Screening of freeze-dried protective agents for the formulation of biocontrol strains, Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40

Authors

  • Y. Zhan,

    1.  Department of Plant Pathology, Engineering Center of Bioresource Pesticide in Jiangsu Province, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
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    • These authors contributed equally to this study and are regarded as joint first authors.

  • Q. Xu,

    1.  Department of Plant Pathology, Engineering Center of Bioresource Pesticide in Jiangsu Province, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
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    • These authors contributed equally to this study and are regarded as joint first authors.

  • M.-M. Yang,

    1.  Department of Plant Pathology, Engineering Center of Bioresource Pesticide in Jiangsu Province, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
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  • H.-T. Yang,

    1.  Biology Institute of Shandong Academy of Sciences, Jinan, China
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  • H.-X. Liu,

    1.  Department of Plant Pathology, Engineering Center of Bioresource Pesticide in Jiangsu Province, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
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  • Y.-P. Wang,

    1.  Department of Plant Pathology, Engineering Center of Bioresource Pesticide in Jiangsu Province, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
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  • J.-H. Guo

    1.  Department of Plant Pathology, Engineering Center of Bioresource Pesticide in Jiangsu Province, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
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Y.-P. Wang, Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Engineering Center of Bioresource Pesticide in Jiangsu Province, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing 210095, China. E-mail: ypwang@njau.edu.cn

Abstract

Aims:  The effects of different freeze-drying protective agents on the viabilities of biocontrol strains Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 were investigated.

Method and Results:  Several concentrations of protective and rehydration media were tested to improve the survival of biocontrol agents after freeze-drying. The subsequent survival rates during storage and rehydration media of freeze-dried biocontrol strains were also examined.

Conclusions:  The results indicated that cellobiose (5%) and d-galactose (5%) gave maximum viability of strains Bu. vietnamiensis B418 and P. agglomerans 2Re40 (98 and 54·3% respectively) while the perfect one (100%) of strain B. cereus AR156 was obtained with sucrose (5%) during freeze-drying, and the highest survival of the three strains was reached when they were rehydrated with 10% nonfat skim milk. In the following storage, the survival rates showed that B. cereus AR156 could still reach 50% after 12 months.

Significance and Impact of the study:  This study showed that freeze-drying could be used to stabilize cells of these three biocontrol strains. Further studies should focus on the scale-up possibilities and formulation development.

Introduction

Biological control using microbial antagonists has attracted much interest as an alternative to chemical methods of controlling pre- and postharvest plant pathogens (Janisiewicz 1988). An acceptable microbial formulation should be easy to distribute to the intended environment, inexpensive to produce, and has a long shelf-life (Melin et al. 2007). Compared with a liquid formulated product, the dry-powder formulation has the advantage on packaging, transportation and storage. Drying of micro-organisms can be accomplished by a number of methods including freeze-drying, fluidized-bed drying, vacuum drying and spray-drying. Freeze-drying is the most convenient and successful method of preserving bacteria, yeasts and sporulating fungi (Larena et al. 2003) and has been frequently used to preserve lactic acid bacterial starter cultures involved in dairy and food fermentations (Kearney et al. 1990). And the advantages of freeze-dried formulation are protection against contamination and infestation during storage of biocontrol agents, long viability, ease of strain distribution and easily reconstituted by rehydration in water (Tang and Pikal 2004). However, the high cost of product manufactures the major obstacles for large-scale application.

Not all strains survive the freeze-drying process, and among those surviving, quantitative viability rates as low as 0·1% have been reported (Miyamoto-Shinohara et al. 2006). A good freeze-drying protective agent (FDPA) should provide the cryoprotection to the cells during the freezing process, also a good matrix to improve stability in storage and ease of rehydration. Various groups of substances have been explored to tests for their protective abilities, including polyols, polysaccharides, disaccharides, monosaccharide, amino acids, protein hydrolysates, proteins, minerals, salts of organic acids and vitamins-complex media (Berny and Hennebert 1991; Martos et al. 2007). However, not all these substances work well, and the protection afforded by a FDPA can vary largely depending on the species of micro-organisms (Palmfeldt et al. 2003).

Rehydration of freeze-dried cells is a critical step in the recovery of freeze-dried micro-organisms. Microbial cells subjected to injury during the freeze-drying may not be able to repair damage if they are rehydrated under inappropriate conditions (Carvalho et al. 2004).

