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Applied and Environmental Microbiology, November 2008, p. 6797-6802, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00518-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Joint Immobilization of Plant Growth-Promoting Bacteria and Green Microalgae in Alginate Beads as an Experimental Model for Studying Plant-Bacterium Interactions{triangledown}

Luz E. de-Bashan and Yoav Bashan*

Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 85721, and Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, BCS 23090, Mexico

Received 3 March 2008/ Accepted 4 September 2008


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ABSTRACT
 
A simple, quantitative experimental model, offering a convenient and basic approach to studies of plant-bacterium interactions, is proposed. This involves immobilizing a unicellular, freshwater microalga, a Chlorella species, serving as the plant, with a plant growth-promoting bacterium, an Azospirillum species, in small alginate beads to allow close interaction and to avoid external interference from bacterial contaminants.


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INTRODUCTION
 
A major obstacle in the study of interactions between prokaryotes and eukaryotes is the complexity of the eukaryote partner, the latter species usually of some economic importance. Studies of basic plant-bacterium interactions of Azospirillum spp., plant growth-promoting bacteria (PGPB), done mainly with cereal roots, are difficult because there are multiple tissue functions and numerous possible interactions with plant roots and there is interference with the soil matrix. Consequently, there is a significantly greater quantity of information on the bacteria than on the interactions (5, 55, 56).

The Arabidopsis thaliana (mouse-ear cress) plant model is widespread, mainly because its 125-Mb genome is small and sequenced (1), and numerous mutants of most of its metabolic pathways are available. The rice plant has a 389-Mb genome (31) and may soon provide research opportunities with PGPB in addition to the extensive research already done with this plant (33, 34, 46, 61). However, larger plant genomes in which these PGPB commonly interact, such as maize (2.5 Gb), oat (11.4 Gb) (2), and wheat (16 Gb) (20), are unlikely to be understood in detail soon, even though some are undergoing sequencing procedures. Microalgae, on the other hand, have the smallest plant genome (~40 Mb), as yet not sequenced (V. Huss, personal communication). To our knowledge, the Chlorella genome is the smallest eukaryotic, photosynthetic microorganism characterized so far (28), which makes it an alternative to higher plants with large genomes interacting with PGPB.

Chlorella spp. (Chlorophyceae) are simple, nonmotile, unicellular, aquatic green microalgae. They were one of the first algae to be isolated as a pure culture in 1890 by Martinus Beijerinck (43). Chlorella has been used in studies of photosynthesis and respiration (30). Much of the knowledge on the synthesis of carbohydrates in microalgae has been obtained with this species (29, 49). Since the late 1940s, attention has been drawn to the potential of mass cultivation of this microalga for the production of high-value, low-volume compounds, such as pigments for food industries, including the health-food market in industrialized countries (52), and application in wastewater treatment (45). Except for symbiotic rhizobia, Azospirillum is the most studied agricultural PGPB (4). It is a rhizosphere-dwelling, N2-fixing bacterium that is very versatile in its nitrogen transformations. In addition to fixing N2 under microaerobic conditions, it denitrifies under anaerobic or microaerophyllic conditions and can assimilate NH4+, NO3–, or NO2– (25) and act as a general PGPB for numerous plant species (5), including Chlorella (21).

Alginate is the most commonly used polymer for microbial cell encapsulation, also called immobilization (54). The polymeric chain is made of mannuronic and guluronic acids in various proportions and sequential arrangements linked by calcium ions by binding consecutive blocks of guluronic acid to form gels (41).

The immobilization of microorganisms trapped in alginate beads is a widely used technique for viable microbial cells (44). It is used in several biotechnological fields: (i) as a practical way to immobilize microbes producing secondary metabolites of commercial value, (ii) as a tool in genetic manipulation to immobilize recombinant bacteria to improve plasmid stability (53), and (iii) to remove inorganic compounds and heavy metals from wastewater (58), where its greatest advantage is that it significantly facilitates harvesting of mass-produced microalgae from the water after treatment (15). Those are the three basic components of the experimental model that we are proposing.

