Screening and optimization of cost-effective substrates for biosurfactant production
Bacillus subtilis SNW3 was previously studied for biosurfactant production using yeast extract as sole source of carbon while in current study conducted number of C/N sources (as mentioned above in methods) were used to enhance the biosurfactant yield. Major type of products produced by this isolate is identified as surfactin in form of C13-, C14-, and C15-surfactin mixture possess applicability in oil recovery [31] and as promising anti-tumour agent [32]. By introducing nitrogen containing compounds urea and yeast extract that exhibits amine groups bring about synthesis of biosurfactants having peptide moieties or enzymes that regulate the synthesis. In current study among nitrogen sources tested preferably urea act as good nitrogen source shows surface tension reduction 31.4 mN/m and of 2 cm (Fig. 1b). It has been reported that supplementation of peptone, urea, sodium nitrate, ammonium nitrate [33] and meat extract [34] increased biosurfactant production. Use of ammonium sulphate (5 g/L) and yeast extract (2 g/L) as nitrogen sources for biosurfactant production was also investigated by [35]. In current study yeast extract was used as control media and selected substrates as carbon sources includes white beans powder, potato peels powder, molasses, and waste frying oil (2%, w/v) were checked for biosurfactant production after 96 h. While single use of these substrates checked for surfactin production by surface tension reduction, emulsification and oil displacement activity shown in Fig. 1a. According to results obtained for 2% potato peels powder and white beans powder individually gave oil displacement value of 1.3 and 2.4 cm with surface tension reduction 41.3 and 33.6 mN/m and E24 55.1 and 57% respectively. Application of cost-effective substrates for biosurfactant production reduce cost of enzymes production on industrial scale. Hence, ideal fermentation medium selection plays an important role for reducing cost of biomolecules [36]. Potato processing produce starch rich waste in form of potato peels, starch rich wastewater and unconsumable potato parts that could be used as substrate for microbial production [37]. Ohno and coworkers [38] investigated use of okara obtained after processing of ground soybeans as substrate for lipopeptide iturin and surfactin production by Bacillus subtilis NB22. Faiza et al. [39] reported use of potato peels for biosurfactant production by DGEF01-06 bacterial strains among which DGEF02 shows highest emulsification value of 70% while in current study conducted 55.1% emulsification was observed with 2% of potato peels waste. In this study, sugar cane molasses and waste frying oil individually indicated maximum E24 of up to 55.3 and 56.3%, ODA i-e 0.9 and 1.8 cm with SFT 41 and 38.2 mN/m. It was previously reported that use of waste frying oil as sole source of carbon and energy lipopeptide production by two Bacillus strains that reduce surface tension up to 36 mN/m these results are consistent with our study that Bacillus strain used produce surfactin while growing on 2% waste frying oil reduce surface tension up to 38 mN/m [40]. De Lima et al. [41] reported rhamnose production by Pseudomonas aeruginosa PACL strain cultivating on waste frying soybean oils results indicate biosurfactant production with 100% emulsification index, surface tension reduction up to 26.0 mN/m and concentration of 3.3 g/L while in current study 56.3% emulsification was observed with 2% waste frying oil. Molasses are co-product obtained from sugar beet and sugar cane industry that are widely used as substrate because of presence of vitamins and other valuable compounds in low cost. Molasses contains various compounds that includes sugars (sucrose 48-56%), non-sugar organic matter (9-12%), inorganic components, proteins and vitamins [42]. Research conducted by Abdel-Mawgoud et al. [43] investigate surfactin production in a cost effective manner with use of 16% molasses and other trace elements that produce surfactin yield of 1.12 g/L. However, it is also stated in many studies that the presence of hydrophobic substrate is essential for production of biosurfactants [44]. In present study, white beans powder and waste frying oil shows improved production of biosurfactant as compared to other substrates tested. Out of all media white beans powder and waste frying oil have good impact on biosurfactant production. Urea and ammonium nitrate have been already used and reported in literature as very cost-effective nitrogen source to produce biosurfactant by Artherobacter paraffineus and various other bacterial species [44]. Beans are considered as rich source of carbohydrates that were further used at different concentration with waste frying oil. Zhu et al. [45] reported use of soybean flour as substrate for surfactin production by Bacillus amyloliquefaciens XZ-173.
