Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

Differences in Exudates Between Strains of Chlorella sorokiniana Affect the Interaction with the Microalga Growth-Promoting Bacteria Azospirillum brasilense : Differences in Exudates Between Strains of Chlorella sorokiniana Affect the Interaction with the Microalga Growth-Promoting Bacteria Azospirillum brasilense.

The microalga Chlorella sorokiniana and the microalgae growth-promoting bacteria (MGPB) Azospirillum brasilense have a mutualistic interaction that can begin within the first hours of co-incubation; however, the metabolites participating in this initial interaction are not yet identified. Nuclear magnetic resonance (NMR) was used in the present study to characterize the metabolites exuded by two strains of C. sorokiniana (UTEX 2714 and UTEX 2805) and A. brasilense Cd when grown together in an oligotrophic medium. Lactate and myo-inositol were identified as carbon metabolites exuded by the two strains of C. sorokiniana; however, only the UTEX 2714 strain exuded glycerol as the main carbon compound. In turn, A. brasilense exuded uracil when grown on the exudates of either microalga, and both microalga strains were able to utilize uracil as a nitrogen source. Interestingly, although the total carbohydrate content was higher in exudates from C. sorokiniana UTEX 2805 than from C. sorokiniana UTEX 2714, the growth of A. brasilense was greater in the exudates from the UTEX 2714 strain. These results highlight the fact that in the exuded carbon compounds differ between strains of the same species of microalgae and suggest that the type, rather than the quantity, of carbon source is more important for sustaining the growth of the partner bacteria.

Soil Type Influences Novel "Milpa" Isolates of Trichoderma virens and Aspergillus tubingensis That Promote Solubilization, Mineralization, and Phytoabsorption of Phosphorus in Capsicum annuum L.

The Capsicum genus has significant economic importance since it is cultivated and consumed worldwide for its flavor and pungent properties. In 2021, Mexico produced 3.3 billion tons on 45,000 hectares which yielded USD 2 billion in exports to the USA, Canada, Japan, etc. Soil type has a dramatic effect on phosphorus (P) availability for plantsdue to its ion retention.In a previous study, novel fungal isolates were shown to solubilize and mineralize P in different kinds of soils with different P retention capacities. The aim of this work was to study the effects of the mineralogy of different kinds of "milpa" soils on the germination, biomass production, and P absorption of chili plants (Capsicum annuum). The germination percentage, the germination speed index, and the mean germination time were significantly increased in the plants treated with dual inoculation. Foliar phosphorus, growth variables, and plant biomass of chili plants grown in a greenhouse were enhanced in different soil types and with different inocula. Correlation studies suggested that the most significant performance in the foliar P concentration and in the growth response of plants was achieved in Vertisol with dual inoculation of 7 × 106 mL-1 spores per chili plant, suggesting this would be an appropriate approach to enhance chili cultivation depending on the soil type. This study stresses the importance of careful analysis of the effect of the soil type in the plant-microbe interactions.

Active indole-3-acetic acid biosynthesis by the bacterium Azospirillum brasilense cultured under a biogas atmosphere enables its beneficial association with microalgae.

AIMS: This study assessed, at the physiological and molecular levels, the effect of biogas on indole-3-acetic acid (IAA) biosynthesis by Azospirillum brasilense as well as the impact of this bacterium during CO2 fixation from biogas by Chlorella vulgaris and Scenedesmus obliquus. METHODS AND RESULTS: IpdC gene expression, IAA production and the growth of A. brasilense cultured under air (control) and biogas (treatment) were evaluated. The results demonstrated that A. brasilense had a better growth capacity and IAA production (105.7 ± 10.3 µg ml-1 ) when cultured under biogas composed of 25% CO2  + 75% methane (CH4 ) with respect to the control (72.4 ± 7.9 µg ml-1 ), although the ipdC gene expression level was low under the stressful condition generated by biogas. Moreover, this bacterium was able to induce a higher cell density and CO2 fixation rate from biogas by C. vulgaris (0.27 ± 0.08 g l-1 d-1 ) and S. obliquus (0.22 ± 0.08 g l-1 d-1 ). CONCLUSIONS: This study demonstrated that A. brasilense has the capacity to grow and actively maintain its main microalgal growth-promoting mechanism when cultured under biogas and positively influence CO2 fixation from the biogas of C. vulgaris and S. obliquus. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings broaden research in the field of Azospirillum-microalga interactions and the prevalence of Azospirillum in environmental and ecological topics in addition to supporting the uses of plant growth-promoting bacteria to enhance biotechnological strategies for biogas upgrading.