Although several technical aspects of this model that show its use for supporting studies on wastewater treatment have previously been published (13), this is the first presentation of the entire model, as a vehicle to study plant-bacterium interactions.

Chlorella vulgaris Beijerinck (UTEX 2714; University of Texas, Austin) and Chlorella sorokiniana Shih. et Krauss (UTEX 1602) were used. The PGPB partners were Azospirillum brasilense Cd (DMS 1843; Brunswick, Germany), A. brasilense Sp6 (3), and Bacillus pumilus ES4 (formerly Bacillus chitinolyticus [48]). The procedures for culturing, immobilizing, and counting these microorganisms were described by de-Bashan et al. (8, 11). The growth promotion, ammonium assimilation, protein content, and activity of the enzyme glutamate dehydrogenase were measured as previously described (8, 12, 14, 21). All experiments were done in triplicate, where an Erlenmeyer flask served as a replicate. Each experiment was repeated two or three times in full. Results between treatments were analyzed by Student's t test at a P value of ≤0.05; differences between cycles of the same treatment were analyzed by one-way analysis of variance (ANOVA) at a P value of ≤0.05 and then by Tukey's post hoc analysis. Computations were made with STATISTICA software, version 6.0 (StatSoft, Tulsa, OK).

The following sequence of events occurs during the interaction between the two microorganisms within the polymeric sphere (Table 1). Initial immobilization of Chlorella spp. with a PGPB strain is described in Fig. 1A and is characterized as random immobilization of particles inside a gel matrix. Nutrients in the surrounding medium freely diffuse in the porous gel. Over time (6 to 48 h), depending on the microalga-bacterium combination, both microorganisms are found in the same cavity within the sphere, mainly just beneath the periphery of the sphere, after small parts of the internal structure of the matrix dissolve or split and separate as internal microcolonies develop and enlarge (Fig. 1B) (36). The bacteria excrete indole-3-acetic acid (IAA) and other undefined signal molecules, possibly reaching the nearby microalgal cells. At this stage, the activities of the microalgal enzymes (two were tested so far, glutamine synthetase and glutamate dehydrogenase) are not enhanced (Fig. 1B). At the next phase of interaction, beginning 48 h after joint immobilization and continuing later, the following happens: glutamate synthetase and glutamate dehydrogenase activities are enhanced, photosynthetic pigment production is enhanced, nitrogen and phosphorus uptake into microalgal organelles is enhanced, and the jointly immobilized system liberates oxygen produced by Chlorella spp. as a by-product of photosynthesis (Fig. 1C). The most noticeable effect of joint immobilization is that the population of microalgae increases two- to threefold over that of immobilized microalgae (21). Experimental testing of the proposed model used several microalga-PGPB pairs. Enhanced growth promotion was demonstrated in the joint immobilization system of C. sorokiniana and B. pumilus (Fig. 1D) and that of C. vulgaris and A. brasilense Sp6 (Table 2). Enhanced ammonium absorption occurred in the same systems (Fig. 1E) at the culture and cellular levels (Table 2). The enhancing effect of several concentrations of exogenous tryptophan (precursor of the phytohormone IAA and the main mechanism by which Azospirillum affects the growth of Chlorella [9]) was tested with a combination of C. vulgaris and A. brasilense Cd (Fig. 1F to I). The increase in the production of protein was evaluated with a combination of C. vulgaris and A. brasilense Sp6 at two ammonium concentrations (Table 2), and the enhancement of the activity of glutamate dehydrogenase, a key enzyme in ammonium assimilation in plants, was evaluated with the same microalgal species but was paired with A. brasilense strain Sp6 (Table 2). Taken as a whole, these additional studies show that this model can serve as an appropriate experimental tool to study events related to growth and nitrogen and phytohormone cycles in eukaryotes.