According to literature different types of oils e.g., vegetable oils, waste cooking oil, glycerol, glucose and diesel has been used as substrate for production of biosurfactant by a fungal species M. circinelloides 11.7 cm ODA showed maximum biosurfactant production by utilizing waste cooking oil as a carbon source [46] showed that 8% (v/v) WCO, biosurfactant production by M. circinelloides was maximum and lesser at higher concentration 10% (v/v) at 72 hours. It has also been reported in literature that Pseudomonas aeruoginosa undergoes productive yield of biosurfactant by using waste cooking oil. In present research white beans powder, waste frying oil and urea was used in different concentrations collectively Fig. 1c. Results obtained showed that the concentrations of white beans powder 6% with combination of waste frying oil 1.5 mL and urea 0.1 g showed significantly maximum surfactin yield indicated improved oil displacement value of (from 2.4 to 4.9 cm) maximum emulsification index (from 57 to 69.8%) and lowest surface tension reduction of (from 33.6 to 28.8 mN/m) Fig. 1c. In previous studies molasses and cheese whey were used in combination by four strains of Bacillus subtilis reduce the surface tension of medium up to 34 to 37 mN/m [47]. The results obtained in present study while using combination of white beans powder and waste frying oil were more significant lowers SFT up to 28.8 mN/m as compared to results described by Joshi et al., [47]. It was reported by Zhu et al. [48] that cell free supernatant based on glycerol produced by B. subtilis N3-4P, showed 27.8 mN/m surface tension and 38.3% emulsification index value in case of emulsifier not good results were obtained while comparing values with current study conducted. It’s conferred by Cooper and Goldenberg [23] that if surface tension value is reduced up to 40 mN/m or below it could be regarded as efficient biosurfactant producer. These reported results give indication about Bacillus subtilis SNW3 as efficient biosurfactant producer. Different environmental habitats like hydrocarbon contaminants, marine and terrestrial environment, reported Bacillus species and related genera as Aeribacillus sp., B. licheniformis and Bacillus subtilis etc. for biosurfactant production [49, 50]. However, until now no reports are showed by Bacillus nealsonii strains for biosurfactant production. Study conducted by Medeot et al. [51] showed high yield of biosurfactant (1.7 mg/mL) while using NH4NO3 and glucose as substrate for production by Bacillus amyloliquefaciens MEP218. In the same way, combination of sucrose and NH4NO3 were used by Fernandes et al. [52] and they reported high yield of biosurfactant (0. 2 g/L) by Bacillus subtilis RI4914. Likewise, study conducted for surfactin production by Abdel-Mawgound et al. [43] reported use of different carbon nitrogen sources and ultimate results showed maximum biosurfactant production by Bacillus subtilis BS5 while using NaNO3 and NH4NO3 as source of nitrogen. Obviously, source of nitrogen shows an important role for production of biosurfactant, but carbon/ nitrogen substrates combination has a crucial role in production.
