Shelf Life of Azospirillum brasilense in Alginate Beads Enriched With Trehalose and Humic Acid

Since abiotic and biotic factors can compromise the survival of bacteria and their viability, encapsulation of cells in biodegradable gel matrices, a biological macromolecule, is one alternative to have their shelf life extended. Here, it was developed a gel-based formulation of the bioinoculant Azospirillum brasilense strain AbV5 and determined the effect of trehalose and humic acid supplementation in viability and survival of bacteria. For each 2 ml of sodium alginate solution (3%), 1 ml of the inoculum was extruded in a solution containing sodium alginate complexed with calcium chloride, forming calcium alginate beads. Supplements were used in a ratio of 2:2:1. Treatments were peat; alginate; alginate + humic acid; alginate + trehalose 0.1 M; alginate + trehalose 1 M. Morphometric aspects, survival rate and viability were determined in 9 storage periods (3, 5, 7, 14, 21, 30, 45, 60, 90 days). As results, beads were able to sustain the growth of A. brasilense for 90 days. Shelf life quality decreased in all treatments and peat remained the best carrier. Encapsulation, despite promoting the greatest losses in the survival of bacteria in the first days, ensured better cell viability. Trehalose in low concentrations (0.1M) improved cell viability during storage, optimizing plant inoculation.


Introduction
World food production is based on the extensive use of chemical fertilizers, which not only pollute the environment but also are expensive due to their non-renewable sources like fossil fuels, used in their exploitation, transportation and application (Schoebitz, López, & Roldán, 2013).Therefore, eco-friendly and economical alternatives have been increasingly demanded.Among some of these alternatives, plant-growth promoting bacteria (PGPB) are sustainable and low-cost biofertilizers, but need specific formulation when used in agronomical practices (Malusa & Vassilev, 2014).
Biofertilizers as inoculants must have 3 fundamental characteristics: to promote bacterial growth; to keep the cells viable for a certain period of time and to release a minimum population of bacteria, which will certainly be associated to plants (Yoav Bashan et al., 2014;Shcherbakova et al., 2018).Microbial survival after soil inoculation depends on both abiotic and biotic factors.The population of the inoculated bacteria declines progressively over time, preventing the accumulation of a bacterial pool in the rhizosphere sufficient to promote beneficial effects (Yoav Bashan, 1998;Sivakumar, Parthasarthi, & Lakshmipriya, 2014).
Nutritional conditions, humidity, temperature, and pH of soil solution are factors that compromise the survival of bacteria in the rhizosphere.In addition, the survival of the inoculated bacteria depends to a large extent on the availability of a specific niche in which competition for nutrients or substrates does not exist.It also depends on the resistance to predation and/or on the mutualistic coexistence with the native microflora, generally more adapted (Reetha, Kumaresan, & John Milton, 2014;Schoebitz et al., 2013).Peat is the most common carrier used in the inoculant industry and it is widely recommended to several plant crops (Yoav Bashan et al., 2014).It has a large water adsorption capacity, promoting a favorable microenvironment to cell growth and maintenance (Kaljeet, Keyeo, & Amir, 2011).However, it has as disadvantages little protection to the stresses caused by storage and environmental stresses caused to the bacteria after inoculation (Yoav Bashan, 1998).
Alternatives that aim the improvement of viability of bacteria and extension of shelf life are important for the emergence of a greater number of types of inoculants that combine with different bacterial species and methods (see review Berninger, Mitter, & Preininger, 2016).One of the most successful, safe and effective methods to add bacteria to the soil is by encapsulation of cells in biodegradable gel matrices (Cassidy, Lee, & Trevors, 1996;Vassilev et al., 2015).The hydrogel is formed by the interaction between sodium alginate and Ca +2 ions via the ionotropic gelation mechanism (Burey, Bhandari, Howes, & Gidley, 2008).Alginate is a natural polymer composed of β-(1→4)-linked D-mannuronic acid and α-(1→4)-linked L-guluronic acid, both produced by brown algae (Macrocystis pyrifera), as well as by bacteria (Pseudomonas sp and Azotobacter sp) (Hay, Rehman, Ghafoor, & Rehm, 2010;Nehra & Choudhary, 2015).
The addition of supplements in the encapsulation procedure could optimize the survival and release of bacteria from the inoculant.Humic acid of high molecular weight and colloidal appearance has been efficient in improving the survival of encapsulated microorganisms (Reetha et al., 2014;Young, Rekha, Lai, & Arun, 2006).Trehalose, in turn, is a disaccharide that can be used as source of energy and as protector against dehydration.Trehalose can increase the viability of freeze-drying cells as 70% of them survived after drying (Leslie, Israeli, Lighthart, L. Crowe, & J. Crowe, 1995), compared to other adjuvants (Pereira, Oliver, Bliss, L. Crowe, & J. Crowe, 2002).Nevertheless, the effect of trehalose and humic acid on the survival and viability of alginate encapsulated cells, in a short period of storage, remains unknown.
The objective of this paper was to develop the gel-based formulation of the bioinoculant Azospirillum brasilense strain AbV5 and to determine if trehalose and humic acid can effectively enhance the viability and survival of bacteria along storage period.