The Azospirillum brasilense type VI secretion system promotes cell aggregation, biocontrol protection against phytopathogens and attachment to the microalgae Chlorella sorokiniana.

The plant-growth-promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole-3-acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in Gram-negative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella-Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of lipids, carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS-dependent bio-control protection to microalgae and plants against bacterial pathogens.

Azospirillum brasilense reduces oxidative stress in the green microalgae Chlorella sorokiniana under different stressors.

In this study, we investigated oxidative stress in the green microalgae, Chlorella sorokiniana, in co-culture with the plant growth promoting bacteria (PGPB), Azospirillum brasilense. This relationship was studied in the absence of an exogenous stressor, under copper stress, and under nitrogen limitation stress. We confirmed that copper and nitrogen limitation induced algal oxidative stress and reductions in chlorophyll content. In all cases, the presence of A. brasilense lowered the accumulation of intracellular reactive oxygen species (ROS) while promoting chlorophyll content. This effect was driven, in part, by A. brasilense's secretion of the auxin hormone, indole-3-acetic acid, which is known to mitigate stress in higher plants. The findings of the present study show that stress mitigation by A. brasilense resulted in suppressed starch accumulation under nitrogen limitation stress and neutral lipid accumulation under copper stress. In fact, A. brasilense could almost completely mitigate oxidative stress in C. sorokiniana resulting from nitrogen limitation, with ROS accumulation rates comparable to the axenic control cultures. The biotechnological implication of these findings is that co-culture strategies with A. brasilense (and similar PGPB) are most effective for high growth applications. A second growth stage may be needed to induce accumulation of desired products.

Soil Type Affects Organic Acid Production and Phosphorus Solubilization Efficiency Mediated by Several Native Fungal Strains from Mexico.

Phosphorus (P) is considered a scarce macronutrient for plants in most tropical soils. The application of rock phosphate (RP) has been used to fertilize crops, but the amount of P released is not always at a necessary level for the plant. An alternative to this problem is the use of Phosphorus Solubilizing Microorganisms (PSM) to release P from chemically unavailable forms. This study compared the P sorption capacity of soils (the ability to retain P, making it unavailable for the plant) and the profile of organic acids (OA) produced by fungal isolates and the in vitro solubilization efficiency of RP. Trichoderma and Aspergillus strains were assessed in media with or without RP and different soils (Andisol, Alfisol, Vertisol). The type and amount of OA and the amount of soluble P were quantified, and according to our data, under the conditions tested, significant differences were observed in the OA profiles and the amount of soluble P present in the different soils. The efficiency to solubilize RP lies in the release of OAs with low acidity constants independent of the concentration at which they are released. It is proposed that the main mechanism of RP dissolution is the production of OAs.

Root-Associated Fungal Communities in Two Populations of the Fully Mycoheterotrophic Plant Arachnitis uniflora Phil. (Corsiaceae) in Southern Chile.

The microbiological interactions of the roots of non-photosynthetic plants in South America have been scarcely explored. This study analyzes culturable fungal diversity associated with the mycoheterotrophic plant Arachnitis uniflora Phil. (Corsiaceae) in southern Chile, growing in two different understoreys of native (Nothofagus-dominated) and mixed forest (native, Cupressus sempervirens, and Pinus radiata). Rhizospheric and endophytic fungi were isolated, cultured, and purified to identify microorganisms associated with A. uniflora roots. We showed the different fungi associated with the plant, and that these distributions are influenced by the sampling site. We isolated 410 fungal strains (144 endophytic and 266 from the rhizosphere). We identified 13 operative taxonomical units from plants sampled in the mixed forest, while 15 were from the native forest. Rhizospheric microorganisms were mainly related to Penicillium spp., whereas some pathogenic and saprophytic strains were more frequent inside the roots. Our results have also shown that the fungal strains are weak for phosphate solubilization, but other pathways such as organic acid exudation and indole acetic acid production can be considered as major mechanisms to stimulate plant growth. Our results point to new fungal associates of A. uniflora plants reported in Andean ecosystems, identifying new beneficial endophytic fungi associated with roots of this fully mycoheterotrophic plant.

