Bioresource Technology 98 (2007) 447–451
Short Communication
EVect of free and encapsulated Pseudomonas putida CC-FR2-4 and Bacillus subtilis CC-pg104 on plant growth under gnotobiotic conditions P.D. Rekha, Wai-An Lai, A.B. Arun, Chiu-Chung Young
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Department of Soil and Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung 40277, Taiwan, ROC Received 30 August 2005; received in revised form 12 January 2006; accepted 17 January 2006 Available online 3 March 2006
Abstract A study was performed to investigate the eYciency of microbial inoculants after encapsulating in alginate supplemented with humic acid on plant growth. Two promising plant growth promoting rhizobacteria were identiWed by 16S rDNA sequencing as Pseudomonas putida CC-FR2-4 and Bacillus subtilis CC-pg104, which were further characterized by biochemical analyses and inoculated to Lectuca sativa L. seedlings as free cells and entrapped in beads. SigniWcant increase in shoot height after 21 days of growth was observed with encapsulated CC-pg104 inoculated plants. Highest increase in root length was observed with CC-pg104 free-cell inoculated plants, followed by plants inoculated with encapsulated CC-pg104. Results clearly demonstrated that inoculation of the encapsulated bacterial isolates promoted plant growth similar to their respective free cells and could be a novel and feasible technique for application in agricultural industry. © 2006 Elsevier Ltd. All rights reserved. Keywords: PGPB; Encapsulation; Gnotobiotic; Root colonization; Alginate; Humic acid
1. Introduction Microbial co-operation in the rhizosphere is the key factor in the maintenance of soil fertility and plant establishment. Soil microbial populations are involved in fundamental activities that ensure the stability and productivity of both agricultural systems and natural systems (Barea et al., 2005). It is well established that introduction of plant growth promoting bacteria (PGPB) to soil improves the plant growth. PGPB promote plant growth directly through the production of plant hormones (Patten and Glick, 2002; Bottini et al., 2004). Symbiotic and nonsymbiotic, free-living soil bacteria promote plant growth by interfering in the soil processes which improves the supply of available nutrients to the plant (Glick, 1995).
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Corresponding author. Tel./fax: +886 4 22861495. E-mail address:
[email protected] (C.-C. Young).
0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.01.009
Microbial survival following introduction to natural soils depends on both abiotic and biotic factors (Van Veen et al., 1997). The population of the inoculated bacteria declines progressively preventing the buildup of a suYciently large PGPB population in rhizosphere (Bashan, 1998). In the soil, the survival of the inoculated bacteria largely depends on the availability of the empty niche, withstand competition with the often better-adapted native microXora and predation by protozoans or by other microinvertebrates. A major role of inoculant formulation is to provide more suitable microenvironment for the prolonged survival in the soil. Inoculum strategies should include application of carrier materials aimed at providing protective niche together with the provision of nutrient sources. It is opined that the encapsulation method helps to increase the survival rate and easy delivery of bacterial cultures. It also helps in segregating the bacterial cells from adverse environment thereby reducing cell loss. Advantages of microencapsulation of PGPB are described extensively by Cassidy et al. (1996).
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To get maximum beneWts of inoculation, the selection of most eVective strains is a prerequisite. Several PGPB isolates from our laboratory collection showed remarkable plant growth promoting traits. Thus, to overcome the various constraints in developing a feasible technology for bacterial inoculum, the present study was designed to develop a cost eVective and mild protocol for the delivery of PGPB and involved the screening of two PGPB strains, their biochemical traits associated with plant growth regulation, importance of inoculum preparation and delivery to enhance the plant growth under gnotobiotic conditions. 