GROWTH PROMOTER EFFECT ON THE B. braunii KUTZING 1849 CULTURE BY SEVERAL DIFFERENT BACTERIAL STRAINS

EFECTO DE PROMOTORES DE CRECIMIENTO EN B. braunii KUTZING 1849 A PARTIR DE DIFERENTES CEPAS BACTERIANAS

Erika J. Obando-Montoya^{2, *3}, Juan C. Gaviria-García^{2, 4}, Andrés A. Arbeláez-Pérez, ^{2, 5} Lucía. Atehortúa-Garces.¹

¹Docente, Instituto de Biología, Universidad de Antioquia. Calle 70 No 52-21, Medellín Colombia A. A. 1226. latehor@gmail.com

²Grupo de Biotecnología, Facultad de Ciencias Exactas, Universidad de Antioquia. A. A. 1226. Medellín (Antioquia), Colombia.

Correos Electrónicos: ^*3anailujakire@gmail.com. ⁴juan.gaviria@udea.edu.co ⁵andresalonsoap@hotmail.com

ABSTRACT

There are several strategies to improve the growth of microalgae in industrial processes. In recent years, one of them have gained strength to achieve this goal: the co-culture with bacteria. Using growth-promoting substances producer bacteria enhances the microalgae biology activity, similarly to how they have been used to promote the successful production of crops. The aim of this study is to evaluate the promoter capacity of strains Bacillus subtilis, Corynebacterium aquatile and Flavobacterium aquatile, their ability to improve the growth rate of microalgae Botryococcus braunii and to optimize the process derived from its cultivation. This study shows that the tested bacteria are able to increase up to 1.7 times the B. braunii growth rate and this promoting ability remains present in cell lysates preparations from the same bacterial strains.

KEY WORDS: Botryococcus braunii, Flavobacterium aquatile, Corynobacterium aquatile, Bacillus subtilis, microbial elicitation and co-cultivation.

RESUMEN

El empleo de bacterias productoras de sustancias promotoras de crecimiento, para mejorar la eficiencia en el crecimiento de las microalgas y así potenciar su actividad en procesos industriales es una práctica que ha tomado fuerza durante los últimos años, de manera análoga a como han sido utilizadas las bacterias para favorecer la producción exitosa de cultivos vegetales. El objetivo de este trabajo fue evaluar la capacidad de las cepas Flavobacterium aquatile, Corynobacterium aquatile y Bacillus subtilis de actuar como promotoras en el crecimiento de la microalga Botryococcus braunii con el fin de mejorar su velocidad de crecimiento y optimizar los proceso derivados de su cultivo. Este estudio muestra que las bacterias evaluadas tienen la capacidad de aumentar hasta 1,7 veces el crecimiento de B. braunii y esta capacidad promotora continúa presente en preparaciones de lisados celulares procedentes de estas mismas cepas bacterianas.

PALABRAS CLAVES: Botryococcus braunii, Flavobacterium aquatile, Corynobacterium aquatile, Bacillus subtilis, elicitación microbiana y cocultivo.

INTRODUCTION

The consortium of algae and bacteria is widely present in nature. For this reason, some researchers have undertaken the task of revealing the relationship among these microorganisms. A study conducted on 326 different species of algae revealed that about 52% of them requires vitamin B12 exogenously, which seems provided by the bacteria in a symbiotic way (Croft et al. 2005)

Regarding Chlorella sp, it is known that this microalga naturally forms consortiums with a variety of bacteria belonging to the genders Acinetobacter, Bacillus, Flavobacterium and Pseudomonas. Chlorella sp. has been found in associations with some fungi too, with which can be attached through the membrane because of the presence of polysaccharides (also this situation could be present with bacteria). Several studies have demonstrated the promotion of growth of the C. pyrenoidosa and C. vulgaris by strains of bacteria such as, Brevundimonas sp., Leptolyngbya sp. and Ralstonia sp. through the production of phytohormones, which also provide resistance to the culture, against the attack of crop contaminating fungi (Tate et al. 2013).

