40, N3 and R43 (Fig. 1c). After autoclaving, the cell-free centrations affected significantly the EA of strains L97b culture supernatants of the tested isolates retained an and R55 (Fig.  3a). Extreme pHs and temperature treat- EA unchanged compared to their corresponding activi- ments significantly affected bacterial BEs performance ties under standard conditions; except for strains N3, in the case of L97b (pH 4), R55 (pH 4, 55  °C, autoclav- R40, L88, R4p and R43 (Fig. 1c). ing), R4p (55  °C, autoclaving), L37 and L88 (pH 12, Bacterial BEs acted in a completely different way when 55 °C, autoclaving), up to complete EA loss in the case hexane was used as solvent (Fig. 2a–c). In the presence of of strains L37, L88 and R4p at 55 °C and strains L88 and salt stress (15%), a significant reduction of bacterial BEs R4p after autoclaving (Fig. 3b, c). performances was observed, except for isolates L37 and Gutiérrez et  al. [33] reported that heat (100  °C) and L45 that showed E I values statistically comparable to acid treatment (0.1  N) increased the relative activity of the corresponding activities under standard conditions, the bioemulsifiers produced by Halomonas sp. Activ - as well as to that of Tween 80 with 15% NaCl. Reaching ity increment was explained by a heating activation of Raddadi et al. Microb Cell Fact (2018) 17:83 Page 6 of 12 Fig. 1 EI (%) of toluene recorded under standard and stressful conditions. EI of toluene under low water activity (ANOVA: p < 0.0001, F = 43.86, df = 43) (a), extremes of pH (ANOVA: p < 0.0001, F = 35.89, df = 32) (b) and different temperatures (ANOVA: p < 0.0001, F = 64.15, df = 43) (c). Results of post hoc analyses of each condition were represented as letters: different letters correspond to statistically different EI (%) results (α = 0.05) 24 Raddadi et al. Microb Cell Fact (2018) 17:83 Page 7 of 12 Fig. 2 EI (%) of hexane recorded under standard and stressful conditions. EI of hexane under low water activity (ANOVA: p < 0.0001, F = 95.13, df = 32) (a), extremes of pH (ANOVA: p < 0.0001, F = 38.18, df = 32) (b) and different temperatures (ANOVA: p < 0.0001, F = 86.43, df = 43) (c). Results of post hoc analyses of each condition were represented as letters: different letters correspond to statistically different EI (%) results (α = 0.05) 24 Raddadi et al. Microb Cell Fact (2018) 17:83 Page 8 of 12 polymeric emulsifiers releasing a higher number of emul - sifying moieties from the biopolymers, enhancing the emulsifying capacity. BEs not affected by salt stress (i.e. NaCl concentra - tions up to 300  g/L) and extremes of pH (2–13) have been previously described from Paenibacillus sp. #510 [30] as well as from marine Bacillus sp., Halomonas sp. and Marinobacter sp. [34]. Dubey et al. [35] reported that bioemulsifiers from Pseudomonas aeruginosa strain-PP2 and Kocuria turfanesis strain-J retained their activities at up to 20% NaCl and 121  °C. Marine Rhodococcus sp. has been reported to produce trehalolipid biosurfactant that formed emulsions stable to a wide range of tempera- tures (20–100  °C), pH (2–10) and NaCl concentrations (5–25% ) [36], and bioemulsifiers highly stable at low w/v water activity and temperature extremes were reported from Marinobacter sp. [34]. EIs relatively stable under extreme pH, temperature, and salinity conditions have been previously reported from Bacillus spp. [23–25, 28]. The long-term stability of emulsions (reported as resid - ual emulsification activity %) under standard screening conditions or after exposure to water, heat or extreme pH stresses was evaluated after a monitoring period of up to 30  months. The emulsions showed a high stabil - ity at room temperature, maintaining up to 100% of the initial EIs under standard conditions (Table 2), while dif- ferent responses have been observed under the differ - ent stresses applied (Tables  3, 4, 5). Interestingly, some strains were able to retain unchanged activity in the pres- ence of up to 15% NaCl (strain L45; Table 3), under acid/ alkaline pHs (strains L45, L97b, R40 and R55; Table  4) and at 4  °C or after autoclaving (strains L45 and R55, respectively; Table 5). Long-term EI stabilities have been previously observed for the bioemulsifiers produced by Pedobacter sp. strain MCC-Z where the emulsions remained stable for 4  months [37]. Extended stability for longer incubation times (18–30  months) under low water activity or after thermal treatments was also observed for bioemulsifi - ers produced by Marinobacter sp. isolates [34], while no reports on long period incubation under pH extremes are available to the best of our knowledge. Water retention and humidity uptake by a BS/BE‑treated sandy soil Based on the characterization of the surface activity of the produced bacterial compounds two isolates (L45 and Fig. 3 EI (%) of hexadecane recorded under standard and stressful R43) were selected for further tests. While L45 produced conditions. EI of hexadecane under different salt concentrations only BEs, R43 was able to produce BEs that significantly (ANOVA: p < 0.0001, F = 28.26, df = 27) (a), extremes of pH (ANOVA: reduced IFT but exhibited low emulsification activ - p < 0.0001, F = 34.44, df = 20) (b) and temperature (ANOVA: p < 0.0001, F = 53.73, df = 20) (c). Results of post hoc analyses of each ity. Their BS/BEs were used for testing their ability to condition were represented as letters: different letters correspond to improve water content and humidity uptake of sandy soil. statistically different EI (%) results (α = 0.05) An additional strain, V3E1 isolated from arid agricultural Raddadi et al. Microb Cell Fact (2018) 17:83 Page 9 of 12 Table 2 Residual emulsification activity (REA%) and 11.67 ± 2.36 for toluene, hexane and hexadecane, after  an  incubation period of  30  months under  standard respectively. conditions The water retention capacity of sandy soils treated with BS/BE solutions was monitored after two irrigation Isolate/surfactant Toluene Hexane Hexadecane events using sterilized tap water. No significant differ - Tween 80 94.74 ± 0.00 90.10 ± 9.58 16.67 ± 8.19 ences between the treatments were observed during the Bacillus sp. L37 95.24 ± 6.73 91.20 ± 4.19 0.00 ± 0.00 first ten hours of monitoring, after the start of the first Bacillus sp. L45 91.71 ± 8.48 94.12 ± 4.71 7.56 ± 0.18 irrigation (Fig.  4a). Based on this, a second irrigation Bacillus sp. L97b 82.40 ± 3.95 94.76 ± 2.69 0.00 ± 0.00 treatment was performed and new measurements were Bacillus sp. L98 26.67 ± 1.33 58.93 ± 7.58 20.00 ± 1.00 carried out (Fig.  4b). Statistical analyses showed that Bacillus sp. R40 97.06 ± 4.16 84.62 ± 4.23 0.00 ± 0.00 after 12.5  h, compared to control (not subjected to BS/ BE treatment) which retained only 9.1 ± 0.9% (Fig.  4b), Triton X-100, R43, L45 and V3E1-treated sandy soil samples significantly reduced water evaporation retain - soil, was also used. The isolate V3E1 was included in the ing 16.3 ± 0.8; 19.0 ± 0.1; 27.0 ± 3.5 and 28.6 ± 1.2% of study since it has been shown to produce extracellu- the initial water, respectively. Hence, comparing the data lar polymeric substances (EPS), and based on literature obtained we can show that after 12 h bacterial BS/BE sig- reports EPS have been found to play an important role nificantly increased water retention, with values up to in water retention in biological soil crusts [8]. The isolate 314.3% (V3E1) higher than the untreated control. This produced surface-active compounds that were able to capability to retain a significantly higher water amount, reduce the