Early Changes in Nutritional Conditions Affect Formation of Synthetic Mutualism Between Chlorella sorokiniana and the Bacterium Azospirillum brasilense.

The effect of three different nutritional conditions during the initial 12 h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth-promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83 h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.

Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus.

Remote effects (occurring without physical contact) of two plant growth-promoting bacteria (PGPB) Azospirillum brasilense Cd and Bacilus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 were studied. The two PGPB remotely enhanced the growth of the microalga, up to six-fold, and its cell volume by about three-fold. In addition to phenotypic changes, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga's physiology. The two bacteria produced large amounts of volatile compounds, including CO2, and the known plant growth-promoting volatile 2,3-butanediol and acetoin. Several other volatiles having biological functions in other organisms, as well as numerous volatile compounds with undefined biological roles, were detected. Together, these bacteria-derived volatiles can positively affect growth and metabolic parameters in green microalgae without physical attachment of the bacteria to the microalgae. This is a new paradigm on how PGPB promote growth of microalgae which may serve to improve performance of Chlorella spp. for biotechnological applications.

Construction of probe of the plant growth-promoting bacteria Bacillus subtilis useful for fluorescence in situ hybridization.

Strains of Bacillus subtilis are plant growth-promoting bacteria (PGPB) of many crops and are used as inoculants. PGPB colonization is an important trait for success of a PGPB on plants. A specific probe, based on the 16 s rRNA of Bacillus subtilis, was designed and evaluated to distinguishing, by fluorescence in situ hybridization (FISH), between this species and the closely related Bacillus amyloliquefaciens. The selected target for the probe was between nucleotides 465 and 483 of the gene, where three different nucleotides can be identified. The designed probe successfully hybridized with several strains of Bacillus subtilis, but failed to hybridize not only with B. amyloliquefaciens, but also with other strains such as Bacillus altitudinis, Bacillus cereus, Bacillus gibsonii, Bacillus megaterium, Bacillus pumilus; and with the external phylogenetic strains Azospirillum brasilense Cd, Micrococcus sp. and Paenibacillus sp. The results showed the specificity of this molecular probe for B. subtilis.

Tryptophan, thiamine and indole-3-acetic acid exchange between Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense.

During synthetic mutualistic interactions between the microalga Chlorella sorokiniana and the plant growth-promoting bacterium (PGPB) Azospirillum brasilense, mutual exchange of resources involved in producing and releasing the phytohormone indole-3-acetic acid (IAA) by the bacterium, using tryptophan and thiamine released by the microalga, were measured. Although increased activities of tryptophan synthase in C. sorokiniana and indole pyruvate decarboxylase (IPDC) in A. brasilense were observed, we could not detect tryptophan or IAA in the culture medium when both organisms were co-immobilized. This indicates that no extra tryptophan or IAA is produced, apart from the quantities required to sustain the interaction. Over-expression of the ipdC gene occurs at different incubation times: after 48 h, when A. brasilense was immobilized alone and grown in exudates of C. sorokiniana and at 96 h, when A. brasilense was co-immobilized with the microalga. When A. brasilense was cultured in exudates of C. sorokiniana, increased expression of the ipdC gene, corresponding increase in activity of IPDC encoded by the ipdC gene, and increase in IAA production were measured during the first 48 h of incubation. IAA production and release by A. brasilense was found only when tryptophan and thiamine were present in a synthetic growth medium (SGM). The absence of thiamine in SGM yielded no detectable IAA. In summary, this study demonstrates that C. sorokiniana can exude sufficient tryptophan and thiamine to allow IAA production by a PGPB during their interaction. Thiamine is essential for IAA production by A. brasilense and these three metabolites are part of a communication between the two microorganisms.

Influence of tryptophan and indole-3-acetic acid on starch accumulation in the synthetic mutualistic Chlorella sorokiniana-Azospirillum brasilense system under heterotrophic conditions.