The strong relationship between the growth of photosynthetic organisms and bacteria presence has been known from long time ago. This phenomenon is also applied to higher plants. Generally, microbial cytokinins produces pathological symptoms in higher plants (Greene 1980). An example of this is Corynebacterium fascians, who is the responsible of fasciation disease in plants, due to the presence of cytokines ribosyl type Z (Einset and Skoog 1977). Treatment of pea seedlings with exogenous CKs shows similar symptoms as those caused by this illness. This is a clear evidence of the direct action of cytokines of microbial origin on plant cells, being these substances fully responsible for the pathogenic effects. On the other hand, it is known that for the virulence generation by Agrobacterium tumefaciens in plant cells, it is necessary the transfection with oncogenes, this genes have been related to the increase in the production of plant hormones, as they induce a high expression of tryptophan monooxygenase enzymes and indole-3-acetamide hydrolase (iaaM and IAAH); both of them intervene in the conversion of tryptophan in indole acetic acid (IAA) and the condensation of adenosine into monophosphate (AMP) by catalysis of protein isopentyl transferase (ipt). AMP is an essential precursor for the production of isoprenoid Cks (Britton et al. 2002). While these two examples in higher plants are referred to pathogenic associations, the knowledge about this process has been exploited to desired elicitation in growth plant and to confer resistance to crops against various stress conditions (Mayak et al. 2004, Nie et al. 2002)

Like those, there are several reported examples of relationships between these types of organisms. Then, the understanding of the events present in interaction networks is necessary and useful to improve the existing biotechnology processes, based on plant cells cultivation; and is that background that has motivated this study. The objective is to evaluate the promoter growth factor of reported cell over B. braunii. In this case, Corynobacterium aquatile and Flavobacterium aquatile (Chirac et al. 1985) were tested; but also other Gram-positive Bacillus subtilis ubiquitous consortia were analyzed.

MATERIALS AND METHODS

The activation of the strains C. aquatile DSM 20146 and F. aquatile DSM 1132 was performed, according to the described procedure in the lyophilized vials insert. The content of the medium for activating the bacteria F. aquatile has a concentration of  5 g/l of  KH₂PO₄, 13 g/l of K₂HPO₄, 0.2 g/l of MgCl₂, 2 g/l of (NH₄)₂SO₄ and 5 g/l of glucose. For C. aquatile the concentration in medium is 10 g/l of glucose, 15 g/l of peptone, 3 g/l of K₂HPO₄, 1 g/l of yeast extract, 2 g/l of NaCl and 0.2g/l of MgSO₄˖7H₂O. Both mediums are stabilized at pH 7 (Chohnan et al. 1997), (Wada et al. 1999). The strain of Bacillus subtilis was already active when donated by the Biocontrol and Environmental Microbiology (BIOME) group of the University of Antioquia. The culture of C. aquatile  and F. aquatile bacteria was developed by using a specific medium for their growth (Wada et al. 1999). For Bacillus  subtilis both nutrients were used as a growth medium (figure 1).  

The growth curve for each bacterium is determined by tracking the bacteria growth every two hours by spectrophotometric measurements at 450 nm; which is related to cell concentration expressed in terms of number of cells/mL, counted in a Neubauer chamber, through a linear regression (Bainbridge 2000)

The B. braunii strain UTEX LB 572 is kept active by weekly culture in 100ml flasks Erlenmeyer containing 50ml of BG11 modified medium (Rao et al. 2007). B. braunii cultures were performed for 16 days in the same medium; its conditions were 200 rpm orbital shaking, 17.6 µmol/m²s intensity of light for 24 hours, room temperature of 20°C and daily blowing by 60 seconds with flows of 70 ml/min of CO₂ to achieve 20% saturation in the atmosphere; which are the control culture conditions.