surface tension of the cultivation medium up compared to control, was statistically confirmed also dur - to 69.81 ± 0.88  mN/m and to emulsify the different sol - ing the two following hours. However, after 16 h, samples vents used exhibiting EI (%) of 55.93 ± 5.76; 5.00 ± 2.36 irrigated with Triton X-100, R43 and water were found Table 3 Residual emulsification activity (REA%) under  low water activity after  an  incubation period of  up  to 30  months under standard conditions Isolate/surfactant Time 8% NaCl 15% NaCl 30% NaCl (months) Toluene Hexane Hexadecane Toluene Hexane Hexadecane Toluene Tween 80 29 2.54 ± 3.60 59.74 ± 7.03 88.89 ± 4.44 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Bacillus sp. L45 30 96.15 ± 3.26 100.00 ± 0.00 0.00 ± 0.00 95.32 ± 4.26 93.75 ± 0.00 0.00 ± 0.00 81.03 ± 7.79 Paenibacillus tarimensis L88 30 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 11.93 ± 4.02 0.00 ± 0.00 0.00 ± 0.00 16.78 ± 1.98 Bacillus sp. L97b 29 66.52 ± 3.16 77.28 ± 6.88 0.00 ± 0.00 10.00 ± 0.50 12.92 ± 0.65 0.00 ± 0.00 0.00 ± 0.00 Bacillus sp. R40 29 64.91 ± 6.95 28.57 ± 1.43 nt 66.25 ± 8.84 na nt 63.65 ± 7.52 Bacillus sp. R55 29 85.74 ± 5.54 46.67 ± 2.33 0.00 ± 0.00 91.70 ± 7.47 na 0.00 ± 0.00 87.94 ± 7.63 nt EI not tested, na not applicable (the stability was not monitored when no EI was recorded) 24 24 Table 4 Residual emulsification activity (REA%) at pH 4 and pH 12 after an incubation period of up to 30 months Isolate/surfactant Time pH 4 pH 12 (months) Toluene Hexane Hexadecane Toluene Hexane Hexadecane Tween 80 29 96.22 ± 2.10 94.44 ± 0.00 89.47 ± 0.00 92.87 ± 2.23 96.22 ± 2.10 87.81 ± 1.15 Bacillus sp. L45 30 100.00 ± 0.00 77.09 ± 2.70 0.00 ± 0.00 66.43 ± 3.96 55.00 ± 7.07 0.00 ± 0.00 Paenibacillus tarimensis L88 30 82.26 ± 8.41 2.97 ± 4.20 0.00 ± 0.00 43.00 ± 8.44 27.38 ± 8.42 0.00 ± 0.00 Bacillus sp. L97b 29 52.04 ± 8.33 100.00 ± 0.00 0.00 ± 0.00 94.44 ± 4.72 98.30 ± 1.66 0.00 ± 0.00 Bacillus sp. L98 29 0.00 ± 0.00 na nt 9.87 ± 1.75 54.25 ± 1.30 nt Bacillus sp. N3 30 86.19 ± 0.67 0.00 ± 0.00 nt 26.30 ± 0.52 70.59 ± 3.53 nt Bacillus sp. R40 29 0.00 ± 0.00 50.00 ± 0.00 nt na 100.00 ± 0.00 nt Bacillus sp. R55 29 100.00 ± 0.00 59.92 ± 2.81 0.00 ± 0.00 87.50 ± 4.38 100.00 ± 0.00 19.33 ± 0.97 nt EI not tested, na not applicable (the stability was not monitored when no EI was recorded) 24 24 Raddadi et al. Microb Cell Fact (2018) 17:83 Page 10 of 12 Table 5 Residual emulsification activity (REA%) under  different thermal treatments after  an  incubation period of  up  to 30 months Isolate/surfactant Time 4 °C 55 °C Autoclave treatment (months) Toluene Hexane Toluene Hexane Hexadecane Toluene Hexane Hexadecane Tween 80 29 91.27 ± 0.28 93.50 ± 1.33 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 90.38 ± 1.29 77.78 ± 0.00 0.00 ± 0.00 Bacillus sp. L45 30 95.03 ± 5.14 100.00 ± 0.00 72.08 ± 0.92 56.41 ± 6.81 51.49 ± 8.00 66.79 ± 6.57 86.15 ± 8.70 0.00 ± 0.00 Paenibacillus tarimen- 30 62.50 ± 8.84 0.00 ± 0.00 0.00 ± 0.00 na na na na na sis L88 Bacillus sp. L97b 29 92.26 ± 1.52 92.72 ± 5.42 75.00 ± 3.75 90.42 ± 0.00 0.00 ± 0.00 28.57 ± 0.00 81.25 ± 4.06 0.00 ± 0.00 Bacillus sp. L98 29 79.41 ± 8.32 83.29 ± 4.13 0.00 ± 0.00 0.00 ± 0.00 nt 0.00 ± 0.00 79.48 ± 6.33 nt Bacillus sp. R4p 29 92.08 ± 2.37 77.05 ± 2.90 0.00 ± 0.00 0.00 ± 0.00 na 0.00 ± 0.00 0.00 ± 0.00 na Bacillus sp. R55 29 83.95 ± 8.72 68.40 ± 8.86 79.40 ± 8.94 77.59 ± 3.88 88.67 ± 4.43 97.94 ± 1.97 100.00 ± 0.00 0.00 ± 0.00 nt EI not tested, na not applicable (the stability was not monitored when no EI was recorded) 24 24 to loose almost completely their water content (Fig.  4). The effect of the bacterial BS/BE on the soil water Those irrigated with L45 or V3E1 BS/BE still retained a retention and humidity absorption highlights the poten- significantly higher amount (6.2 ± 2.4 and 8.2 ± 0.0%, tial of bacterial strains typical of arid environments to respectively) of the initial water, confirming the BS/BE improve the hydrological properties of sandy soils, even capacity to improve water retention in sandy soil. All though we must consider that these results were obtained the samples lost their water content after 18  h from the with a single soil type. Different soils may present differ - start of the second irrigation (Fig.  4b). It is of note that ent responses to bacterial BS/BE and a further research the sandy soil samples treated with BE produced by L45 is needed to improve the knowledges regarding the and V3E1, which did not reduce IFT, retained higher response of soils to the use of surfactants for improving water content compared to those treated with Triton its hydrological properties. X-100 or BS from isolate R43, which instead was able to reduce the IFT. These results suggest that L45 and V3E1 produce high molecular weight polymers with bioemulsi- Conclusions fication activity that can improve sandy soil compactness We showed that bacteria from arid environments can and/or absorb high water amounts, finally determin - produce polyextremotolerant bioemulsifiers that are ing higher water retention and reduction of evaporation functional in broad ranges of pH and temperature and in rates. Improvement of the water retention capacity was the presence of 30% NaCl. The emulsions were stable w/v observed in induced biological soil crusts as a function of up to 30  months incubation under several conditions. their total carbohydrate content [8]. The partially purified BS/BE produced by isolates L45, The capability of BS/BE-treated sandy soil to absorb R43 and V3E1 significantly improved water retention humidity from the surrounding environment was also and humidity uptake of a sandy soil compared to Triton evaluated after 36 h of incubation at 30 °C and high rela- X-100 or tap water. The data offer insights into the bio - tive humidity. Samples treated with tap water or Triton technological properties of BS/BE from bacteria inhabit- X-100 absorbed the same amounts of humidity, which ing non-conventional environments and their potential was statistically different from the quantity of water role for environmental protection and the improvement vapor absorbed by the sand samples treated with L45, of soil hydrological properties in arid regions. Indeed, the R43 and V3E1. The BS/BE produced by these bacteria use of BS/BE as additives in the irrigation water would significantly improved the amount of water absorbed be an interesting approach to promote plant stress to by the sandy soil. Data reported in Fig.  4c showed that drought as an alternative to the conventional inoculation L45-treated sand sample was able to absorb the highest of living microbial cells, overcoming potential limitations amount of water vapor, i.e. 607.7% higher than the con- related to over competition of the inoculated bacteria by trol treated with tap water. Similarly, V3E1 and R43 BS/ the rhizosphere microbioma. However, the application of BE determined an uptake of humidity 259.1 and 254.9%, BS/BE should be carefully evaluated taking into consid- respectively, higher than control (Fig.  4c). Interestingly, eration the characteristics of the soil to be irrigated since the strain L45, that showed the highest humidity uptake BS/BE–soil interaction can vary, resulting in a decrease rate, exhibited also the ability to highly emulsify different or an increase of the soil hydrophobicity. solvents (Table 1). Raddadi et al. Microb Cell Fact (2018) 17:83 Page 11 of 12 Additional files Additional file 1:Table S1. List of the 23 bacterial strains used in this work. Additional file 2: Figure S1. Phylogenetic