For establishment of environmental parameters (temperature, pH, agitation and amount of inoculum) were checked by oil displacement activity that significantly influence surfactin production (Fig. 2). It was observed that 30 ˚C temperature was the most suitable temperature for maximum biosurfactant production by Bacillus subtilis SNW3 shown in Fig. 2a. At 30 ˚C the clear zone of about 1.26 cm was observed that was also reported by Bonilla et al. [53] in their research for maximum biosurfactant production. In study reported by Bertrand et al. [54] Bacillus mycoides and Bacillus brevis strains were used for maximum biosurfactant production at temperature that ranges between 35–40 °C. Though this temperature range is good for minimizing the production cost. According to Sahoo et al. [55] biosurfactant production by Pseudomonas aeruginosa OCD1 is more efficient at 30 °C temperature. Also, it was reported by Najafi et al. [56] that 30 °C is the optimum temperature for biosurfactant production. These results are in correspondence with results obtained by our research. For monitoring inoculum size maximum production rate was observed with 1% inoculum size that gives a clear zone of 2.1 cm shown in Fig. 2b. On the other hand, inoculum size of 2% and 2.5% showed a very low yield of biosurfactant with a zone size of only 1.3 cm and 0.7 cm. Inoculum size of 0.5% and 1.5% gives a zone of 1.6 cm and 1.9 cm of oil displacement zone. In current study at agitation 150 rpm biosurfactant production was observed maximum ODA 1.2 cm as compared to other tested agitation speed shown in Fig. 2c. At static condition, no significant biosurfactant production was observed as well-known importance of oxygenation for biosurfactant production, while with increase in rpm productivity of biosurfactant reduced that’s also reported for Candida lipolytica [57]. However, at pH 6 high values for ODA of 2.1 cm were observed regarding optimization of biosurfactant production. Results obtained showed that pH of culture media have significant effect on biosurfactant production (Fig. 2d). At acidic condition productivity of biosurfactant reduced indicates that bacterial growth is sensitive to acidic conditions. Same in previous studies at pH 7 Stenotrophomonas maltophilia NBS-11 shows maximum production of biosurfactant [58].
To study surfactin production on optimized media and its growth kinetics at optimized conditions (30 °C, 150 rpm, 1% inoculum size, pH 6, yeast extract (2%, w/v) and white beans powder, waste frying oil and urea (6:1.5:0.1%, w/v), in 1L shake flask fermentation setup revealed a growth-associated production (Fig.1d) under optimum conditions shows maximum surfactin production with surface tension reduction value 28.5 mN/m, ODA 5.53 cm emulsifying activity E24 70.6%, biomass 4.6 g/L, surfactin concentration of 1.17 g/L attaining preferable media position to replace costly yeast extract media.
Characterization of biosurfactant produced.
Crude biosurfactant produced by Bacillus subtilis SNW3 was analyzed by thin-layer chromatography (TLC) that indicates product nature as lipopeptide surfactin with retention factor (Rf) value of 0.68 through band observed on plate in comparison to standard surfactin as illustrated in Fig. 3. Likewise, Rf value of 0.76 was observed by [59] produced by Bacillus subtilis that indicates presence of surfactin. Similar results for Rf values were observed by [60] using Bacillus subtilis UMAF6619, UMAF6614, UMAF8561, UMAF6639 and Bacillus amyloliquefaciens PPCB004 for fengycin, iturin and surfactin as 0.9, 0.3 and 0.7 respectively. Similarly, Ramyabharathi and co-workers obtained results for surfactin and iturin production by Bacillus subtilis Bbv57 confirmed by TLC analysis showed Rf value for surfactin and iturin as 0.3 and 0.7 respectively making comparison with standard from Sigma-Aldrich [61]. In same way, Yánez-Mendizábal and co-authors showed Rf value of 0.3 for surfactin and 0.7 for iturin [62]. Similar TLC pattern was observed by Joy et al. [26] obtained 0.55 and 0.72 Rf values for lipopeptide nature of biosurfactants that was produced by Bacillus specie (SB2).