Encapsulation and Gel-based Bioinoculant Formulations
A. brasilense strain AbV5 was maintained by continuous cultivations in NFb Lactate solid medium at 28 °C.The pre-inoculum was prepared by transferring a bacteria colony into a 5 mL of NFb Lactate medium at 32 °C in a shaker incubator.After 24 h, 1 mL of pre-inoculum was transferred to 50 mL of NFb-Lactate medium, constituting the inoculum, which had been maintained under the same conditions as described before.The log phase of cell growth was measured by turbidimetry at 600 nm.
The encapsulation of A. brasilense cells in beads was performed according to the protocol proposed by Reetha et al. (2014) with modifications.The proportion was 2:1 in order to obtain beads (for each 2 mL of sodium alginate solution (3%), 1 mL of the bacterial suspension was added).The ratio was 2:2:1 when the supplement was used.To avoid fungal and other bacterial contamination, Maxim'sTM (0.01% v/v) and nalidixic acid (20 μg mL -1 ) were added.
Before extrusion, the mixture was kept under gentle stirring for 30 min in sterile conditions for complete homogenization.The mixture was extruded through a Pasteur pipette into a beaker containing sterile 0.1 M CaCl 2 solution, under gently stirring at room temperature.The macrobeads were maintained in CaCl 2 solution for 2 h so that solid beads of homogeneous size could be formed.The CaCl 2 solution was drained and the beads washed twice with sterile water.After washing, the beads were incubated in NFb-Lactate liquid medium for 24 h in a shaker at 120 rpm and 32 °C to allow bacteria to multiply inside them.Afterwards, the beads were washed again twice with autoclaved distilled water, collected and left under air stream for 30 min.Aliquots of approximately 7 g of beads each were packed into 3 mm-thick plastic bags.In total, 30 packets of beads were made from each treatment.
Innocuous peat (Nitro1000 TM ) served as control, where 30 packets of 7 g were inoculated with 1 mL of peptone solution containing A. brasilense (10 9 CFU mL -1 ).All packages or aliquots containing the different formulations were stored in a dry place in the dark and at a temperature of 21±2 °C.
Beads were diametrically measured (mm) with a graduated ruler and weighted (mg) in an analytical balance in triplicate after the encapsulation of the bacterial cells.

Electronic Scan Microscopy (SEM)
External surfaces of the spheres were scanned using the Scanning Electron Microscope (SEM) technique (FEI Quanta 440).The bead-shaped samples were subjected to fixation with 0.2M sodium cacodylate buffer (pH = 7.2), followed by dehydration with increasing concentrations of ethanol (50% for 15 min, 60% for 15 min, 70% for 15 min, 80% for 15 min, 90% for 15 min and 100% for 15 min).After dehydration, the supernatant was discarded and the beads received 1.5 mL of cool acetone.Samples were placed on the sample set, which contained a double-sided carbon tape, and were subsequently dried and metallized with a thin layer of gold on the surface so that a photo could capture.

Evaluation of the Survival of Viable Cells or Microbial Counting
Three packages containing aliquots of beads from each treatment in different periods of storage were analyzed.From each aliquot, 10 beads were taken and dissolved in a falcon tube containing 10 mL of potassium phosphate buffer (0.25 M, pH 6.8±0.1).The tubes were kept in a BOD incubator for 16-24 h at 30±2 °C.After this period, and for complete solubilization of beads, tubes were shaken for 1 min in a vortex.Serial dilutions (10×) and plating were performed following the protocol of Romeiro (2001) that is by counting bacterial colonies that were visible on nutrient agar plates after 24 h of inoculation.