affiliation of partial 16S rRNA gene of the 23 bacterial isolates obtained from chott and desert in the south of Tunisia constructed using MEGA6 package. Neighbor-Joining phylogenetic tree was built using MEGA 6, computing the evolutionary distances using the Kimura 2-parameter model. Authors’ contributions NR isolated the bioemulsifier-producing strains, designed the study and wrote the manuscript. LG performed the studies related to bioemulsifiers produc- tion, functional characterization and participated in the water retention/ uptake assays. RM isolated the strain V3E1 and participated in the manuscript preparation. DD, AC and FF participated in the design of the study and the analyses of data. All authors read and approved the final manuscript. Author details Department of Civil, Chemical, Environmental and Materials Engineer- ing (DICAM), Alma Mater Studiorum University of Bologna, via Terracini 28, 40131 Bologna, Italy. Biological and Environmental Sciences and Engineer- ing Division, King Abdullah University of Science and Technology (KAUST ), Thuwal 23955-6900, Saudi Arabia. LR Biotechnology and Bio-Geo Resources Valorization, Higher Institute for Biotechnology, Biotechpole Sidi Thabet, University of Manouba, 2020 Ariana, Tunisia. Acknowledgements Not applicable. Competing interests The authors declare that they have no competing interests. Availability of data and materials Partial 16S rDNA sequences obtained within this work are available at GenBank under the accession numbers listed in the text. All data generated or analyzed during this study are included in this published article (and its Additional files 1 and 2). Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Fig. 4 Results of water retention and moisture uptake assays by a Funding sandy soil treated with a BS/BE solution, Triton X-100 or tap water. a This work was financially supported by the EU in the frame of the FP-7 Project Trend of water content during the first cycle of irrigation; b results No. 312139 “Integrated Biotechnological Solutions for Combating Marine Oil of water retention assay recorded after 12.5 h of the onset of the Spills”–KILL·SPILL. second irrigation. Analyses of variance performed on water content after 12.5, 14, 15, 16 and 17 h (ANOVA 12.5 h to ANOVA 17 h), Publisher’s Note showing statistically significant differences among the samples Springer Nature remains neutral with regard to jurisdictional claims in pub- (ANOVA 12.5 h: p < 0.0001, F = 139.41, df = 4; ANOVA 14 h: p < 0.0001, lished maps and institutional affiliations. F = 138.19, df = 4; ANOVA 15 h: p < 0.0001, F = 127.16, df = 4; ANOVA 16 h: p < 0.0001, F = 72.06, df = 4; ANOVA 17 h: p < 0.0001, Received: 24 January 2018 Accepted: 22 May 2018 F = 17.80, df = 4; ANOVA 18 h: p = 0.15, F = 2.00, df = 4). According to post–hoc analysis (α = 0.05), means sharing the same letter are not significantly different from each other; c results of moisture uptake assay. ANOVA showing statistically significant differences among References the samples (p < 0.0001; F = 148.70; df = 4). According to post–hoc 1. 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Appl fast, convenient online submission Microbiol Biotechnol. 2015;99:7907–13. thorough peer review by experienced researchers in your field 21. Wang M, Yang M, Zhou G, Luo X, Zhang L, Tang Y, Fang C. Paenibacillus tarimensis sp. nov., isolated from sand in Xinjiang, China. Int J Syst Evol rapid publication on acceptance Microbiol. 2008;58:2081–5. support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Microbial Cell Factories Springer Journals

Bacterial polyextremotolerant bioemulsifiers from arid soils improve water retention capacity and humidity uptake in sandy soil

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Chemistry; Applied Microbiology; Biotechnology; Microbiology; Microbial Genetics and Genomics; Enzymology; Genetic Engineering
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Abstract

Background: Water stress is a critical issue for plant growth in arid sandy soils. Here, we aimed to select bacteria pro- ducing polyextremotolerant surface-active compounds capable of improving water retention and humidity uptake in sandy soils. Results: From Tunisian desert and saline systems, we selected eleven isolates able to highly emulsify different organic solvents. The bioemulsifying activities were stable with 30% NaCl, at 4 and 120 °C and in a pH range 4–12. Applications to a sandy soil of the partially purified surface-active compounds improved soil water retention up to 314.3% compared to untreated soil. Similarly, after 36 h of incubation, the humidity uptake rate of treated sandy soil was up to 607.7% higher than untreated controls. Conclusions: Overall, results revealed that polyextremotolerant bioemulsifiers of bacteria from arid and desert soils represent potential sources to develop new natural soil-wetting agents for improving water retention in arid soils. Keywords: Polyextremotolerant bioemulsifiers, Desert sandy soil, Water retention, Humidity uptake, Water stress Background BE and BS are used in agriculture for several applica- Biosurfactants (BS) are amphipathic compounds pro- tions ranging from the improvement of the quality of duced by a variety of microorganisms. They can be low- polluted soils, to the control of plant pathogens or for molecular weight, generally glycolipids or lipopeptides, favoring plant–microbe interactions [5]. The potential of or high-molecular weight compounds, which are mainly exopolysaccharides (EPS) with bioemulsifying properties lipopolysaccharides, lipoproteins, or a combination of have been considered for the promotion of plant growth both. The high-molecular weight BS, also called bioemul - by bacterial producers [6, 7] or for their effect on the sifiers (BE), are capable of producing stable emulsions, hydrological behavior of biological soil crusts [8]. Appli- but do not always determine decreases of surface or cation of surfactants to soil has been included among interfacial tensions [1]. There is a growing interest in twelve strategies for the remediation of soil water repel- microbial biosurfactants owing to several advantages lency [9], even though the effect of biosurfactants on soil over conventional surfactants, including biodegradability, water repellency are controversial. Some studies have low toxicity and production from renewable substrates reported that fungi may produce hydrophobins that favor [2–4]. the development of water repellency [10]. One aspect that, to our knowledge, has not been con- sidered in the literature on BS/BE is their potential for *Correspondence: noura.raddadi@unibo.it improving soil water retention [8], especially in arid and Department of Civil, Chemical, Environmental and Materials Engineering desert sandy soils that have limited content of organic (DICAM), Alma Mater Studiorum University of Bologna, via Terracini 28, 40131 Bologna, Italy matter and experience extreme environmental conditions Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Raddadi et al. Microb Cell Fact (2018) 17:83 Page 2 of 12 of limited water supply and nutrient content, high tem- index and the drop collapse activity as well as by measur- peratures, irradiation and evaporation rates and high ing the interfacial surface tension (IFT). salinity [11, 12]. The emulsification activity (EA) of the supernatant was We hypothesize that, by living under extreme con- determined as follows. Two milliliters of culture super- ditions, bacteria in arid and desert soils should