FTIR analyses of crude surfactin obtained showed presence of carboxylic functional groups and aliphatic amines that represent peptide bonds characteristic of lipopeptide biosurfactant nature. In current study Bacillus subtilis SNW3 showed various absorbance bands, characterized by aliphatic amines at 1023 cm-1 and 972 cm-1 in standard surfactin (Fig. 4a) and crude biosurfactant (Fig. 4b) respectively resulting in stretching vibrations of C-N bonds. Moreover, bands formation at 1045.92 and 862.03 cm−1 are associated with stretching vibrations that are observed for glycosidic linkage [63]. At 1243 cm-1 and 1240 cm-1 in standard and sample respectively, in range of 1250–1020 cm-1 indicates presence of C–N stretch aliphatic amines. Joshi et al. [47] also reported similar pattern of aliphatic and peptide moieties presence indicates lipopeptide biosurfactant nature. Peaks observed at 1453.40 and 1124.36 cm−1 suggest about stretching bands between carbon atoms and hydroxyl groups in sugar moiety structure [54]. The C=O stretch mode of 1762 cm-1 and 1757 cm-1 among standard surfactin and crude extract respectively ranging from 1690-1762 cm-1 corresponds to ester carbonyl group characterized as peptide component also reported by Joshi et al. [47]. Likewise, stretch at 1721 cm−1 that indicates presence of lactone carbonyl group observed by Faria [64] in biosurfactant product produced by Bacillus subtilis isolate LSFM-05. The C–H stretch at 2942 cm-1, standard surfactin (Fig. 4a) and 2925 cm-1 in crude extract (Fig. 4b) were analysed as alkanes. Another peak ranging from 3500–3200 cm-1 gave indication about alcohols and phenols O–H stretch, H–bonded presence. In current study absorption bands that are prominent obtained at 2925 cm-1, 1240 cm-1 and 1378 cm-1 indicates about (CH2 and CH3) alkyl and aliphatic chains presence in biosurfactant. Another peak observed between 3800 cm−1 and 3100 cm−1 shows about N–H and C–H stretch vibrations in sample. The above results obtained are also reported previously in literature that presence of peptides and aliphatic hydrocarbons gives indication about lipopeptide class of biosurfactants [64].
Critical micelle concentration (CMC) and critical micelle dilution (CMD) determination
The critical micelle concentration (CMC) is the minimum biosurfactant concentration needed to achieve lowest surface tension value after that point micellar aggregates formation starts [65]. The surfactin from Bacillus subtilis SNW3 showed reduction in surface tension of water from 70 to 36 mN/m by increasing surfactin concentration with CMC value of 0.58 mg/mL (Fig. 5a). After that point increase in surfactin concentration did not result in more reduction in water surface tension, gave indication that the CMC had been obtained. These results were efficient as compared to commonly used synthetic surfactants sodium dodecyl sulfate (SDS), attains CMC value at 2100 mg/L [66]. The CMC value for partially purified surfactin obtained in current study that was found to be 580 mg/L that was consistent with those previously reported for surfactin from Bacillus subtilis with CMC 200 and 1500 mg/L in cell free broth by using waste frying cooking oil as substrates reported by Oliveira and Garcia-Cruz, [67]. In present study CMC results were agreed with results obtained by Datta et al. [28] for biosurfactant produced by Bacillus subtilis MG495086. Estimation of surfactin concentration produced in medium could be done by critical micelle dilution. In current study surfactin produced seems to be more competent that remains stable with surface tension reduction values from 29 mN/m to 32 mN/m after making 3-fold dilutions shown in Fig. 5b.
Functional characterization of surfactin by antibiogram activity
The antibiogram of surfactin produced by Bacillus subtilis SNW3 and antibiotics used against multi drug resistant Escherichia coli is shown in Fig. 6. Appearance of clear zone around well was monitored, and diameter (mm) was calculated thrice to get mean value. It was observed that maximum inhibitory zone was with combined synergistic effect of surfactin with antibiotics used. Surfactin in combination with ciprofloxacin (Fig. 6a) and clarithromycin (Fig. 6b) displayed 30 mm inhibitory zone when used in combination in comparison to 27 mm for surfactin and 18 and 20 mm respectively when applied individually.