For the co-culture of B. braunii with three bacteria, three factors that could affect the response variable (g/l of dry biomass at the end of culture) were evaluated: 1) Viability of the bacteria, 2) Species of bacteria (B. subtilis, C.aquatile and F. aquatile) and 3) the presence or absence of B. braunii cells in the culture medium and likewise the interactions among these factors (figure 2 and 3). Co-culture assays were settled at control culture conditions; we performed four control cultures, one for each strain. It is important to highlight that the bacterial species needed two sets of culture, alive and lysed cells.

To test co-culture with dead bacteria was necessary to lyse the bacterial cells, submitting them to an autoclave cycle of 121°C, 15 psi for 20 minutes. Cell viability was verified by all bacteria cultures with diacetate fluorescein (FDA) test (Barer and Harwood 1999). After the contact by 10 minutes of 1 mL of culture with 20 µl of 0.5% acetone FDA solution (Gaurav 2011), performance was estimated by fluorescence microscopy of cells. The absent of fluorescence in green filter means complete death cells.

Final biomass of the cultures were collected by centrifugation at 11090 RCF (relative centrifugal force) and was dried in a convection oven. The dry weight was determined by gravimetric measurement and it is expressed in terms of g/l (Dayananda et al. 2005). The increase in cell biomass was compared with the dry weight of the culture of each alive-and-lysed bacteria strain independently grown, as well as the dry weight of the culture of B. braunii.

The statistical analysis were performed using the program StatGraphics XVI version 16.1.17 and graphics were performed in Prism 6.0 for Windows version 6.03. In all cases the basic assumptions of the model, normality, homoscedasticity and independence of the data were verified. To assess the statistical significance of differences between means Multiple Range Test was applied via the method of maximum difference significant (LSD) of Fisher; p ≥ 0.05 value was considered not significant, and p <0.05 value as significant.

RESULTS

Initially, the data are analyzed by a multifactorial design 3×2×2 (The used strain B. subtilis, C. aquatile and F. aquatil × intact or lysed state of bacterial cells × absent of presence of B. braunii cells) to determine the promoting effect of bacteria-microalgae co-cultive. The response variable is the amount of biomass obtained at the end of cultivation defined in terms of dry weight (g/l). The performed analysis of variance indicates that the used type of bacterial species and the cell state have no effect on the amount of final biomass in dry weight g/l. The presence or absence of B. braunii cells in the culture medium does show an effect on the amount of final biomass obtained. There is no interaction among factors (table 1).

It is verified that effect of bacteria is only shown in the presence of cells of B. braunii. Then, an analysis is conducted using a single categorical individual factor design, with the intention of checking whether there is a statistical difference in the final biomass obtained among control cultures, containing only B. braunii cells and the co-cultivation with bacteria. The multiple range test shows that final culture biomass with any of the bacterial treatment is statistically superior to that obtained in the control cultures. However, there is no statistical difference on the average final biomass obtained among the different co-cultures (figure 4).