have natant and an equal volume of the tested organic solvent evolved capacities and strategies that allow an efficient (n-hexane, n-hexadecane or toluene) were placed in a use of water, including production of BS/BE highly stable test tube that was vortexed at high speed for 2  min and under extreme conditions of temperature and irradiation then allowed to settle for 24  h. The emulsification index and that can facilitate access/retention of low-availa- (EI %) was estimated as the height of the emulsion layer, ble water and nutrients. On the plant leaf surface that divided by the total height, multiplied by 100. experiences analogous extremes such as those in desert The surface qualitative drop collapse activity test was sandy soils [13], epiphytic bacteria have been reported to carried out as follows: 40 µL of the cell free supernatant exploit biosurfactants to increase wettability of the leaf was aliquoted as a droplet onto Parafilm (Parafilm M, and to enhance nutrients diffusion through the wax cuti - Germany); the flattening and the spreading of the droplet cle [14]. Similarly, bacteria inhabiting arid environments on the Parafilm surface was followed over 10  min and and especially sandy soils could take advantage from recorded by visual inspection. The assay was scored nega - BS/BE to access limited nutrient supplies and withstand tive or positive if the drop remained beaded or collapsed, fluctuations in moisture availability in a water-deficient respectively. environment. The IFT of the cell-free culture supernatant was meas - In this study, we have screened a collection of bacte- ured with a Drop Shape Analyzer—DSA30 (KRUSS ria from arid Tunisian soils including (i) an inland saline GmbH, Germany) working in the pendant drop mode. system and sand from the Sahara desert and (ii) an arid The drops were produced by a syringe equipped with a vineyard in the North of the country, for BS/BE activities 2.098  mm needle diameter, were left to equilibrate close under different conditions. We have also tested a sub - to the rupture point and IFT values (mN/m) were cal- group of selected BS/BE bacterial isolates for their poten- culated by the instrument software using the Young– tial to improve water retention and humidity uptake in Laplace equation: sandy soils. �p = σ ∗ 1 r1 + 1 r2 ; Methods where ∆p is the differential pressure between the inside Bacterial strains and outside of the drop, σ is the IFT value and r1 and The 24 bacterial isolates used in this study include 23 r2 are the main radii of the drop curvature. Measure- strains obtained from sediment samples recovered from ments were performed at room temperature, at least in the inland saline system Chott El Fejej and desert sand triplicates. A Tween 80 solution at a final concentration from Douz in the South of Tunisia (Additional file  1: of 0.5% (in water), was used as positive control while w/v Table S1); and one (V3E1) isolated from the root system deionized water and non-inoculated growth medium of grapevine plants growing in an arid soil of Northern with 2% glucose were used as negative controls. All w/v Tunisia (Mornag area). The strains were identified based screening assays were performed in duplicate as inde- on partial 16S rRNA gene sequences (database accession pendent experiments. numbers MG594617-MG594638 and MG637030). Stability of bioemulsifiers Screening for biosurfactant/bioemulsifier production Emulsion stability studies were performed using cell-free Cells were inoculated into glucose mineral salts medium culture supernatants. The stability of the bioemulsifiers (GMSM) (g/L: 20.0 glucose; 0.7 K H PO ; 0.9 Na HPO ; activity was determined by investigating the effect of var - 2 4 2 4 2.0 NaNO ; 0.4 MgSO ·7H O; 0.1 C aCl ·2H O; 2  mL ying NaCl concentration, temperature or pH on the EA. 3 4 2 2 2 of trace elements [per liter, 2.0  g F eSO ·7H O, 1.5  g In order to assess the effect of salinity on the bioemul - 4 2 MnSO ·H O, 0.6  g (NH ) Mo O ·4H O]; pH = 6.72). sifier activity, culture supernatants were supplemented 4 2 4 6 7 24 2 The flasks were incubated at 30  °C on a rotary shaker with different NaCl concentrations (8–30% ) and the w/v (150  rpm). The surface activity of cell-free supernatants emulsifying activity was measured as described above. To was tested on culture samples every 24 h for a period of evaluate the stability of the bioemulsifier at different tem - up to 4 days. The cell-free culture supernatants recovered peratures, culture supernatants were subjected to cooling by centrifugation (10,000 rpm, 10 min, 4 °C) followed by (4 °C, 2 h), heating (55 °C, 2 h) or autoclaving (121 °C for filter-sterilization (0.22  µm) were used for evaluation of 20 min) followed by cooling to room temperature before the surface activity by determining the emulsification performing the EI assay. The measured emulsification Raddadi et al. Microb Cell Fact (2018) 17:83 Page 3 of 12 indexes were compared to the corresponding values samples in a sealed desiccator, in which the dishes were obtained using culture supernatants not subjected to the placed on the platform and the space under the platform different treatments. The EA of hexadecane was not eval - was filled with tap water instead of desiccant in order to uated at 4 °C since the melting point of the solvent was of create a high (100%) relative humidity in the environ- 18  °C and hence at lower temperature the emulsion was ment. After 36  h of incubation at constant temperature subjected to solidification. The pH stability was studied (30  °C), the samples were weighed in order to evaluate by adjusting the cell-free culture supernatants to different their weight increase due to humidity uptake. pH values (4–12) using HCl or NaOH solutions, and then the EA was measured as previously described. Further- Statistical analyses more, the stability of the emulsions produced under the Data related to the evaluation of bioemulsifiers stabil - different conditions was monitored for up to 30 months. ity under stressful conditions and to water retention and All the experiments were carried out in duplicate. The moisture uptake assays were statistically assessed using results were reported as residual emulsification activity the analysis of variance (ANOVA) via MATLAB software (REA) percentage (%) expressed as follows: (Version R2017a, The MathWorks Inc, Natick, USA). The statistical analyses aimed to highlight the statisti - REA (%) = EI EI ∗ 100; t 24 cally significant differences among EI % values obtained where EI and EI are the EI (%) values at incubation under standard and stressful conditions, and among the t 24 time t and after 24  h, respectively; and compared with amounts of retained water and uptaken humidity by those obtained with the positive control. sandy soil samples treated with the bioemulsifiers solu - tions, Triton X-100 or tap water. The significance of the Water retention and moisture uptake assays by a sandy soil data was determined by Tukey honestly significant differ - For BS/BE production, selected bacterial strains were ent test. Statistically significant results were depicted by p grown in GMSM and incubated at 30  °C. Cells were values < 0.05. removed by centrifugation (8000 rpm, 10 min, 4 °C) and the collected supernatant was acidified with 6  N hydro - Results and discussion chloric acid solution to pH 2.0 ± 0.3. The precipitate Screening of surface‑active strains and their identification which contained BS/BE was allowed to settle at 4  °C Among the 23 isolates screened (Additional file1: overnight. The precipitated BS/BE were collected by cen - Table  S1, Additional file2: Figure S1), 9 were capable to trifugation (12,000  rpm, 20  min, 4  °C) and resuspended significantly emulsify (EI % > 45%) at least one of the in sterile tap water at a final concentration of 25 g/L. Tri - solvents tested (Table  1). On the basis of the partial 16S ton X-100 applied at a final concentration corresponding rRNA gene sequences, all isolates had between 99 and to its CMC (0.14 g/L) and sterilized tap water were used 100% sequence similarities with their closest relative as controls. Before using in the experiments, the sandy type strain in the databases (Table 1). Bacillus mojavensis soil sample was oven-dried (105 °C, 4 h) and subjected to IFO15718 isolated from Mojave desert [15, 16] was the sterilization by tyndallization followed by drying (55  °C, closest relative species of the R4p, R43 and R39 strains, overnight) and acclimated at 30 °C for 24 h. while Bacillus endophyticus strain 2DT isolated from the The water retention assay was performed as follows: inner tissues of cotton plants [17] was the closest relative 10  g of sandy soil were placed in a glass Petri dish in of L45, L97b and L37 isolates. The partial 16S rRNA gene duplicate and subjected to wetting with 10  ml of a solu- sequence from strain N3 showed 99% homology with tion of BS/BE to be tested or tap water, followed by dry- different B. subtilis including the subsp. spizizenii TU-B- ing at 30  °C until a constant weight was achieved. The 10 isolated from soil collected near Nefta, Tunisia [18]. samples have been thoroughly mixed manually and then Bacillus licheniformis strain DSM 13 was the closest rela- subjected to two cycles of irrigation with 2.5  mL steri- tive of L98 strain, and Bacillus frigoritolerans DSM 8801 lized tap water and drying until constant weight was isolated from arid soil in Morocco was that of R55 and achieved. During the first drying cycle, each sample was R40 strains. The last isolate among those obtained from weighed immediately after irrigation with tap water and chott and that is positive for BS/BE production was pre- every 60  min up to 12.5  h of incubation and then each viously classified as Paenibacillus tarimensis (strain L88) 12  h until constant weight was achieved. Monitoring of [19], while the grapevine rhizosphere isolate V3E1was the samples weight after the second irrigation was per- assigned to Rhizobium sp.. formed at the time of irrigation, after 12.5 h of incubation The eleven desert/chott isolates capable of produc - and then every 60 min until complete drying. ing BS/BE were spore-formers, a group of bacteria well The moisture uptake assay was performed by incubat - adapted to the arid conditions of the desert primarily ing the Petri dishes containing 10  g of dried sandy soil for their capacity to produce spores that are resistant Raddadi et al. Microb Cell Fact (2018) 17:83 Page 4 of 12 Table 1 List of  BS/BE producers obtained from  the  screening of  23 desert bacterial isolates grown on  MSM with  2% w/v glucose as  carbon source. Emulsification index (EI %) and  interfacial surface tension (IFT) values are expressed and mean value ± SD of two and three replicates, respectively, while drop collapse results as positive (+) or negative (−) Isolate ID 16SrDNA Closest type strains 16S rDNA Drop collapse IFT (mN/m) EI24 (%) Accession (GenBank Accession identity Toluene Hexane Hexadecane No No) (%) Bacillus sp. L37 MG594627 Bacillus endophyticus 99 + 69.28 ± 0.31 57.63 ± 4.24 56.07 ± 2.18 61.11 ± 1.92 (NR_025122) Bacillus sp. L45 MG594628 Bacillus endophyticus 99 − 70.00 ± 0.47 52.53 ± 1.14 59.67 ± 1.48 53.33 ± 9.43 (NR_025122) Paenibacillus tarimensis KF111690 Paenibacillus tarimensis 99 − 74.45 ± 0.16 44.64 ± 2.06 50.93 ± 1.07 48.32 ± 6.27 L88 (NR_044102) Bacillus sp. L97b MG594634 Bacillus endophyticus 99 − 72.48 ± 0.36 57.64 ± 3.67 53.11 ± 5.13 47.36 ± 8.45 (NR_025122) Bacillus sp. L98 MG594631 Bacillus licheniformis 99 + 55.07 ± 0.26 48.82 ± 6.06 50.83 ± 2.18 46.43 ± 5.05 (NR_118996) Bacillus sp. N3 MG594629 Bacillus subtilis 99 + 36.86 ± 1.11 50.81 ± 2.17 34.84 ± 3.21 51.67 ± 7.07 subsp. spizizenii (NR_112686) Bacillus sp. R4p MG594635 Bacillus mojavensis 99 + 28.99 ± 0.35 55.67 ± 6.95 44.91 ± 4.96 28.57 ± 0.00 (NR_112725) Bacillus sp. R39 MG594626 Bacillus mojavensis 99 + 28.36 ± 0.63 11.15 ± 0.00 5.30 ± 2.26 0.00 ± 0.00 (NR_112725) Bacillus sp. R40 MG594633 Bacillus frigoritolerans 99 − 70.25 ± 0.46 52.12 ± 7.54 44.90 ± 4.52 58.33 ± 2.36 (NR_115064) Bacillus sp. R43 MG594630 Bacillus mojavensis 99 + 28.57 ± 0.76 26.67 ± 1.33 32.69 ± 6.78 11.51 ± 2.59 (NR_112725) Bacillus sp. R55 MG594632 Bacillus frigoritolerans 99 − 71.10 ± 0.85 59.98 ± 2.54 49.20 ± 2.92 53.77 ± 8.08 (NR_115064) to heating, desiccation and irradiation [20]. Several of culture supernatants did not exhibit a remarkable sur- the strains were affiliated to species previously isolated face tension reduction but were able to highly emulsify from soil and sand from other desert ecosystems, such the tested organic solvents. Indeed, the formation of as Bacillus mojavensis [15] and Paenibacillus tarimensis emulsions is typical of BE while BS are the compounds [21]. that are able to significantly reduce the surface tension Even though it should be confirmed by a larger range of of aqueous media to around 40 mN/m [14, 22]. isolates and ecosystems, it can be noted that comparing Bacillus spp. are well known BS/BE producers and the bacterial BS/BE producers with the initial collection their applications were related to hydrocarbon (crude of isolates (Additional file1: Table S1), almost all of them oil) degradation and microbial enhanced oil recovery (10 out of 11) belong to the genus Bacillus. This obser - [23–25]. Among others, B. endophyticus isolate TSH42 vation suggests that that BS/BE activities are important was reported to produce surfactin, fengycin and iturin features in the Bacillus genus for the adaptation to arid lipopeptides that exhibit fungal biocontrol activity [26]; conditions. however their ability to reduce the growth medium The maximum emulsifying activity was recorded surface tension or to emulsify organic solvents has between 24 and 96  h of incubation. The significant not been evaluated previously. B. mojavensis has been highest emulsification index was observed using hexa - previously studied for lipopeptide biosurfactant pro- decane as solvent (61.11 ± 1.92% by Bacillus endophyti- duction [27] but not for bioemulsification activities. B. cus L37; Table 1). Cell-free culture supernatants of four licheniformis was the only one previously characterized isolates (Bacillus sp. isolate N3, Bacillus sp. isolates for its production of lipopeptide biosurfactants capa- R4p, R39 and R43) significantly decreased the medium ble to reduce the medium surface tension to 28 mN/m surface tension from 74.66 ± 0.21  mN/m, with a low- [28] and to determine bioemulsification activity [24, est value of 28.36 ± 0.63  mN/m (Table  1). These results 25]. Interestingly, while Paenibacillus sp. isolates have suggest that under these experimental conditions, most been previously studied for their BS/BE production and of the isolates (8 out of 11) produced BE since their activity [29–32], no data regarding B. frigoritolerans were available in literature. Raddadi et al. Microb Cell Fact (2018) 17:83 Page 5 of 12 Stability of bioemulsifiers the maximum salt stress tested (30%), a total absence of Based on BS/BE activities, ten bioemulsifying isolates activity was recorded for all