Stability Studies
After production of surfactin under optimum concentration, it was extracted in ethyl acetate and stability of surfactin was tested at different temperature, pH and salt concentrations that fluctuates depending on conditions. Surface tension reduction was used as an indicator to test stability of biosurfactant produced. Biosurfactant activity produced by Bacillus subtilis SNW3 was tested over various pH range (1, 3, 5, 7, 9, 11) pH affects the stability at very lower and higher values. From pH 1 to 3 and above 7 the activity of surfactin was low as shown in Fig. 7. SFT values were good from pH 5 to 7. However, the lowest surface tension value (28.3 mN/m) and achieved at pH 7. Decreased stability of surfactin at acidic pH could be due to precipitation of surfactin at lower pH. Surfactin produced by Bacillus subtilis SNW3 was thermostable up to 100 °C at different temperature ranges, but was most stable at 40 °C, so the surface tension of 28.9 mN/m. Similarly, it was reported by Moussa and Azeiz [68] that only minor variations occurred in biosurfactant stability that was produced by Bacillus methylothrophicus and Rhodococcus equi strains while analyzed at temperature ranged between 20–120 °C. It was noticed by Hatef and Khudeir [69] while doing experiments on biosurfactant stability check produced by Pseudomonas putida PS6 that that biosurfactant produced remains stable in temperature ranged between 20–70 °C, while after that with increase in temperature above 70 °C it starts to decrease in stability. Effect of salt concentration was observed by adding NaCl in different concentration (1, 2, 4, 6, 8 and 10%) into surfactin produced by all the three strains and best results were observed at 1-2% concentration of NaCl that shows lowest value of surface tension reduction at 30 mN/m while at higher salt concentration up to 8% decrease in stability of surfactin was observed. Reasons for decrease in stability of biosurfactant at increased NaCl concentration is due to ion-dipole interactions between salt and water which are stronger than interactions between salt and gaseous phase, that is avoid solute molecules to reach at interface to lowers surface tension. In a previous study conducted by Isty Adhitya Purwasena [70] biosurfactant produced shows good stability regarding emulsification at high temperature of 120 °C, pH of 4-10 and NaCl concentration of 10% (w/v) that are consistent with this study.
Effect of biosurfactant produced on seed germination and plant growth.
Percent germination
The analysis of the stimulation effect of surfactin on the seed germination ability was studied in the first step and then growth was studied. It was observed that in all seeds tested germinated better when applied with surfactin than in control. Solanum lycopersicum, Pisum sativum, Capsicum annuum and Lactuca sativa all showed good percent seed germination after seven days by increasing the concentration of surfactin (Fig. 9a, b). Better results were obtained at higher concentrations of surfactin than lower concentrations. All surfactin concentrations showed significant effect (P<0.05) on seed germination of tomato, chilli, pea and lettuce seeds. The germination of Solanum lycopersicum seeds was not significantly affected at lower concentrations of surfactin tested, however at higher concentration (0.7 g/100 mL) effect germination significantly. Among all seeds tested greatest stimulation was observed for Solanum lycopersicum seeds at 0.7 g/100 mL concentration with percent germination of 68.75% in comparison to control water at which shows germination of 56.25%. Among other seeds, there was also a significant difference in germination (P<0.05) Fig. 8a. Among surfactin concentrations that were tested for Capsicum annuum it was observed that at lowest (0.1 and 0.3 g/100 mL) did not affect germination significantly in comparison to control. However, at high concentrations of surfactin (0.5 and 0.7 g/100 mL) there was significantly difference in number of seed germination. In Capsicum annum seeds, surfactin concentration of 0.5 g/100 mL effect the germination percent of seeds 51.7% while compared with control MilliQ water that showed germination of 21.6%. In contrast, among all seeds surfactant resulted in increase in germination percent, the Capsicum annum seeds showed significantly increase in germination speed almost double with respect to control MilliQ and at lower concentration of 0.5 g/100mL water grouped seeds Fig. 8a. Almost all surfactin concentrations verified accelerated germination of Pisum sativum seeds, with highest stimulation observe with increase in surfactin concentration that is at 0.7 g/100 mL, and percent germination of seeds on average were 37.2% as compared to control water that shows 19.43% Fig. 8a. For Lacuta sativa, seed germination was significantly affected (P<0.05) to some extent equally at all concentrations of surfactin added and there was not much difference in germination as compared to control. The experimental data about effect on seed germination and plant growth shown in (Table. 1).