Bacillus cereus strain AR156 and Pantoea agglomerans 2Re40 are biocontrol agents used against root-knot nematode caused by Meloidogyne incognita (Kofoid & White) and rice disease caused by the fungal pathogen Rhizoctonia solani and Magnaporthe grisea in our laboratory (Yang et al. 2008). Burkholderia vietnamiensis strain B418 is a biocontrol agent against wheat disease caused by the fungal pathogen Rhizoctonia cerealis (Yang et al. 2007). This study was designed to investigate the protection efficiency of different FDPAs and rehydration media on the viability of three biocontrol agents, B. cereus AR156, Bu. vietnamienssi B418 and P. agglomerans 2Re40, after freeze-drying and during storage

Materials and methods

Bacterial strains and cell preparation

The B. cereus strain AR156 used in this study was isolated from the rhizosphere of tomato plants in Nanjing, China, and the P. agglomerans 2Re40 was isolated from phyllosphere of rice in Zhenjiang, China, which were identified based on 16S rDNA sequences (Yang et al. 2008). The Bu. vietnamiensis strain B418 was obtained from the Biology Institute of Shandong Academy of Sciences in Shandong Province of China.

The three biocontrol strains were all cultured with 250-ml Luria-Bertani (LB) medium in a 1-l conical flask at 30°C and 200 rev min−1. Cells were harvested at the beginning of the stationary phase (24 h) by centrifugation at 5000 g for 5 min at 20°C in an Avanti-TM J-25I centrifuge (Beckman, Palo Alto, CA, USA). The cell paste was resuspended in 500-ml sterile distilled water and dispersed into the protective medium later.

Freeze-drying and storage

The three bacterial suspensions were distributed (400 μl) in 1·5-ml Eppendorf tubes and placed at −20°C for 24 h. After storage in the freezer, samples were desiccated in a freeze-dryer (Thermo Fisher Scientific, Inc., Asheville, NC, USA) at a condenser temperature of −45°C for 48 h (pressure < 1 Pa) (Carvalho et al. 2003; Schoug et al. 2006; Pehkonen et al. 2008). Another 40-ml sample of each bacterial strain was deposited in 50-ml vials, freeze-dried and stored at 25°C with 30% of humidity to test the survival periodically.

Rehydration

After freeze-drying, the samples were resuspended to the volume before freeze-dried (400 μl) with different rehydration media. After 10 min on the bench, the serial dilution technique was employed to examine the colony-forming units on LB agar plates. The survival rate was calculated using the ratio between the colony forming units (CFU) of viable cells after freeze-drying and the CFU of viable cells before freeze-drying (Hamoudi et al. 2007).

Freeze-drying protective agent used in assay

Solutions of the FDPA were prepared with distilled water. The FDPA used in this study were (w/v): (i) 10, 5 and 1% glucose; (ii) 10, 5 and 1% fructose; (iii) 10, 5 and 1%d-galactose; (iv) 10, 5 and 1% sucrose; (v) 4, 2 and 1% trehalose; (vi) 10, 5 and 1% cellobiose; (vii) 0·5, 0·25 and 0·05 mol l−1l-glutamic acid; (viii) 2, 1 and 0·5% soluble starch; (ix) 0·5, 0·25 and 0·05% carmellose sodium; (x) 4, 2 and 1 mol l−1glycerol; (xi) 4, 2 and 1% sorbitol; (xii) 4, 2 and 1% peptone; (xiii) 10, 5 and 1% nonfat skim milk (NFSM), and distilled water was used as a control. These FDPAs were selected on the basis of previous studies (Champagne et al. 1991; Hubalek 2003). All the FDPAs were confected in twofold concentration in preparation. The NFSM and glucose were sterilized at 115°C for 15 min, which were denaturalized at 121°C, and other FDPAs were sterilized at 121°C for 20 min. Then, the protectants were mixed with an equal volume of micro-organism without LB medium. The prepared samples obtained initial concentration of 7·6 × 108–8·1 × 108 CFU ml−1 on B. cereus AR156, 5·6 × 109–6·7 × 109 CFU ml−1 on Bu. vietnamiensis B418, 1·2 × 1010–1·7 × 1010 CFU ml−1 on P. agglomerans 2Re40, respectively. The procedure of cell preparation, freeze-drying and rehydration was as described earlier. After freeze-drying, all samples were rehydrated with phosphate buffer (0·05 mol l−1, pH6·8) to 400 μl, and the level of survival was evaluated. The experiment was repeated three times.