the culture supernatants as were selected and the stability of their EA was evaluated well as for Tween 80 (Fig. 2a). In the presence of a lower under different conditions. The activity of the crude BEs NaCl concentration (8%) the hexane E A was main- was evaluated from cell-free culture supernatants after tained statistically unchanged compared to the standard exposure to low water activity conditions as well as to conditions for Tween 80 and strains L37, L45, L97b, R4p extremes of pH or temperature (Figs. 1, 2, 3). In presence and R55; while it was completely lost for R43 and L98 of toluene under standard conditions, six of the isolates and significantly reduced for the remaining isolates (L88, (L37, L45, L97b, R4p, R40 and R55) did not exhibit a sta- N3, R40). Considering pH as stressful condition, under tistically significant decrease of their BE activity com - acidic conditions EA was significantly reduced for iso - pared to the positive control (Tween 80, Fig.  1a), while lates R40 and R55 with a complete loss for strain L98 at EI (%) of the remaining isolates (L88, L98, N3 and pH 4 (Fig. 2b). In general, alkaline pH did not affect EA 24 24 R43) were significantly reduced. Different trends were with the exception of the strain R40 (Fig.  2b). Looking observed when salt stresses were applied to the solution. to the temperature, also in this case for some strains the With the increase of NaCl concentration (up to 30%), autoclaving procedure resulted in a reduction (N3, R4p) only five isolates (L37, L45, L88, N3 and R55) showed EI or complete loss (L88 and R40) of the EA . The incuba - 24 24 values statistically comparable to those obtained under tion at 55  °C resulted in a significant reduction of EA standard conditions, suggesting how stress can strongly for the isolates L37, L88, R40 and R55 and a complete affect the activity of bacterial BEs. Remarkably, while loss for N3 and R43, while the activity remained statisti- the EA was significantly reduced for the strains R40 cally stable for the other strains. The reduction of EA at 24 24 and L97b, it was completely lost for R4p, L98 and R43 55  °C (although it was stable at higher temperature, i.e., (Fig. 1a). to autoclaving), for example in the case of isolates L37, When the stress applied was determined by extreme R43 and R55, could be ascribable to partial degradation pH (4 and 12), a different scenario was observed (Fig.  1b). of the bioemulsifiers as a result of the activation of extra - Under these conditions, no significant changes in EA cellular hydrolytic enzymes (for example proteases and/ values were reported for L45, L97b, R55, N3, R4p and or lipases) present in the culture supernatant of these iso- L37, as well as for the positive control (Tween 80), com- lates. Indeed, the cell free culture supernatants were pre- pared to standard conditions. The toluene EA by culture heated and further incubated at 55  °C once the organic supernatant of isolate P. tarimensis L88 was significantly solvents to be tested were added, which could has led the enhanced at pH 4, while remained statistically equiva- bioemulsifier polymeric compounds degradation. The lent to standard conditions at pH 12. On the contrary, activity of extracellular hydrolytic enzymes at relatively a significant EA reduction/loss was recorded for iso- high temperature (55 °C) can be expected considering the lates R40 (pH 4 and pH 12), R43 (pH 12) and L98 (pH 4) origin of the isolates from a hot desert. On the contrary, a (Fig. 1b). With the exception of isolates N3, R43 and R40, low temperature (4 °C) reduced EA of R55, R40 and N3 all the remaining cell-free culture supernatants retained but did not affect that of the Tween 80 or the remaining EA at low temperature (4  °C), which were statistically strains (Fig. 2c). identical to that of Tween 80 (Fig.  1c). When the tem- The stability of the emulsification activity in hexade - perature was increased to 55 °C, isolates L97b, L88, L98, cane under stressful conditions was evaluated only for R4p, R55 and L45 showed E A comparable to the cor- isolates exhibiting a significant stability in hexane and responding standard conditions and to Tween 80, while toluene solvents (L37, L45, L88, L97b, R4p and R55) as a reduction/loss of activity was recorded for strains L37, reported in Fig.  3a–c. The presence of different salt con - R40, N3 and R43 (Fig. 1c). After autoclaving, the cell-free centrations affected significantly the EA of strains L97b culture supernatants of the tested isolates retained an and R55 (Fig.  3a). Extreme pHs and temperature treat- EA unchanged compared to their corresponding activi- ments significantly affected bacterial BEs performance ties under standard conditions; except for strains N3, in the case of L97b (pH 4), R55 (pH 4, 55  °C, autoclav- R40, L88, R4p and R43 (Fig. 1c). ing), R4p (55  °C, autoclaving), L37 and L88 (pH 12, Bacterial BEs acted in a completely different way when 55 °C, autoclaving), up to complete EA loss in the case hexane was used as solvent (Fig. 2a–c). In the presence of of strains L37, L88 and R4p at 55 °C and strains L88 and salt stress (15%), a significant reduction of bacterial BEs R4p after autoclaving (Fig. 3b, c). performances was observed, except for isolates L37 and Gutiérrez et  al. [33] reported that heat (100  °C) and L45 that showed E I values statistically comparable to acid treatment (0.1  N) increased the relative activity of the corresponding activities under standard conditions, the bioemulsifiers produced by Halomonas sp. Activ - as well as to that of Tween 80 with 15% NaCl. Reaching ity increment was explained by a heating activation of Raddadi et al. Microb Cell Fact (2018) 17:83 Page 6 of 12 Fig. 1 EI (%) of toluene recorded under standard and stressful conditions. EI of toluene under low water activity (ANOVA: p < 0.0001, F = 43.86, df = 43) (a), extremes of pH (ANOVA: p < 0.0001, F = 35.89, df = 32) (b) and different temperatures (ANOVA: p < 0.0001, F = 64.15, df = 43) (c). Results of post hoc analyses of each condition were represented as letters: different letters correspond to statistically different EI (%) results (α = 0.05) 24 Raddadi et al. Microb Cell Fact (2018) 17:83 Page 7 of 12 Fig. 2 EI (%) of hexane recorded under standard and stressful conditions. EI of hexane under low water activity (ANOVA: p < 0.0001, F = 95.13, df = 32) (a), extremes of pH (ANOVA: p < 0.0001, F = 38.18, df = 32) (b) and different temperatures (ANOVA: p < 0.0001, F = 86.43, df = 43) (c). Results of post hoc analyses of each condition were represented as letters: different letters correspond to statistically different EI (%) results (α = 0.05) 24 Raddadi et al. Microb Cell Fact (2018) 17:83 Page 8 of 12 polymeric emulsifiers releasing a higher number of emul - sifying moieties from the biopolymers, enhancing the emulsifying capacity. BEs not affected by salt stress (i.e. NaCl concentra - tions up to 300  g/L) and extremes of pH (2–13) have been previously described from Paenibacillus sp. #510 [30] as well as from marine Bacillus sp., Halomonas sp. and Marinobacter sp. [34]. Dubey et al. [35] reported that bioemulsifiers from Pseudomonas aeruginosa strain-PP2 and Kocuria turfanesis strain-J retained their activities at up to 20% NaCl and 121  °C. Marine Rhodococcus sp. has been reported to produce trehalolipid biosurfactant that formed emulsions stable to a wide range of tempera- tures (20–100  °C), pH (2–10) and NaCl concentrations (5–25% ) [36], and bioemulsifiers highly stable at low w/v water activity and temperature