Germination of seed begins after entrance of water in seed through seed coat, that helps in activation of metabolic processes in seed. Permeability of embryonic tissues is the key factor for water entrance [71] when applied on external wrapping tissues facilitate germination process by increasing seeds permeability, as described by [72] for surfactants and in present study reason for chilli seeds germination almost double to control at lower concentration could be due to improved permeability. Small cracks in the cuticle of the palisade layer cause soybean cultivars to display fast wicking, which is the layer responsible for the permeability of water in this species [73]. Evolutionary strategies of this species may enhance the permeability of tissues, therefore did not affect soybean germination. Diffusion of released nutrients at suitable rates is carried out by liquid‐filled intercellular spaces in the seed coat as observed in current study among all species with addition of biosurfactant germination increase as compared to control could be due to more nutrients diffusion. Penetration of rhamnolipids in soybean and sunflower seeds, help to mobilize oleaginous reserve tissue from these species, consequently supporting seedling development. Biopreparations are widely used in enhancement of seed quality these days. Application of biosurfactant to improve plant germination is mostly done on contaminated soil. Some of biopreparation are used as nutrients for plants and helps in germination [74]. Plants enrichment with nitrogen and stimulation of plants height are benefits of Azotobacter sp. Genus in planting crop seeds or for vaccination of roots seedling fertilizers. Increase in germination proportion for pea seeds was observed after treatment with surfactin. This synchrony of germination plays a key role in farming, at harvest time it reduces cost and optimize the producer work because plants were present at same development stage [75]. It was observed in this study that development of seeds varies and depends on cultivars type and concentration of surfactin used. Positive impact of surfactin use on seed germination and development of seedling aids in farming practices at sites that goes through bioremediation practices.
Dry biomass
After germination growth was second parameter to be observed. When evaluating the dry biomass of L. sativa and C. annum seedling it was observed that increase in surfactin concentration (0.5 and 0.7 g/100 mL) did not favoured greater accumulation of dry biomass while there was increase in biomass relative to control indicates mean weight of 0.24 g and 0.20 g at 0.7 g/100 mL in comparison to control seedling of 0.055 g and 0.058 g respectively. Although a positive effect was noted for Pisum sativum seedling, addition of surfactin significantly increase (P<0.05) dry biomass of 2.21 g at 0.7 g/100 mL in relative to control MilliQ water Fig. 8b. In contrast while analysing Solanum lycopersicum seedling dry weight subjected to surfactin showed slight difference in values at all concentrations tested while there was increase in seedling dry biomass of 0.19 g at 0.7 g/100 mL relative to control group 0.078 g was observed. Positive impact of surfactin on seedling germination was observed that indirectly also increase biomass of seedling analysed after treatments. As for rhamnolipids, the hypothesis is that lipopeptides have the ability to create some disturbance in the plant plasma membrane and could consequently activate a cascade of molecular events leading to the activation of defence mechanisms [76].