Rehydration media used in assay

In this assay, the initial concentrations used were 1·1 × 109–1·3 × 109 CFU ml−1 (B. cereus AR156), 1·4 × 109–2·2 × 10CFU ml−1 (Bu. vietnamiensis B418) and 1·4 × 1010–2·1 × 1010 CFU ml−1 (P. agglomerans 2Re40), with 5% sucrose as the FDPA. Freeze-dried samples were rehydrated with the following rehydration media to 400 μl: 10, 5 and 1% NFSM, 10, 5 and 1% peptone, 10, 5 and 1% sucrose and distilled water and phosphate buffer (0·05 mol l−1, pH6·8), both as controls. The procedure of cell preparation, freeze-drying and rehydration were applied as described earlier, and the experiment was repeated for three times.

Data analysis

The resulting colonies from the samples taken before and after freeze-drying were counted, and the surviving percentages of the three strains in the following storage were also estimated. When anova detected significant (P < 0·05) difference between treatment means, Duncan multiple range test for one-way anova was used for mean comparison using Statistical Product and Service Solutions for Windows (version 16.0; SPSS Inc, Chicago, IL, USA).

Results

Assay of FDPA

Using a range of FDPA, significant differences in the viability of cells of B. cereus AR156, Bu. vietnamiensis B418 and P. agglomerans 2Re40 after freeze-drying were observed (Table 1). Compared with B. cereus AR156 and Bu. vietnamiensis B418, the effect of all the FDPA on P. agglomerans 2Re40 was not significant (P > 0·05). The best protection was obtained from saccharides. Cellobiose (5%) was the best FDPA for Bu. vietnamiensis B418 giving viability 98·0% with 6·2 × 109 CFU ml−1 of cells, and d-galactose (4%) was the one for P. agglomerans 2Re40 giving viability 54·3% with 7·9 × 109 CFU ml−1 of cells. The viability of B. cereus AR156 was 100% or more with sucrose (5%), which gave the greatest protection for B. cereus AR156 with 7·9 × 108 CFU ml−1 of cells.

Table 1.   Comparative effect of different FDPAs on viability (%) and concentration of Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 after freeze-drying
FDPAViability after freeze-drying (%)
AR156B4182Re40
  1. Numbers followed by the same letter in the same column are not significantly different at P = 0·05 according to Duncan multiple range test.

  2. *Means standard deviation.

  3. †NFSM, nonfat skim milk.

  4. ‡No cell survived in l-glutamic acid after freeze-drying. Data are means for four replications.

Sucrose (5%)102·3 ± 3·8a*84·5 ± 2·7c38·6 ± 2·6c
Trehalose (4%)85·2 ± 4·5bc93·2 ± 7·5ab37·4 ± 1·3c
Cellobiose (5%)68·3 ± 4·4de98·0 ± 2·7a50·5 ± 1·2a
Glucose (5%)78·2 ± 2·0cd55·0 ± 2·5d42·9 ± 2·6b
Fructose (5%)91·9 ± 6·0b78·5 ± 5·2c49·3 ± 1·9a
d-galactose (5%)66·0 ± 1·6e85·3 ± 2·3bc54·3 ± 2·0a
NFSM (5%)†64·2 ± 4·3ef15·5 ± 1·7ef13·1 ± 1·6de
Peptone (2%)62·8 ± 2·9ef17·9 ± 2·4e15·5 ± 0·2d
Soluble starch (1%)60·5 ± 2·7efg6·1 ± 0·3g3·0 ± 0·2hi
Carmellose sodium (0·25%)53·5 ± 3·2fgh7·5 ± 0·3fg4·8 ± 0·2gh
Sorbitol (2%)51·5 ± 2·0gh2·1 ± 0·3g11·6 ± 0·8def
Glycerol (2 mol l−1)43·6 ± 4·0h0·1 ± 0·0g10·0 ± 1·1ef
l-glutamic acid (0·5 mol l−1)21·5 ± 3·3i–‡
Distilled water2·3 ± 0·1j6·5 ± 0·6g8·5 ± 0·5fg

The peptides, polymers and polyols substances rendered less protection to the biocontrol agents than saccharide. Cell viability with l-glutamic acid was significantly lower, only 21·5% (1·9 × 108 CFU ml−1 of cells) for B. cereus AR156, and survival was under the limit of detection after the freeze-drying when l-glutamic acid was used as the FDPA for Bu. vietnamiensis B418 and P. agglomerans 2Re40 (Table 1).