extremes were reported from Marinobacter sp. [34]. EIs relatively stable under extreme pH, temperature, and salinity conditions have been previously reported from Bacillus spp. [23–25, 28]. The long-term stability of emulsions (reported as resid - ual emulsification activity %) under standard screening conditions or after exposure to water, heat or extreme pH stresses was evaluated after a monitoring period of up to 30  months. The emulsions showed a high stabil - ity at room temperature, maintaining up to 100% of the initial EIs under standard conditions (Table 2), while dif- ferent responses have been observed under the differ - ent stresses applied (Tables  3, 4, 5). Interestingly, some strains were able to retain unchanged activity in the pres- ence of up to 15% NaCl (strain L45; Table 3), under acid/ alkaline pHs (strains L45, L97b, R40 and R55; Table  4) and at 4  °C or after autoclaving (strains L45 and R55, respectively; Table 5). Long-term EI stabilities have been previously observed for the bioemulsifiers produced by Pedobacter sp. strain MCC-Z where the emulsions remained stable for 4  months [37]. Extended stability for longer incubation times (18–30  months) under low water activity or after thermal treatments was also observed for bioemulsifi - ers produced by Marinobacter sp. isolates [34], while no reports on long period incubation under pH extremes are available to the best of our knowledge. Water retention and humidity uptake by a BS/BE‑treated sandy soil Based on the characterization of the surface activity of the produced bacterial compounds two isolates (L45 and Fig. 3 EI (%) of hexadecane recorded under standard and stressful R43) were selected for further tests. While L45 produced conditions. EI of hexadecane under different salt concentrations only BEs, R43 was able to produce BEs that significantly (ANOVA: p < 0.0001, F = 28.26, df = 27) (a), extremes of pH (ANOVA: reduced IFT but exhibited low emulsification activ - p < 0.0001, F = 34.44, df = 20) (b) and temperature (ANOVA: p < 0.0001, F = 53.73, df = 20) (c). Results of post hoc analyses of each ity. Their BS/BEs were used for testing their ability to condition were represented as letters: different letters correspond to improve water content and humidity uptake of sandy soil. statistically different EI (%) results (α = 0.05) An additional strain, V3E1 isolated from arid agricultural Raddadi et al. Microb Cell Fact (2018) 17:83 Page 9 of 12 Table 2 Residual emulsification activity (REA%) and 11.67 ± 2.36 for toluene, hexane and hexadecane, after  an  incubation period of  30  months under  standard respectively. conditions The water retention capacity of sandy soils treated with BS/BE solutions was monitored after two irrigation Isolate/surfactant Toluene Hexane Hexadecane events using sterilized tap water. No significant differ - Tween 80 94.74 ± 0.00 90.10 ± 9.58 16.67 ± 8.19 ences between the treatments were observed during the Bacillus sp. L37 95.24 ± 6.73 91.20 ± 4.19 0.00 ± 0.00 first ten hours of monitoring, after the start of the first Bacillus sp. L45 91.71 ± 8.48 94.12 ± 4.71 7.56 ± 0.18 irrigation (Fig.  4a). Based on this, a second irrigation Bacillus sp. L97b 82.40 ± 3.95 94.76 ± 2.69 0.00 ± 0.00 treatment was performed and new measurements were Bacillus sp. L98 26.67 ± 1.33 58.93 ± 7.58 20.00 ± 1.00 carried out (Fig.  4b). Statistical analyses showed that Bacillus sp. R40 97.06 ± 4.16 84.62 ± 4.23 0.00 ± 0.00 after 12.5  h, compared to control (not subjected to BS/ BE treatment) which retained only 9.1 ± 0.9% (Fig.  4b), Triton X-100, R43, L45 and V3E1-treated sandy soil samples significantly reduced water evaporation retain - soil, was also used. The isolate V3E1 was included in the ing 16.3 ± 0.8; 19.0 ± 0.1; 27.0 ± 3.5 and 28.6 ± 1.2% of study since it has been shown to produce extracellu- the initial water, respectively. Hence, comparing the data lar polymeric substances (EPS), and based on literature obtained we can show that after 12 h bacterial BS/BE sig- reports EPS have been found to play an important role nificantly increased water retention, with values up to in water retention in biological soil crusts [8]. The isolate 314.3% (V3E1) higher than the untreated control. This produced surface-active compounds that were able to capability to retain a significantly higher water amount, reduce the surface tension of the cultivation medium up compared to control, was statistically confirmed also dur - to 69.81 ± 0.88  mN/m and to emulsify the different sol - ing the two following hours. However, after 16 h, samples vents used exhibiting EI (%) of 55.93 ± 5.76; 5.00 ± 2.36 irrigated with Triton X-100, R43 and water were found Table 3 Residual emulsification activity (REA%) under  low water activity after  an  incubation period of  up  to 30  months under standard conditions Isolate/surfactant Time 8% NaCl 15% NaCl 30% NaCl (months) Toluene Hexane Hexadecane Toluene Hexane Hexadecane Toluene Tween 80 29 2.54 ± 3.60 59.74 ± 7.03 88.89 ± 4.44 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Bacillus sp. L45 30 96.15 ± 3.26 100.00 ± 0.00 0.00 ± 0.00 95.32 ± 4.26 93.75 ± 0.00 0.00 ± 0.00 81.03 ± 7.79 Paenibacillus tarimensis L88 30 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 11.93 ± 4.02 0.00 ± 0.00 0.00 ± 0.00 16.78 ± 1.98 Bacillus sp. L97b 29 66.52 ± 3.16 77.28 ± 6.88 0.00 ± 0.00 10.00 ± 0.50 12.92 ± 0.65 0.00 ± 0.00 0.00 ± 0.00 Bacillus sp. R40 29 64.91 ± 6.95 28.57 ± 1.43 nt 66.25 ± 8.84 na nt 63.65 ± 7.52 Bacillus sp. R55 29 85.74 ± 5.54 46.67 ± 2.33 0.00 ± 0.00 91.70 ± 7.47 na 0.00 ± 0.00 87.94 ± 7.63 nt EI not tested, na not applicable (the stability was not monitored when no EI was recorded) 24 24 Table 4 Residual emulsification activity (REA%) at pH 4 and pH 12 after an incubation period of up to 30 months Isolate/surfactant Time pH 4 pH 12 (months) Toluene Hexane Hexadecane Toluene Hexane Hexadecane Tween 80 29 96.22 ± 2.10 94.44 ± 0.00 89.47 ± 0.00 92.87 ± 2.23 96.22 ± 2.10 87.81 ± 1.15 Bacillus sp. L45 30 100.00 ± 0.00 77.09 ± 2.70 0.00 ± 0.00 66.43 ± 3.96 55.00 ± 7.07 0.00 ± 0.00 Paenibacillus tarimensis L88 30 82.26 ± 8.41 2.97 ± 4.20 0.00 ± 0.00 43.00 ± 8.44 27.38 ± 8.42 0.00 ± 0.00 Bacillus sp. L97b 29 52.04 ± 8.33 100.00 ± 0.00 0.00 ± 0.00 94.44 ± 4.72 98.30 ± 1.66 0.00 ± 0.00 Bacillus sp. L98 29 0.00 ± 0.00 na nt 9.87 ± 1.75 54.25 ± 1.30 nt Bacillus sp. N3 30 86.19 ± 0.67 0.00 ± 0.00 nt 26.30 ± 0.52 70.59 ± 3.53 nt Bacillus sp. R40 29 0.00 ± 0.00 50.00 ± 0.00 nt na 100.00 ± 0.00 nt Bacillus sp. R55 29 100.00 ± 0.00 59.92 ± 2.81 0.00 ± 0.00 87.50 ± 4.38 100.00 ± 0.00 19.33 ± 0.97 nt EI not tested, na not applicable (the stability was not monitored when no EI was recorded) 24 24 Raddadi et al. Microb Cell Fact (2018) 17:83 Page 10 of 12 Table 5 Residual emulsification activity (REA%) under  different thermal treatments after  an  incubation period of  up  to 30 months Isolate/surfactant Time 4 °C 55 °C Autoclave treatment (months) Toluene Hexane Toluene Hexane Hexadecane Toluene Hexane Hexadecane Tween 80 29 91.27 ± 0.28 93.50 ± 1.33 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 90.38 ± 1.29 77.78 ± 0.00 0.00 ± 0.00 Bacillus sp. L45 30 95.03 ± 5.14 100.00 ± 0.00 72.08 ± 0.92 56.41 ± 6.81 51.49 ± 8.00 66.79 ± 6.57 86.15 ± 8.70 0.00 ± 0.00 Paenibacillus tarimen- 30 62.50 ± 8.84 0.00 ± 0.00 0.00 ± 0.00 na na na na na sis L88 Bacillus sp. L97b 29 92.26 ± 1.52 92.72 ± 5.42 75.00 ± 3.75 90.42 ± 0.00 0.00 ± 0.00 28.57 ± 0.00 81.25 ± 4.06 0.00 ± 0.00 Bacillus sp. L98 29 79.41 ± 8.32 83.29 ± 4.13 0.00 ± 0.00 0.00 ± 0.00 nt 