Root length
Almost all surfactin concentrations tested influenced root elongation. Surfactin concentration and root development are directly related, increase in surfactin concentration results in better development of root length. It was observed that higher concentration 0.7 g/100 mL of surfactin enhance root growth at maximum. Surfactin effect root elongation more in P. sativum and L. sativa than other plant seeds tested. The root length of Lactuca sativa seeds that were stimulated with surfactin treatment showed somewhat increase with initial minimum concentration of surfactin while greater development of root length of 2.74 cm at 0.7 g/100mL. In contrast while Pisum sativum seeds subjected to surfactin treatment the development of roots length increases in uniformly with increase in concentration showed 2.89 cm at 0.7 g/100 mL relative to control. However, for Capsicum annum seeds it was noticed that at high concentration (0.3,0.5,0.7 g/100 mL) had significant effect (P<0.05) on elongation of root gave 1.87 cm at 0.7 g/100 mL in comparison to control group seeds mean root length of 1 cm. The use of surfactin for Solanum lycopersicum also increase root development seen with increase in surfactin concentration added Fig. 8c. Resistance of seed coating is main factor that limits the root axis extension for development of roots thus applying surfactin helps in decreasing strength of wrapping tissues that favour characterisation of germinate on through protrusion of radical [71]. While considering root length of lettuce seeds after application of surfactin at different concentrations it was observed that before root region leaf portion emerged therefore less growth was observed for root as compared to other seeds. This atypical leaf emergence before roots development is possibly due to more reduction of resistance in leaf axis region of seed coat.
Height of plant
Surfactant stimulation affects height of seedlings in comparison to control specimen (Fig. 9c). The stimulation impact on height of C. annum and P. sativum plants was revealed more and differ significantly (P<0.05) with increase in surfactin concentration as compared to control. The average height of C. annum and P. sativum plant increase 8.06 mm and 12.66 mm respectively at 0.7 g/100 mL relative to control. When L. sativa plants were checked for height revealed at lower concentration of surfactin (0.1 g and 0.3 g/100 mL) application was not much significant but at higher concentrations (0.5 and 0.7 g/100 mL) showed significant (P<0.05) increase as compared to control Fig. 8d. All results showed that seed germination is highly affected by adding various concentrations of surfactin and are significantly different relative to control, but plant growth was comparatively less by adding surfactin. It was demonstrated by these results that applying surfactin shows positive effect for plants tested and could be used in future for growing such species to replace use of synthetic surfactant. Research done for biosurfactant impact on growth of plant is not more. It is believed there is indirectly promotion of plant growth through microbial surfactants by increasing hydrophobic compounds bioavailability to microbes living in region of rhizosphere [25]. In a recent study conducted surfactin produced by Bacillus isolates studied about ISR (induced systemic resistance) inducer where a strong relationship was analyzed between concentration of surfactin use and induction of defence activity among plant that indicates that with increase in concentration of surfactin introduce to plant also increase in systemic resistance [77]. In this study at higher concentration from 0.5 g/100 mL to 0.7 g/100 mL of surfactin added there is a weaker stimulation in plant growth that may be due to increase in hydrophobic compounds present in environment, which makes difficult for rhizosphere microorganisms to assimilate all these compounds or through release of some inhibitory compounds from soil that results in growth inhibition of these microorganisms. About research for biosurfactants effect on plants development examine about heavy metals and hydrocarbon polluted environments. Our study creates opportunity for use of these biological surfactants for plant growth promotion in agricultural field in cost effective and environment friendly manner. However, some research gaps are still required to be filled for explanation of mechanism that shows effect of biological surfactants on plants growth and development. Biosurfactant induced different mechanism recommended for plant growth promotion like reduction in seed microflora [78] incidence increase in IAA phytohormones production [79] and increase in amylase activity [80] that helps in improvement of plant growth better option for development in agricultural field.
Table 1: Statistical Mean (M), Std. Deviation (SD), Std. Error (SE) and P value for relative seed germination, dry biomass, root length and plant height at four different concentrations of biosurfactant produced by Bacillus subtilis SNW3 used for four different plant species.