Assay of rehydration media

Based on the assay results of the FDPA, sucrose was selected as the FDPA for the assay of rehydration media. Viability percentages of the three biocontrol agents with different rehydration media are shown in Fig. 1. The results indicated that there were some differences in viabilities of the bacteria depending on the rehydration media. As shown in Fig. 1, there were no significant difference between most of the tested rehydration media and phosphate buffer for the viabilities of P. agglomerans 2Re40 and B. cereus AR156. 10% NFSM, as a rehydration medium, gave the best recovery of B. cereus AR156 and Bu. vietnamiensis B418 (99·4 and 99·1%) among the rehydration media tested and 5% NFSM gave the best recovery of P. agglomerans 2Re40 (45·0%).

Figure 1.

 Effect of rehydration media on the viability of Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 cells freeze-dried with 5% sucrose as freeze-drying protective agent. (a) B. cereus AR156, (b) Bu. vietnamiensis B418, (c) P. agglomerans 2Re40; the separation of means was conducted according to Duncan multiple range test. Columns with different letters indicate significant differences (P < 0·05). Numbers are means for three replications.

Peptone and sucrose showed no benefit to the recovery of the cells of B. cereus AR156 or P. agglomerans 2Re40, and even had inhibitory effect compared with the control (87·7% with phosphate buffer, 89·0% with water) (Figs 1a and 2c). While 10% peptone could improve the recovery of Bu. vietnamiensis B418 better compared with the sucrose and the control, and rehydration in distilled water had the lowest survival rate (73·9%) (Fig. 1b).

Figure 2.

 Survival of Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans agglomerans 2Re40 with different freeze-drying protective agents during freeze-dried storage when 5% nonfat skim milk was used as rehydration medium. (a) B. cereus AR156, (b) Bu. vietnamiensis B418, (c) P. agglomerans 2Re40; 5% sucrose (◊); 4% trehalose (□); 5% fructose (Δ); 5% NFSM (○). N0: CFU ml−1 initial; Nf: CFU ml−1 final.

Storage stability of the three bacteria with FDPA

Significant differences (P < 0·05) in the viability of cells of B. cereus AR156, Bu. vietnamiensis B418 and P. agglomerans 2Re40 in store were observed depending on the FDPA used that included 5% sucrose, 4% trehalose, 5% fructose and 5%NFSM (Fig. 2). It demonstrated that saccharide, which showed a significant effect on the viability of cells during freeze-drying, also had better storage stability in store.

It can be concluded from Fig. 2 that the survival in storage was significantly (P < 0·05) dependent on the bacterium tested. The survival of B. cereus AR156 could be kept for 12 months, which could be maintained at 3·9 × 108 CFU ml−1 after 360 days. In contrast, the viability of Bu. vietnamiensis B418 and P. agglomerans 2Re40 decreased significantly during the storage, and after 3 months there was no cell detected.

After 1 month of subsequent storage, the viability of Bu. vietnamiensis B418 declined from 5·9 × 109–1·0 × 109 CFU ml−1 on trehalose (4%), which was the best stabilizer for Bu. vietnamiensis B418. After 60 days of storage, the highest viability declined to 4·3 × 108 CFU ml−1 on 4% trehalose (Fig. 2b). After 60 days of storage, the viability of P. agglomerans 2Re40 declined to 1·8 × 108 CFU ml−1 on fructose (5%), which was the best stabilizer of P. agglomerans 2Re40 (Fig. 2c). Other saccharides gave little stability, while polymers, polyols and peptides were all ineffective on storage stability of Bu. vietnamiensis B418 and P. agglomerans 2Re40 (data not shown).

Discussion

This study showed the impact of FDPA and rehydration media on viabilities of biocontrol agents after freeze-drying and storage. It was the first investigation of the different viabilities of the three biocontrol agents from different genera in the process of freeze-drying and subsequent storage. The freeze-dried formulation of the biocontrol agents prepared from B. cereus and Bu. vietnamiensis was also first reported.