2. Methods 2.1. Bacterial isolates CC-pg104 strain was isolated from a compost sample and CC-FR2-4 was isolated from the rhizosphere of Ficus religiosa L. tree on nutrient agar. Both the strains showed increased germination rate in seed germination bioassay using lettuce seeds. The isolates were identiWed based on 16S rDNA sequencing by the methods described earlier (Young et al., 2005). 2.2. Biochemical characterization and identiWcation of PGPB In vitro auxin production by the isolates was studied by a method described by Patten and Glick (2002). Detection of siderophore production was carried out in chrome azurol (CAS) agar plates (Milagres et al., 1999). Mineral phosphate solubilization (MPS) activity of the isolates was detected on tricalcium phosphate agar (Nautiyal, 1999). Nitrogen Wxing activity was assessed on nitrogen free solid plate in addition to acetylene reduction assay (ARA) (Rennie, 1981). Cellulase, protease, amylase, and pectinase were determined on solid agar plate method as outlined in Shaw et al. (1995), Plazinski and Rolfe (1985), Magnelli et al. (1997), respectively. Enzyme activities of isolates were characterized with API-ZYM-test strips (bioMerieux) according to the manufactures instructions. 2.3. Encapsulation of isolates For encapsulation, both bacterial isolates were grown in 100 ml of nutrient medium (HIMEDIA, India) for 72 h at 30 °C. The cells were harvested at log phase (108 cfu¡1 ml) by centrifugation (4 °C at 5000g). Cell pellet was mixed with 2 ml of 1% alkaline humic acid (extracted from peat as described in Young et al., 2004) and 8 ml of 2% aqueous solution (w/v) reagent grade sodium alginate (KISHIDA Chemical Reagents, Japan) and vortexed for uniform dispersion of the cells in the polymer mixture. This mixture was extruded from sterile syringe (26 G) into 1.5% CaCl2 solution with mild stirring. Instantaneously formed beads were cured at the room temperature for 6 h and were washed in sterile normal saline solution. The beads were
designated as AH-CC-FR2-4 and AH-CC-pg104. Ten beads were solubilized for cell counts in 0.2 M phosphate buVered saline (PBS) pH 7.0. 2.4. EVect of encapsulated and free cells of CC-pg104 and CC-FR2-4 on plant growth and root colonization A quartz sand jar system as described by Simons et al. (1996) was employed to evaluate the growth promotion and root colonization using lettuce plant as a model system. Lettuce seeds were surface sterilized with 0.05% NaOCl for 30 min, rinsed 5 times in sterile water and germinated on the double layered wet Wlter paper sheets (Whatman No. 1). Four diVerent types of treatments in quadruplets (in 3 sets for weekly interval samplings) were designed as follows; two treatment regimes contained, 1 ml of CC-pg104 free cells suspension (2 £ 108 cells ml¡1) and CC-FR2-4 free cell suspension (2 £ 108 cfu ml¡1) while, the other two treatments included, 100 mg beads each of two bacterial strains. Beads were carefully placed below the surface of quartz sand. Moisture content of the jar was maintained at 50%. Three pre-germinated seeds were planted in each tube and were trimmed after 5 days to retain a single plant. Five milliliter of sterilized Hoagland solution (Hogland and Arnon, 1950) provided nutrients to the plants in the jar. A control received all the above treatments with an exception of bacterial inoculant in any form. Jars were arranged randomly in growth chamber maintained at 28 °C with 12 h photoperiod. At every 7 days interval after inoculation, plant and adhering sand was carefully removed from the jars. Root colonization by the introduced bacteria was studied as outlined in Matthysse and McMahan (1998). For the measurement of root length, fresh roots were aseptically cut into 10 mm pieces and spread at random in a tray marked with gridlines and was calculated as described by Newman (1966). 2.5. Statistical analyses One-factor analysis of variance (ANOVA) followed by student t-test (Post-hoc analysis) (Stat Soft, Inc., 1995) was performed to assess the diVerence between the test and control treatments in the shoot length and root length. 3. Results 3.1. Biochemical characteristics of bacterial strains CC-pg 104 and CC-FR2-4 The isolate CC-pg104 was identiWed as Bacillus subtilis (99% identity) and CC-FR2-4 as Pseudomonas putida (98% identity) by 16S rDNA sequencing and was submitted to NCBI Gen Bank (CC-pg104 as AY149235 and CC-FR2-4 as DQ193603). Both the isolates presented traits that were favorable for plant growth (Table 1). CC-FR2-4 showed comparatively better enzyme activities than CC-pg104.