|
|
Control
|
0.1g
|
0.3g
|
0.5g
|
0.7g
|
|
|
M
|
SD
|
SE
|
P
|
M
|
SD
|
SE
|
P
|
M
|
SD
|
SE
|
P
|
M
|
SD
|
SE
|
P
|
M
|
SD
|
SE
|
p
|
Root length
|
Chilli
|
1.01
|
.43
|
.13
|
.00
|
.93
|
.32
|
.08
|
.00
|
1.72
|
.49
|
.12
|
.00
|
1.87
|
.45
|
.08
|
.00
|
1.87
|
.53
|
.09
|
.00
|
Tomato
|
2.15
|
.46
|
.08
|
|
2.53
|
.38
|
.07
|
|
2.67
|
.42
|
.074
|
|
2.86
|
.36
|
.06
|
|
3.08
|
.51
|
.08
|
|
Pea
|
1.56
|
1.05
|
.26
|
|
2.15
|
.42
|
.13
|
|
2.45
|
.36
|
.11
|
|
2.59
|
.49
|
.12
|
|
2.89
|
.53
|
.14
|
|
Lettuce
|
1.50
|
.29
|
.51
|
|
1.81
|
.23
|
.04
|
|
1.91
|
.34
|
.06
|
|
2.39
|
.34
|
.06
|
|
2.74
|
.46
|
.08
|
|
% Germination
|
Chilli
|
21.6
|
15.47
|
5.85
|
.00
|
28.21
|
17.31
|
6.54
|
.02
|
26.07
|
18.65
|
7.05
|
.00
|
51.79
|
28.16
|
10.64
|
.03
|
48.21
|
27.72
|
10.48
|
.02
|
Tomato
|
56.25
|
24.69
|
10.08
|
|
55.83
|
24.88
|
10.16
|
|
62.50
|
24.19
|
9.87
|
|
62.92
|
26.00
|
10.61
|
|
68.75
|
27.92
|
11.39
|
|
Pea
|
19.43
|
13.25
|
5.49
|
|
24.40
|
15.84
|
6.47
|
|
29.42
|
13.42
|
5.48
|
|
32.75
|
22.34
|
9.12
|
|
37.20
|
16.39
|
6.69
|
|
Lettuce
|
20.50
|
12.93
|
5.28
|
|
22.50
|
13.11
|
5.35
|
|
25.83
|
12.82
|
5.23
|
|
24.00
|
11.69
|
4.77
|
|
26.50
|
12.18
|
4.97
|
|
Plant height
|
Chilli
|
5.35
|
.61
|
.16
|
.00
|
5.27
|
.94
|
.25
|
.00
|
5.99
|
.54
|
.13
|
.00
|
8.08
|
.39
|
.07
|
.00
|
8.06
|
.39
|
.07
|
.00
|
Tomato
|
5.97
|
.66
|
.12
|
|
6.37
|
.50
|
.09
|
|
6.92
|
.43
|
.08
|
|
7.28
|
.61
|
.10
|
|
7.65
|
.96
|
.16
|
|
Pea
|
10.62
|
.32
|
.12
|
|
11.22
|
.24
|
.07
|
|
11.54
|
.39
|
.11
|
|
12.58
|
.39
|
.09
|
|
12.65
|
.48
|
.12
|
|
Lettuce
|
5.26
|
.61
|
.12
|
|
5.58
|
.40
|
.07
|
|
5.73
|
.37
|
.06
|
|
6.00
|
.29
|
.05
|
|
6.22
|
.37
|
.06
|
|
Dry biomass
|
Chilli
|
.06
|
.02
|
.01
|
.00
|
.07
|
.03
|
.01
|
.00
|
.14
|
.03
|
.01
|
.00
|
.20
|
.03
|
.00
|
.00
|
.21
|
.03
|
.01
|
.00
|
Tomato
|
.08
|
.03
|
.00
|
|
.11
|
.02
|
.00
|
|
.13
|
.02
|
.01
|
|
.16
|
.03
|
.005
|
|
.19
|
.03
|
.006
|
|
Pea
|
1.52
|
.19
|
.06
|
|
1.68
|
.18
|
.06
|
|
1.98
|
.23
|
.07
|
|
2.02
|
.26
|
.07
|
|
2.21
|
.22
|
.06
|
|
Lettuce
|
.06
|
.03
|
.004
|
|
.06
|
.02
|
.004
|
|
.15
|
.04
|
.006
|
|
.23
|
.06
|
.01
|
|
.25
|
.12
|
.02
|
|