This study measured the relations between tryptophan production, the phytohormone indole-3-acetic acid (IAA) and the metabolism and accumulation of starch during synthetic mutualism between the microalgae Chlorella sorokiniana and the microalgae growth-promoting bacteria Azospirillum brasilense, created by co-immobilization in alginate beads. Experiments used two wild-type A. brasilense strains (Cd and Sp6) and an IAA-attenuated mutant (SpM7918) grown under nitrogen-replete and nitrogen-starved conditions tested under dark, heterotrophic and aerobic growth conditions. Under all incubating conditions, C. sorokiniana, but not A. brasilense, produced tryptophan. A significant correlation between IAA-production by A. brasilense and starch accumulation in C. sorokiniana was found, since the IAA-attenuated mutant was not producing increased starch levels. The highest ADP-glucose pyrophosphorylase (AGPase) activity, starch content and glucose uptake were found during the interaction of A. brasilense wild type strains with the microalgae. When the microalgae were grown alone, they produced only small amounts of starch. Supplementation with synthetic IAA to C. sorokiniana grown alone enhanced the above parameters, but only transiently. Activity of α-amylase decreased under nitrogen-replete conditions, but increased under nitrogen-starved conditions. In summary, this study demonstrated that, during synthetic mutualism, the exchange of tryptophan and IAA between the partners is a mechanism that governs several changes in starch metabolism of C. sorokiniana, yielding an increase in starch content.

Accumulation of intra-cellular polyphosphate in Chlorella vulgaris cells is related to indole-3-acetic acid produced by Azospirillum brasilense.

Accumulation of intra-cellular phosphate, as polyphosphate, was measured when the microalga Chlorella vulgaris was immobilized in alginate with either of two wild-type strains of the microalgae growth-promoting bacterium Azospirillum brasilense or their corresponding IAA-attenuated mutants. Wild type strains of A. brasilense induced higher amounts of intra-cellular phosphate in Chlorella than their respective mutants. Calculations comparing intra-cellular phosphate accumulation by culture or net accumulation by the cell and the amount of IAA that was produced by each of these strains revealed that higher IAA was linked to higher accumulations of intra-cellular phosphate. Application of four levels of exogenous IAA reported for A. brasilense and their IAA-attenuated mutants to cultures of C. vulgaris enhanced accumulation of intra-cellular phosphate; the higher the content of IAA per culture or per single cell, the higher was the amount of accumulated phosphate. When an IAA-attenuated mutant was complemented with exogenous IAA, accumulation of intra-cellular phosphate at the culture level was even higher than phosphate accumulation with the respective wild type strains. When calculating the net accumulation of intra-cellular phosphate in the complementation experiment, net intra-cellular phosphate induced by the IAA-attenuated mutant was completely restored and was similar to the wild strains. We propose that IAA produced by A. brasilense is linked to polyphosphate accumulation in C. vulgaris.

Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris.

Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoAcarboxylase, temperature, and co-immobilization with Azospirillum brasilense [corrected].

The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

Enhanced activity of ADP glucose pyrophosphorylase and formation of starch induced by Azospirillum brasilense in Chlorella vulgaris.

ADP-glucose pyrophosphorylase (AGPase) regulates starch biosynthesis in higher plants and microalgae. This study measured the effect of the bacterium Azospirillum brasilense on AGPase activity in the freshwater microalga Chlorella vulgaris and formation of starch. This was done by immobilizing both microorganisms in alginate beads, either replete with or deprived of nitrogen or phosphorus and all under heterotrophic conditions, using d-glucose or Na-acetate as the carbon source. AGPase activity during the first 72h of incubation was higher in C. vulgaris when immobilized with A. brasilense. This happened simultaneously with higher starch accumulation and higher carbon uptake by the microalgae. Either carbon source had similar effects on enzyme activity and starch accumulation. Starvation either by N or P had the same pattern on AGPase activity and starch accumulation. Under replete conditions, the population of C. vulgaris immobilized alone was higher than when immobilized together, but under starvation conditions A. brasilense induced a larger population of C. vulgaris. In summary, adding A. brasilense enhanced AGPase activity, starch formation, and mitigation of stress in C. vulgaris.