P.D. Rekha et al. / Bioresource Technology 98 (2007) 447–451 Table 1 Selected biochemical and physiological traits of two PGPB strains used in this study Pseudomonas putida (CC-FR2-4)
Ca-P solubilization Protease Amylase Cellulase Pectinase N2 Wxation C2H2(m/h¡1) Alkaline phosphatase Acid phosphatase Esterase Esterase lipase Lipase Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin -chymotrypsin Napthol-AS-BI phosphohydrolase -glucosidase -glucosidase IAA production (g/ml) Siderophore production
+ + + + + 31 + + + + ¡ ¡ ¡ ¡ ¡ ¡ +
+ + + + + 30 + + + + + + + + + + +
+ + 4.0 +
¡ ¡ 42.8 +
CC-FR2-4 CC-pg104 Control
120
Shoot length (mm)
Bacillus subtilis (CC-pg104)
140 AH-CC-FR2-4 AH-CC-pg104
100 80 60 40 20
(a)
0 7 days
140
14 days CC-FR2-4 CC-pg104 Control
120
Root length (mm)
Biochemical characteristics
449
21 days AH-CC-FR2-4 AH-CC-pg104
100 80 60 40 20
+, positive activity; ¡, negative activity. 0 7 days
3.2. Characteristic of the beads Average diameter of the alginate beads ranged between 1.8 and 2.2 mm with an average weight of single fresh bead as 9.74 and 9.33 mg and initial cell densities as 7 £ 108 and 3 £ 109 cfu g¡1 for AH-CC-pg104 and AH-CC-FR2-4, respectively. 3.3. Jar experiments for the assessment of plant growth promotion Plant growth promotion was evidenced by the increased shoot and root length under diVerent treatments compared to the control treatment. Free-cells inoculated plants showed signiWcant (p < 0.001) increase in the shoot length compared to the control after 7 days of inoculation (Fig. 1a). The CC-pg104 and CC-FR2-4 free-cells inoculated plants showed 381% and 248% increase in the shoot length than the control, respectively. Similarly, plants inoculated with AH-CC-FR2-4 showed a signiWcant (p < 0.05) increase of 95% in the shoot length while, AH-CC-pg104 inoculated plants showed only 57% increase compared to control plants. A diVerent trend was observed in the growth of free and encapsulated cells treated plants after 14 days wherein, AH-CC-FR2-4 inoculated plants showed maximum growth (369% of control) which was signiWcantly higher (p < 0.01) than its respective free-cell inoculated plants (267% of control). Eventhough, the CC-pg104 inoculated plants showed comparatively lower growth promotion (in terms of shoot height) than the CC-FR2-4 treated plants; there was a signiWcant (p < 0.001) increase in shoot
(b)
14 days
21 days
Growth after germination
Fig. 1. (a, b) Shoot and root growth of Lactuca sativa (L.) plants inoculated with CC-FR2-4 and CC-pg104 free cells and encapsulated AH-CCFR2-4 and AH-CC-pg104.
height compared to the control. Response of the plants to CC-FR2-4 and CC-pg104 inoculation at the end of 21 days of growth was contrary to the 7 and 14 days results. CCFR2-4 free-cell inoculated plants did not show any signiWcant increase in shoot length compared to the control. There was 10% increase in shoot length of AH-CC-FR2-4 inoculated plants. But interestingly, CC-pg104 free cells and AH-CC-pg104 inoculated plants showed highly signiWcant (p < 0.001) increase of 29% and 65% more than the control plants. Root development was also positively responded to the PGPB inoculation. Initial root growth (7 days) was signiWcantly (p < 0.01) higher in plants inoculated with CC-pg104, CC-FR2-4 and AH-CC-pg104 (Fig. 1b). After 14 days of growth, signiWcant (p < 0.001) increase in the root lengths of all the bacterial inoculated plants was observed. Maximum increase (473% of control) in root lengths was evidenced in the plants treated with AH-CC-FR2-4, followed by CCFR2-4 (424% of control). AH-CC-pg104 and CC-pg104 treated plants showed 177% and 66% increment, respectively in the root length. The signiWcant (p < 0.001) increase in the root length was also observed after 21 days with CCpg104 (207% of control) and its respective encapsulated cell inoculated plants (176% of control). While, no signiWcant increase was observed between the CC-FR2-4 inoculated and control plants, but, 26% increase in root length was
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observed in the AH-CC-FR2-4 inoculated plants compared to control plants. The diVerence between CC-pg104 and AH-CC-pg104 inoculated plants was signiWcant only at p < 0.05 level. Results of root colonization studies showed the presence of inoculated bacteria in the rhizosphere, rhizoplane and root interior in all free-cell and bead inoculated root systems. 