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  		TABLE 1. Studies of paired microalga-PGPB interactions using the proposed model


Figure 1
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  		FIG. 1. Conceptual model for jointly immobilizing microalgae and PGPB in alginate spheres and testing of the model. (A) Both microorganisms are randomly immobilized in the polymeric matrix. (B) After cocultivating for a short period of time, the two microorganisms share the cavities within the sphere. Here, the PGPB excrete IAAs (and possibly other plant hormones) that enhance the growth of microalgae. (C) Nutrients from the medium diffuse into the sphere and are assimilated into cell components of the microalgae, leading to increasing population biomass and releasing oxygen to the medium. The size is not to scale. (D) Promotion of growth of C. sorokiniana by B. pumilus. (E) Enhancement of assimilation of ammonium in C. sorokiniana by B. pumilus. (F to I) Promotion of growth of C. vulgaris by A. brasilense Cd at four concentrations of tryptophan. The points on each curve denoted with different lowercase letters differ significantly at a P value of ≤0.05 by one-way ANOVA, according to Tukey's post hoc analysis. The points denoted by different capital letters at each cycle of incubation differ significantly at a P value of ≤0.05 by Student's t test. Columns denoted by different capital letters at each cycle of incubation differ significantly at a P value of ≤0.05 by one-way ANOVA. Whisker bars represent standard errors. The absence of a bar in panel E indicates a negligible value. GS, glutamine synthetase; GDH, glutamate dehydrogenase.


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  		TABLE 2. Effects of joint immobilization of Chlorella and Azospirillum on growth rate, absorption of ammonium, protein content, and glutamate dehydrogenase activity incubation for 48 ha

The usefulness of a simple experimental model for basic studies of complex interactions between plants and bacteria is obvious. This study suggests such a model. This model involves interaction between two well-studied microorganisms, Azospirillum and Chlorella, entrapped in a gel matrix.

The microorganisms Chlorella and Azospirillum were chosen for their unique characteristics. Chlorella possesses one of the smallest plant genomes (28) and has been one of the best-studied microalgae for almost a century. Azospirillum is the most studied associated PGPB (5, 25). There are numerous available mutants of these genera (3, 6, 7, 18, 24, 32, 35, 56, 59), and many species and strains are easily obtained from most culture collections. Both Chlorella and Azospirillum are easy to cultivate and maintain. Growth conditions for both organisms are straightforward and can be either autotrophic or heterotrophic. Immobilization inside a harmless and nontoxic alginate gel matrix ensures that this artificial pairing of the microorganisms limits the movement of the motile Azospirillum strain; consequently, the two usually affect each other, although immobilization has only a facilitating role in the interaction. The immobilization and liberation of both microorganisms from alginate beads is common microbial inoculant technology. The alginate from the giant kelp Macrocystis pyrifera is commonly used in food, medicine and dentistry, and pigment production and is easy to handle and very inexpensive (41).

These favorable characteristics notwithstanding, the model is not restricted to the Chlorella vulgaris-Azospirillum brasilense interactions that have comprised most of the studies done so far. Other PGPB, such as B. pumilus, and other microalgae, such as C. sorokiniana, were successfully tested (Table 1). These options create opportunities for endless combinations of microalgae and PGPB. Similarly, different alginates and derivatives from many macroalgae are commercially available (41, 60), even from bacteria (19), for entrapment combination schemes, as needed. Because the immobilization of microorganisms is also commonly used with other polymers (44), this model is not restricted to alginates, but each polymer has its own advantages and disadvantages.

Apart from easy technical handling of this experimental model, the logic of choosing a microalga as the plant partner is simple. The most basic definition of a green plant is that it contains chlorophylls a and b, starch as storage material inside the chloroplast, and a cell wall made of cellulose (51). Systematically, higher plants and algae are part of the same group (Chlorobionta). There is 70 to 98% genetic similarity between land plants and algae (16). The size of the organism, the number of cells, and differentiation into organs are not defining parameters of a plant. Consequently, single-celled microalgae are considered plants. The bacterial partner, Azospirillum, was chosen because, although its origin and main effects were studied in conjunction with agricultural plants (5), it also affects the growth of microalgae; hence, it has been designated a growth-promoting bacterium for microalgae (8, 9, 10, 11, 12, 13). It is likely that these bacteria should have a significant effect on many metabolic pathways during their interaction with microalgae.