DISCUSSION

An example of artificial linkages is given between bacteria and Chlorella sp., which has been evaluated with the aim to improve its growth and incidentally improve too the wastewater treatment, whose process has the greatest application of the microalgae. Taking advantage of properties in nitrogen fixation by some bacteria. Another example is given by C. sorokiniana culture and C. vulgaris conducted in partnership with an immobilized manner Azospirillum brasilense, in which an increase in dry weight was found, in the number of cells, in algal cluster size and in the pigment levels number as chlorophyll a and b, violaxanthin, lutein, and β-carotene, as well as an increase in the amount and the profile of produced  lipid by the appearance of four new fatty acids (De-Bashan et al. 2002), demonstrating thereby that presence of this bacterium induces significant changes in metabolism of the microalgae. This promoter activity has been explained in part by the same authors, due to production of growth promoting substances like auxin, confirmed by performing co-cultures with mutated A. brasilense strains deficient in the production of IAA, presenting a clear decrease in promoter activity compared to cultures performed when using the wild strain, which is restored by the exogenous supplementation of auxin (De-Bashan et al. 2008). There are also associations between Chlorella sp. and fungi, an example is given with Rhodotorula glutinis, which shows that cultures in a mixed medium of molasses, not only have the ability to increase the production of lipids by the microalgae, but also the production per se of characteristic fatty acids of plants (Cheirsilp et al. 2011). The evaluations in this study show that the eliciting effect of the bacteria biomass can increase up to 1.7 times the final dry weight after 15 days of culture. Contrary to all reported about the formation of consortia between species of microalgae and bacteria, the eliciting effect seems to be unrelated to the viability and the active metabolite of bacteria, because the elicitation was statistically identical as well using lysed cell by an autoclaving process as viable bacteria cells. Similarly, elicitation is not peculiar to those bacterial species closely related to the formation of "blooms" by the green microalgae, but instead it is also given by B. subtilis which is not related in the same way with the microalgae. This unspecificity in growth promoting activity could be caused by the previously reported growth inducing capacity of plants by B. subtilis (Zablotowicz et al. 1991), achieved by various strategies, including the production of phytohormones, especially of the type auxin IAA and gibberellins as AG3 (López-Valdez et al. 2011), (Chowdappa et al. 2013)

Although reports relating to C. aquatile and F. aquatile bacteria with production of phytohormones are unknown, they could produce thermo resistant phytohormones, such as the auxin indolebutyric acid (IBA) or K, BAP cytokinins, that keep unaltered even after autoclave lysis process (Torres et al. 2010).

We could also think that growth promoting effects shown with the use of viable or dead bacteria on the cultivation of B. braunii may be due to other mechanisms such as those carried out by the group of plant growth promoting rhizobacteria; within them there are representatives of the genera Bacillus and Flavobacterium; these have the capacity to solubilize phosphate and other minerals enhancing thereby their bioavailability. Therefore the biomass of these bacteria is used as a fertilizer in agriculture (Yao et al. 2006); (Rodríguez and Fraga 1999).

Moreover, one recent compilation (Subashchandrabose et al. 2011) reveals that mixed cultures of bacteria and algae have an optimal degradation of organic and inorganic contaminants form bacteria activity. This degradation is carried out due to the large amount of dissolved oxygen. The increasing oxygen concentration has its origin in photosynthesis realized by algae. Both factors improve the efficiency of CO₂ fixation, keeping the optimal dissolved CO₂/O₂ ratio constant. Also, this phenomenon reduces the algae inhibition growth due to accumulation of toxic metabolites during the culture time (Subashchandrabose et al. 2011), this could contribute to the explanation why these bacteria promote the growth of B. braunii.

Additionally, some works over B. braunii have revealed its capacity of photo, mixo, or/and heterotrophically, growing preferably by using glucose as carbon source (Zhang et al. 2011); (Tanoi et al. 2011). The possibility of development mixo or heterotrophic process, in the assays of the present study, could explain the increased algae growth by using bacterial biomass as carbon source. However, this would imply a superior competitive capacity in the case where the co-cultures were carried on, using viable bacteria solutions. Then it would be necessary to develop studies to confirm this behavior

CONCLUSIONS

Bacterial cells of C. aquatile and F. aquatile, do not exhibit a particular interaction as they are grown with B. braunii, their biomass as that of B. subtilis cells, have the ability to produce a growth promoting effect on the microalgae, reflected on the increase in the dry weight obtained at the end of the culture. The clear growth promoting activity, obtained by the bacteria on the culture of B. braunii, opens a clear interest in the evaluation of its influence and eliciting effect on the production of valuable metabolites from microalga origin. The lack of specificity of this effect is remarkable for the production of B. braunii biomass, converting bacterial biomass into a low cost item to promote growth in industrial processes associated with this microorganism, which at the moment represents a better cost benefit alternative, than the use of phytohormones.

ACKNOWLEDGEMENTS

The authors thank the sub-direction of Research and Business Development and Energy of Empresas Públicas de Medellín (EPM) and the University of Antioquia

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