Biological deterioration of alginate beads containing immobilized microalgae and bacteria during tertiary wastewater treatment.

Secondary treatment of municipal wastewater affects the mechanical stability of polymer Ca-alginate beads containing the microalgae Chlorella vulgaris that are jointly immobilized with Azospirillum brasilense as treating agents whose presence do not affect bead stability. Nine strains of potential alginate-degrading bacteria were isolated from wastewater and identified, based on their nearly complete 16S rDNA sequence. Still, their population was relatively low. Attempts to enhance the strength of the beads, using different concentrations of alginate and CaCl2 or addition of either of three polymers (polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose), CaCO3, or SrCl2, failed. Beads lost their mechanical strength after 24 h of incubation but not the integrity of their shape for at least 96 h, a fact that sustained successful tertiary wastewater treatment for 48 h. In small bioreactors, removal of phosphorus was low under sterile conditions but high in unsterile wastewater. Alginate beads did not absorb PO4 (-3) in sterile wastewater, but in natural wastewater, they contained PO4 (-3). Consequently, PO4 (-3) content declined in the wastewater. A supplement of 10 % beads (w/v) was significantly more efficient in removing nutrients than 4 %, especially in a jointly immobilized treatment where >90 % of PO4 (-3) and >50 % ammonium were removed. Tertiary wastewater treatment in 25-L triangular, airlift, autotrophic bioreactors showed, as in small bioreactors, very similar nutrient removal patterns, decline in bead strength phenomena, and increase in total bacteria during the wastewater treatment only in the presence of the immobilized treatment agents. This study demonstrates that partial biological degradation of alginate beads occurred during tertiary wastewater treatment, but the beads survive long enough to permit efficient nutrient removal.

Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: I. Autotrophic conditions.

The effect of the microalgae-growth promoting bacterium Azospirillum brasilense on accumulation of total carbohydrates and starch in two species of Chlorella (Chlorella vulgaris and Chlorella sorokiniana), when the bacterium and each microalga were jointly immobilized in alginate beads was studied under autotrophic conditions for 144 h in synthetic medium. The interaction of the bacterium with the microalgae enhanced accumulation of total carbohydrate and starch. Cells of Chlorella accumulated the highest amounts of carbohydrate after incubation for 24h. Yet, this did not coincide with the highest affinity and volumetric productivity measured in these cultures. However, after incubation for 72 h, mainly in jointly immobilized treatments of both microalgae species, the cultures reached their highest total carbohydrate content (mainly as starch) and also the highest affinity and volumetric productivity. These results demonstrate the potential of A. brasilense to affect carbohydrates and starch accumulation in Chlorella spp. when both microorganisms are co-cultured, which can be an important tool for applications of microalgae.

Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: II. Heterotrophic conditions.

The effect of the bacterium Azospirillum brasilense jointly immobilized with Chlorella vulgaris or C. sorokiniana in alginate beads on total carbohydrates and starch was studied under dark and heterotrophic conditions for 144 h in synthetic growth medium supplemented with either d-glucose or Na-acetate as carbon sources. In all treatments, enhanced total carbohydrates and starch content per culture and per cell was obtained after 24h; only jointly immobilized C. vulgaris growing on d-glucose significantly increased total carbohydrates and starch content after 96 h. Enhanced accumulation of carbohydrate and starch under jointly immobilized conditions was variable with time of sampling and substrate used. Similar results occurred when the microalgae was immobilized alone. In both microalgae growing on either carbon sources, the bacterium promoted accumulation of carbohydrates and starch; when the microalgae were immobilized alone, they used the carbon sources for cell multiplication. In jointly immobilized conditions with Chlorella spp., affinity to carbon source and volumetric productivity and yield were higher than when Chlorella spp. were immobilized alone; however, the growth rate was higher in microalgae immobilized alone. This study demonstrates that under heterotrophic conditions, A. brasilense promotes the accumulation of carbohydrates in two strains Chlorella spp. under certain time-substrate combinations, producing mainly starch. As such, this bacterium is a biological factor that can change the composition of compounds in microalgae in dark, heterotrophic conditions.