0.00 ± 0.00 79.48 ± 6.33 nt Bacillus sp. R4p 29 92.08 ± 2.37 77.05 ± 2.90 0.00 ± 0.00 0.00 ± 0.00 na 0.00 ± 0.00 0.00 ± 0.00 na Bacillus sp. R55 29 83.95 ± 8.72 68.40 ± 8.86 79.40 ± 8.94 77.59 ± 3.88 88.67 ± 4.43 97.94 ± 1.97 100.00 ± 0.00 0.00 ± 0.00 nt EI not tested, na not applicable (the stability was not monitored when no EI was recorded) 24 24 to loose almost completely their water content (Fig.  4). The effect of the bacterial BS/BE on the soil water Those irrigated with L45 or V3E1 BS/BE still retained a retention and humidity absorption highlights the poten- significantly higher amount (6.2 ± 2.4 and 8.2 ± 0.0%, tial of bacterial strains typical of arid environments to respectively) of the initial water, confirming the BS/BE improve the hydrological properties of sandy soils, even capacity to improve water retention in sandy soil. All though we must consider that these results were obtained the samples lost their water content after 18  h from the with a single soil type. Different soils may present differ - start of the second irrigation (Fig.  4b). It is of note that ent responses to bacterial BS/BE and a further research the sandy soil samples treated with BE produced by L45 is needed to improve the knowledges regarding the and V3E1, which did not reduce IFT, retained higher response of soils to the use of surfactants for improving water content compared to those treated with Triton its hydrological properties. X-100 or BS from isolate R43, which instead was able to reduce the IFT. These results suggest that L45 and V3E1 produce high molecular weight polymers with bioemulsi- Conclusions fication activity that can improve sandy soil compactness We showed that bacteria from arid environments can and/or absorb high water amounts, finally determin - produce polyextremotolerant bioemulsifiers that are ing higher water retention and reduction of evaporation functional in broad ranges of pH and temperature and in rates. Improvement of the water retention capacity was the presence of 30% NaCl. The emulsions were stable w/v observed in induced biological soil crusts as a function of up to 30  months incubation under several conditions. their total carbohydrate content [8]. The partially purified BS/BE produced by isolates L45, The capability of BS/BE-treated sandy soil to absorb R43 and V3E1 significantly improved water retention humidity from the surrounding environment was also and humidity uptake of a sandy soil compared to Triton evaluated after 36 h of incubation at 30 °C and high rela- X-100 or tap water. The data offer insights into the bio - tive humidity. Samples treated with tap water or Triton technological properties of BS/BE from bacteria inhabit- X-100 absorbed the same amounts of humidity, which ing non-conventional environments and their potential was statistically different from the quantity of water role for environmental protection and the improvement vapor absorbed by the sand samples treated with L45, of soil hydrological properties in arid regions. Indeed, the R43 and V3E1. The BS/BE produced by these bacteria use of BS/BE as additives in the irrigation water would significantly improved the amount of water absorbed be an interesting approach to promote plant stress to by the sandy soil. Data reported in Fig.  4c showed that drought as an alternative to the conventional inoculation L45-treated sand sample was able to absorb the highest of living microbial cells, overcoming potential limitations amount of water vapor, i.e. 607.7% higher than the con- related to over competition of the inoculated bacteria by trol treated with tap water. Similarly, V3E1 and R43 BS/ the rhizosphere microbioma. However, the application of BE determined an uptake of humidity 259.1 and 254.9%, BS/BE should be carefully evaluated taking into consid- respectively, higher than control (Fig.  4c). Interestingly, eration the characteristics of the soil to be irrigated since the strain L45, that showed the highest humidity uptake BS/BE–soil interaction can vary, resulting in a decrease rate, exhibited also the ability to highly emulsify different or an increase of the soil hydrophobicity. solvents (Table 1). Raddadi et al. Microb Cell Fact (2018) 17:83 Page 11 of 12 Additional files Additional file 1:Table S1. List of the 23 bacterial strains used in this work. Additional file 2: Figure S1. Phylogenetic affiliation of partial 16S rRNA gene of the 23 bacterial isolates obtained from chott and desert in the south of Tunisia constructed using MEGA6 package. Neighbor-Joining phylogenetic tree was built using MEGA 6, computing the evolutionary distances using the Kimura 2-parameter model. Authors’ contributions NR isolated the bioemulsifier-producing strains, designed the study and wrote the manuscript. LG performed the studies related to bioemulsifiers produc- tion, functional characterization and participated in the water retention/ uptake assays. RM isolated the strain V3E1 and participated in the manuscript preparation. DD, AC and FF participated in the design of the study and the analyses of data. All authors read and approved the final manuscript. Author details Department of Civil, Chemical, Environmental and Materials Engineer- ing (DICAM), Alma Mater Studiorum University of Bologna, via Terracini 28, 40131 Bologna, Italy. Biological and Environmental Sciences and Engineer- ing Division, King Abdullah University of Science and Technology (KAUST ), Thuwal 23955-6900, Saudi Arabia. LR Biotechnology and Bio-Geo Resources Valorization, Higher Institute for Biotechnology, Biotechpole Sidi Thabet, University of Manouba, 2020 Ariana, Tunisia. Acknowledgements Not applicable. Competing interests The authors declare that they have no competing interests. Availability of data and materials Partial 16S rDNA sequences obtained within this work are available at GenBank under the accession numbers listed in the text. All data generated or analyzed during this study are included in this published article (and its Additional files 1 and 2). Consent for publication Not applicable. Ethics approval and consent to participate Not applicable. Fig. 4 Results of water retention and moisture uptake assays by a Funding sandy soil treated with a BS/BE solution, Triton X-100 or tap water. a This work was financially supported by the EU in the frame of the FP-7 Project Trend of water content during the first cycle of irrigation; b results No. 312139 “Integrated Biotechnological Solutions for Combating Marine Oil of water retention assay recorded after 12.5 h of the onset of the Spills”–KILL·SPILL. second irrigation. Analyses of variance performed on water content after 12.5, 14, 15, 16 and 17 h (ANOVA 12.5 h to ANOVA 17 h), Publisher’s Note showing statistically significant differences among the samples Springer Nature remains neutral with regard to jurisdictional claims in pub- (ANOVA 12.5 h: p < 0.0001, F = 139.41, df = 4; ANOVA 14 h: p < 0.0001, lished maps and institutional affiliations. F = 138.19, df = 4; ANOVA 15 h: p < 0.0001, F = 127.16, df = 4; ANOVA 16 h: p < 0.0001, F = 72.06, df = 4; ANOVA 17 h: p < 0.0001, Received: 24 January 2018 Accepted: 22 May 2018 F = 17.80, df = 4; ANOVA 18 h: p = 0.15, F = 2.00, df = 4). According to post–hoc analysis (α = 0.05), means sharing the same letter are not significantly different from each other; c results of moisture uptake assay. ANOVA showing statistically significant differences among References the samples (p < 0.0001; F = 148.70; df = 4). According to post–hoc 1. 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