Efficiency of Encapsulation
It was proposed to measure the encapsulation efficiency by the ratio between the log-UFC mL -1 obtained at the inoculation day (day 0) and the log after encapsulation (1st day).

Viability and Release of A. brasilense After Inoculation of Wheat Seeds
In vitro assay was performed with 40 wheat seeds (cv CD 104) in each treatment.Seeds were washed according to a protocol suggested by Neiverth et al. (2014).Subsequently, they were placed in agar-water medium at 30±2 ºC and kept there for 3 days for complete germination.At the 4th day, the inoculum was prepared with 20 beads diluted in 3 mL of potassium phosphate solution (0.25 M, pH 6.8±0.1).Around 20 pre-germinated wheat seeds were immersed in the inoculum for 3 h at 30±2 °C.Test tubes containing 25 mL of distilled water and 5 cm of polypropylene pellets were prepared to support the inoculated seeds.Each pre-germinated and inoculated seed was transferred to the test tubes, randomly arranged under a photoperiod of 16 h/8 h of light/dark, respectively, at temperature of 25±2 °C, where the seeds stayed for 7 days.

Counting of Epiphytic Bacterial Population
The epiphytic bacterial population was evaluated after 7 days of inoculation.Three plants had their roots washed 3 times with distilled and autoclaved water, placed in tubes containing NaCl solution (0.9%) and sonicated for 20 s.Serial dilutions (10×) and plating followed the protocol of Romeiro (2001).The plates were kept at 30±2 °C for 48 h.The evaluation was done in triplicate.The colony-forming units (CFU) obtained were counted using a stereoscopic magnifying glass (Quimis).
One single bacterial colony, obtained after growth in NFb-L medium, was transferred to a PCR microtube and resuspended in 20 μL of ultrapure water.Cells were lysed through heating at 96 °C for 6 min.The supernatant was separated from the cell lysate by brief centrifugation.PCR was performed in a volume of 20 μL using 2 μL of DNA, 1× PCR Buffer, 1.5 mM MgCl 2 , 0.4 mM dNTP mix, 1 unit of Taq DNA Polymerase (4G Research and Development), and 0.2 μM of each primer.The reaction was conducted in a Bioer Life Express model MJ96 thermal cycler, with cycling conditions as follows: initial denaturation at 94 °C for 3 min, followed by 25 cycles of 94 °C for 30 s, 58 °C for 1 min, 72 °C for 1 min, with final extension of 72 ºC for 5 min.PCR products were visualized in agarose gels 1.5% stained by 0.5 μg mL -1 ethidium bromide in 1× TBE Buffer (90 mM Tris-base; 90 mM boric acid; 2 mM EDTA pH 8.0) and photo documented with the equipment Loccus Biotechnology model L.PIX.The standard molecular weight of 100 bp (NorgenTM) was used.jas.ccsenet.