Additionally, the use of a green microalga as the plant partner in studies of plant-bacterium interactions has practical advantages and has already been tested: (i) phytohormones that affect plants affect microalgae, including IAA, indole-3-butyric acid, jasmonic acid, gibberellins, and cytokinins (17, 40, 43, 57); (ii) enzymes associated with the nitrogen cycle, such as glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, are similar in plants and green microalgae; (iii) the processes of lipid accumulation are similar; (iv) photosynthetic pigments and photosynthesis are similar (Table 1); and (v) many species and strains of microalgae are available for experimentation.

The usefulness of molecular and biochemical studies can be illustrated with a study using various IAA-attenuated mutants of several Azospirillum species. These mutants failed to promote the growth of the microalgal cells compared to wild types (9). Similarly, slight changes in the enzymatic activities of glutamate dehydrogenase and glutamine synthetase could be accurately measured following joint immobilization of the two microorganisms (12). There are additional hypothesized possibilities for this model, including the study of bacterial interactions with cell walls of plants. The cell walls of green microalgae are related and have many structural features in common with those of land plants. Both types of cell walls contain cellulose, hemicellulose, pectin, and glycoproteins, although the chemical composition of green microalgal cell walls is more diverse (38, 39, 42, 47). The general similarity raises the potential usefulness of this model to study the invasive entry of PGPB, such as Azospirillum and Azorhizobium, known for harmless invasive entry through cracks in the roots (23, 37, 50) that do not harm the host plant or its defense responses. Thus, the Chlorella cell wall remains a valid representation of cell walls of higher plants that are addressed by this model. Because thousands of cells were needed to measure minute changes in the growing population, their enzymatic activities, and cell-cell interactions, a model that allows for the growth, harvesting, and counting of populations of single cells is advantageous. Furthermore, a potential assessment of this model as a preliminary screening tool of candidates for agricultural and forestry inoculants was only initiated but can be expanded. For example, two PGPB strains, Bacillus pumilus ES4 and RIZO1, first screened as potential PGPB by this model (Table 1), were later used as inoculants for sorghum (M. E. Puente, E. Ortiz, and Y. Bashan, unpublished data) and eroded land reforestation by legume-family trees (Y. Bashan, B. Salazar, M. E. Puente, and R. Linderman, unpublished data). With this line of reasoning, the proposed model, involving the immobilization of two species, might significantly facilitate molecular and physiological studies on cell-cell recognition and attachment, cell wall receptors, chemotaxis, proton extrusion, metabolic pathways of eukaryotes affected by prokaryotes, and photosynthesis that have not yet been pursued.

The practical and analytical aspects of this model are considerable. All ingredients are inexpensive, and the microorganisms are easy to cultivate and test in standard microbiology facilities. The results are available on a microbial time scale (days to weeks). Reproducibility is very high, and the replicate is merely an Erlenmeyer flask, allowing as many replicates as needed in a small space and in a soil-free system. By analyzing hundreds of published results using this system, it appears that the standard error is very low and allows detection of minute effects between the interacting organisms. So far, we have not observed any disadvantages in experiments conducted over the past 10 years.

In summary, Chlorella responds to interaction with Azospirillum in ways that are very similar to the interaction responses of higher plants by enhancing its growth and changing its metabolism. The system of immobilization is easy to handle, is inexpensive, and produces rapid results. For basic studies of the physiology and molecular biology of plant-bacterium interactions, we recommend a practical experimental model involving an immobilization system containing two microorganisms.


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ACKNOWLEDGMENTS
 
We thank Delia de la Toba and Juan Pablo Hernandez for technical assistance.

Experimental work on the model was largely supported by the Consejo Nacional de Ciencia y Tecnología of Mexico (CONACYT; contract no. 50560-Z) and the Secretaria de Medio Ambiente y Recursos Naturales of Mexico (SEMARNAT; contract no. 23510). Additional support came from The Bashan Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, BCS 23090, Mexico. Phone: 52-612-123-8484. Fax: 52-612-125-4710. E-mail: bashan{at}cals.arizona.edu Back

{triangledown} Published ahead of print on 12 September 2008. Back


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Applied and Environmental Microbiology, November 2008, p. 6797-6802, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00518-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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