In previous studies, not all strains survived the lyophilization, and the protection afforded by FDPA during freeze-drying varied with species of micro-organisms. In our study, it was demonstrated that B. cereus AR156 and Bu. vietnamiensis B418, whose viabilities can be preserved more than 80% with suitable FDPA, are highly resistant to freezing and osmotic dehydration during freeze-drying (Table 1). The strain of P. agglomerans 2Re40 is more sensitive than B. cereus AR156 and Bu. vietnamiensis B418 to lyophilization. The difference in survival might be reflected through structural diversity in the cell wall and the cell membrane composition of the micro-organisms (Miyamoto-Shinohara et al. 2006). The stress withstanding characteristics of the microbes could be attributed to: (i) differences in genetic constitution that may lead to differences in phenotypes among various strains (Selmer-Olsen et al. 1999); and (ii) differences in cell wall and membrane composition, with different melting points of its phospholipids, which may cause differences among strains (Valdez et al. 1985).

Saccharides have been reported to provide good protection for bacteria during freeze-drying in the previous studies. The saccharides, especially disaccharides, replace structural water in membranes after dehydration (Clegg 1986) and prevent unfolding and aggregation of proteins by hydrogen bonding with polar groups of proteins (Carpenter et al. 1990). Different effects exhibited by saccharides in our study might be related to their water-binding capacity and prevention of the formation of intracellular and extracellular ice crystal (Burke 1986). In previous work, sucrose has been used as FDPA successfully (Costa et al. 2000) and is consistent with our study. Moreover, sucrose has a low cost, which could be used widely and economically. The previous work also found that trehalose was one of the best FDPA. In Costa’s work (2000), 80% viability of freeze-dried P. agglomerans could be obtained with trehalose. However, trehalose has no excellent performance in our study. In our study, the monosaccharides, such as d-galactose and fructose, also provided favourable protection, even better than some disaccharides. This might be related to the stabilization of the monosaccharides on bacterial proteins. The structure-stabilizing effect on the proteins would decrease with the increasing molecular weight of saccharide, and larger saccharide molecules should have less ability to form effective molecular interaction with proteins by the increasing steric hindrance and possible phase separation in the freeze concentrates (Izutsu et al. 2004). The viability of B. cereus AR156 with sucrose (5%) was more than 100%, and the viabilities greater than 100% after freeze-drying were also obtained by To and Etzel (1997) with Streptococcus thermophilus, and by Costa et al. (2000) with P. agglomerans CPA-2. The reason for this was still unclear.

In this study, the effects of all the FDPA were not improved along with the increased concentration, and it might be hypothesized that the water activity, which is one of thermophysical properties for freeze-drying; decreased in the FDPA with increased concentrations, and then the protections of the substances were weakened (Schoug et al. 2006).

The high recovery obtained by NFSM might be related to the hydration rate of the samples. NFSM could supply a large variety of nutrients that would enhance the recovery of cell after freeze-drying. Furthermore, the concentration of the rehydration medium plays an important role in restoring water to the dried cell. There should be some finite rate of rehydration at which the damage to the dried cells could be minimized (Choate and Alexander 1967).

There are few studies about storage and the corresponding viability of freeze-dried biocontrol agents’ formulation, because the shelf-life of products is generally regarded as a commercial secret. In our study, effective FDPA are consistent between freeze-drying process and storage in essence. The strain of B. cereus AR156 has more remarkable stabilization in store than the other two strains (Fig. 2). The main reason should be that AR156 is classified as Bacillus, which can form spores when it suffers extreme environment conditions. The loss of viability during storage could be explained by the remaining moisture, for water used to be reported to contribute to protein degradation (Garzon-Rodriguez et al. 2004). Temperature is another reason, as the storage temperature increases, mortality also increases, which leads the period of storage to be reduced (Costa et al. 2002).

This work showed that freeze-drying can be used to preserve biocontrol agents, especially the Bacillus; nevertheless, we should continue our research on the factors above and obtain an optimized system of these factors that is necessary for the industrial development of the biocontrol agent formulation.

Acknowledgements

This research was supported by Program for New Century Excellent Talents in University (NCET-06-0492), National Natural Science Foundation of China (30971956), Special Fund for Agro-scientific Research in the Public Interest (No. 200903052), Governmental Scientific Ministrant Project of Beijing Agricultural Council (BJNY2007-03-02) and Jiangsu Special Guiding Fund Project for Scientific Innovation and Technology Transfer (BY2009157).

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