Morph
The encap preserving The effici efficiency,

Discussion
To promote bacteria survival and viability is the main challenge for the inoculation technology, once shelf life is influenced by several parameters: bacterial species, culture medium, physiological condition of the microorganisms, dehydration process, storage temperature and water concentration in the inoculum (Schoebitz et al., 2013;Sivasakthivelan & Saranraj, 2013).
In order to bacteria function as promoters of plant growth, they must reach the root, survive for a certain period of time to associate with plants, and compete with other bacteria in the rhizosphere.Therefore, formulations should provide protection to bacteria longevity (Vassilev et al., 2015).
In relation to the morphological appearance, some beads exhibited striations, agreeing with other authors who observed grooves in the surface of the encapsulated beads (Berninger et al., 2016).The humic acid beads presented dark spots, probably caused by deposition of the humic acid or other impurities.The wrinkled aspect, originated from invaginations, could explain the weight gain in some treatments.This result corroborates with those obtained by other authors who observed by microphotography a wrinkled and grooved area (Bashan, Carriers, & Bashan, 1986;Sivakumar et 2014).The formulation of alginate enriched with humic acid presented higher porosity, similar to what Young et al. (2006) found.On the other hand, the treatment using trehalose formed beads of smoother appearance, without invaginations, which would explain their lower weights.(Ivanova, Teunou, & Poncelet, 2005) reported another protocol in which the beads size ranged from 1 to 5 mm, and observed that by increasing the spheres size, bacteria survival enhanced 36%.Reetha et al. (2014) obtained encapsulated particles of smaller size (1.3 to 3.2 mm) and lower weight (0.5 to 10.3 mg) compared to our data, which is explained by the type of pipetting instrument used during the extrusion of the inoculum.
Study by Amiet-Charpentier, Gadille, Digat, and Benoit (1998) showed that alginate matrix was not toxic or incompatible with Pseudomonas cells.However, in all treatments the CFU mL -1 decreased significantly 24 h after encapsulation.This evidence suggests that extrusion caused a harmful or stressing effect on the bacterial population encapsulated (Figures 2A-2D).After 48h of encapsulation, alginate motivated a sharp decline in the CFU of about 44% in relation to the 1 st day (Table 1) and 35% compared to peat during the same period.This result corroborates the research by Ivanova et al. (2002) who observed greater decrease in the initial phase, after extrusion (7 days).Bashan et al. (2002) observed that the microencapsulation process affects the survival of bacteria due to the cross-linking of the alginate-calcium complex with the cell membrane of the bacteria, killing many of them.
Among all formulations, peat demonstrated the best capacity in maintaining bacterial survival throughout storage period.According to Kaljeet et al. (2011), peat was the only carrier that maintained 10 7 CFU mL -1 of rhizobia for up to 8 weeks of storage.Our results are distinct from those obtained by Reetha et al. (2014), who observed a reduction in the microbial population of around 57% in only 6 days of evaluation, but it shifted after 90 days, when the population increased to 10 × 10 9 CFU mL -1 .Young et al., (2006) had also tested alginate supplemented with humic acid, but did not detect loss of microbiota (2 × 10 8 CFU g -1 of beads) after 24 h of encapsulation or even after 5 months of storage.The great difference between their data and ours can be partially explained by the instrument they used during the extrusion, a 26-gauge needle, smaller than one used in this work, thus forming much smaller beads (1 to 2 mm).Smaller beads would be preferable for their survival than the larger ones.
In a similar protocol, but with a concentration of 2.5% of sodium alginate, humic acid promoted a smaller reduction in the CFU mL -1 of A. lipoferum (Reetha et al., 2014).The authors reported that the beneficial effect occurred because humic acid increased porosity, enhancing oxygenation and access to nutrients, facilitating cellular metabolism within the beads.
O'Callaghan (2016) reported that the greatest benefits of supplements in the encapsulation formulations are to provide the bacteria better life conditions to withstand stresses as well as to improve cell vigor.However, data did not show improvement of CFU during periods longer than 3 weeks of storage compared to peat.
The best performance of peat regarding cell survival and viability after 90 days of storage can be explained by its high moisture holding capacity, since there may have been loss of water during encapsulation (Kaljeet et al., 2011).The calcium alginate matrix, on the other hand, is rich in water (97-98%), meaning that it would fail to provide cell protection, stated Bashan et al. (2002).Schoebitz et al. (2013) demonstrated that the addition of starch to alginate reduced the water concentration to 65% and improved significantly bacterial survival.Another factor that could explain the success of peat as carrier is that it provides better oxygenation.The inconvenience, however, is the great rates of contamination: without nalidixic acid and fungicide, it was not possible to carry out the evaluations using peat as control.
Results of viability (CFU mL -1 ) polynomial regression analyses showed that all treatments can be represented by the common regression equation (Y = 8.21 + 0.011X -0.000025X 2 , Figure 5B).The treatments with peat (p-value 0.0188) and alginate + trehalose 0.1M (p-value 0.0018) showed linear increases in viability, and the combination of alginate + trehalose 0.1M presented the highest mean value of viability, suggesting a positive effect of this disaccharide in helping encapsulated cells become vigorous until reaching the rhizosphere and associate with plants.The results obtained with trehalose at low concentrations (Figures 2C and 5B) can open up the potential for new formulations using some PGPB strains that strive to survive, even without encapsulation.
Medium and higher concentrations of trehalose in inoculants formulations should be tested to check possible improvements in bacterial survival.The positive results of increase in cell survival and viability, found in the formulations with trehalose mainly right after encapsulation, confirm the protective effect of trehalose on proteins and components of the cell membrane at the initial phase of bacteria growing inside the beads (Leslie et al., 1995).

Conclusions
The gel-based formulation of Azospirillum brasilense developed with sodium alginate (3%) was competent to produce beads of uniform size that sustained bacteria growth and viability along 90 days of storage.Peat was the best carrier to support bacteria survival.Encapsulation in a gel matrix provided higher cell viability, mainly when low concentration (0.1M) of trehalose was added.These findings can optimize plant inoculation.