4. Discussion Immobilized microorganisms improve the productivity of bioreactors and provide several advantages over free cells in industrial fermentation, environmental and agricultural applications (Park and Chang, 2000). Calcium alginate gelled by ionic bond swells and dissolves in a solution containing chelating agents such as phosphate, which is suitable for the soil applications (Vassilev et al., 2001a,b). For the successful survival of the encapsulated microbial cells the choice of polymer is very important. Although a variety of biomaterials are available alginate was preferred as the microbial cells density in alginate beads can be maintained as high as 1010–1011 cfu g¡1 of bead. The method being mild and the beads are formed instantaneously by one step gelling, the cell loss can be reduced (Chang et al., 1996). Survival of the microbes within the beads is dependent on the enrichment of beads with nutrients. The choice of humic acid was made to serve as a carbon source to the encapsulated bacteria. This might also helped in survival of bacteria in beads upon storage as evidenced by little or minimum cell loss for a storage period of 6 months (data not presented). Additionally, initial colonization of the released bacteria following the bead inoculation might be beneWted by the humic acid supplement. It has been well documented that, humic acid when applied to the soil serves as the plant nutrient and aid in early plant growth (Young and Chen, 1997). It also serves as a source of nitrogen (Tomonori and Akira, 2004). Recently, an interesting phenomenon of nitrogen Wxation by humic acid under laboratory conditions was also proposed (Young et al., 2004). Other advantage of humic acid includes, darkening of beads which merges with the soil background, being a complex molecule might also form chemical bonding with alginate giving more strength and stability and, is economically viable. Plant growth promotion by PGPB is well documented under laboratory and Weld conditions (Wu et al., 2005; Bottini et al., 2004) while, only a few reports on advantages of encapsulated PGPB inoculation are available. Vassilev et al. (2001a) recorded tomato plant growth promotion after inoculation with free and encapsulated yeast and arbuscular mycorrhizal fungus. The two isolates, B. subtilis CC-pg104 and P. putida CCFR2-4 in the present study showed remarkable plant growth promotion with the former being better than the latter. Encapsulation did not inhibit the beneWcial eVects of these PGPB isolates. Eventhough initial plant growth was vigorous with free-cell inoculation it was stabilized after
14 days of growth. In contrast, the inoculation with encapsulated cells resulted in an initial lag in the growth which after 14 days showed remarkable increase. This may be due to the slow and controlled release of the cells from the beads. Such controlled release will help in the long-term survival and establishment of the inoculated PGPB in the soil. For enhancing the initial eVects on plant growth the beads can be forced to release the cells by mechanical crushing, while, the intact beads can serve for slow release. The free cells though showed excellent results under gnotobiotic conditions their survival following inoculation to soil is more critical than the encapsulated cells. There is a worldwide demand for biofertilizers and organic fertilizers to reduce the chemical fertilizers input to achieve environmental sustainability. Isolating, identifying, and characterizing the free-living rhizobacteria helps in the selection of potential candidates as plant inoculants. This study demonstrates the feasible technology of inoculum formulation for soil applications. The eVectiveness of these encapsulated PGPB strains are being tested presently in the greenhouse and Weld conditions. Acknowledgements This research work was kindly supported by grants from the National Science Council of Taiwan, ROC and Council of Agriculture, Executive Yuan, Taiwan, ROC. We thank F.-T. Shen and W.-S. Huang for their assistance in identiWcation of isolates. References Barea, J.M., Pozo, M.J., Azcon, R., Azcon-Aguilar, C., 2005. Microbial cooperation in rhizosphere. J. Exp. Botany 56, 1761–1778. Bashan, Y., 1998. Inoculants of plant growth-promoting bacteria for use in agriculture. Biotechnol. Adv. 4, 729–770. Bottini, R., Cassan, F., Piccoli, P., 2004. Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase. Appl. Microbiol. Biotechnol. 65, 497–503. Cassidy, M.B., Lee, H., Trevors, J.T., 1996. Environmental application of immobilized cells. J. Ind. Microbiol. 16, 79–101. Chang, H.N., Seong, G.H., Yoo, I.K., Park, J.K., Seo, J.H., 1996. Microencapsulation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid core alginate capsules. Biotechnol. Bioeng. 51, 157–162. Glick, B.R., 1995. The enhancement of plant growth by free-living bacteria. Can. J. Microbiol. 41, 109–117. Hogland, D.R., Arnon, D. 1950. The water culture method for growing plants without soil. California Agriculture Experiment station circular No. 347, pp. 39. Magnelli, P.E., Martinez, A., Mercuri, O.A., 1997. Simple method for determining cellulolytic activity in fungi. Rev. Arg. Microbiol. 29, 210– 214. Matthysse, G.A., McMahan, S., 1998. Root colonization by Agrobacterium tumefaciens is reduced in cel, attB, attD and attR mutants. Appl. Environ. Microbiol. 64, 2341–2345. Milagres, A.F.M., Machuca, A., Napoleao, D., 1999. Detection of siderophore production from several fungi and bacterial by a modiWcation of chrome azurol S (CAS) agar plate assay. J. Microbiol. Methods. 37, 1–6. Nautiyal, C.S., 1999. An eYcient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265–270.
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