Experimental Study and Modeling the Metabolism of Hydrogen Production in Algae-Bacteria Consortia

El hidrógeno molecular (H2) se considera una fuente de energía limpia y de alto contenido energético. La producción fotobiológica de H2 por algas verdes puede ser un método limpio y renovable para la generación de este gas. Chlamydomonas reinhardtii (Chlamydomonas) es una microalga verde unicelular capaz de llevar a cabo la foto-producción de H2. En este trabajo se estudia el efecto que la intensidad lumínica y diferentes fuentes de carbono y nitrógeno tienen en la producción de H2 en co-cultivos de Chlamydomonas con diferentes bacterias. El objetivo general es obtener un mejor conocimiento sobre cómo las interacciones alga-bacteria pueden mejorar la producción de H2. Primero, se estudiaron co-cultivos de Chlamydomonas con diferentes bacterias (Pseudomonas spp., Escherichia coli y Rhizobium etli) en medios ricos, con acetato como fuente de carbono y a tres intensidades de luz diferentes (12, 50 y 100 PPFD). El aumento de 0.87 a 18.2 ml de H2/L de cultivo fue la mayor mejora en la producción de H2 que se obtuvo cuando Chlamydomonas se cocultivó con Pseudomonas putida 12264 bajo 100 PPFD. Se obtuvieron mejoras en la producción de H2 en co-cultivos respecto a los monocultivos del alga. Estas mejoras estaban claramente relacionadas con la menor capacidad de los co-cultivos para consumir el ácido acético de los medios. Cuanto más tiempo permaneció el ácido acético en el medio, mayor fue la producción de H2. En el segundo estudio se observó que en co-cultivos incubados con azúcares como única fuente de carbono, la foto-producción de H2 por parte del alga es posible cuando las bacterias producían ácido acético. También observamos que el cocultivo de Chlamydomonas con Escherichia coli condujo a una producción sinérgica de H2 que produjo un 60% más de H2 en cocultivos en comparación con la suma de la producción en monocultivos de alga y bacteria. La acumulación de ácido acético es uno de los principales inconvenientes de la producción fermentativa de H2 llevada a cabo por bacterias. Sin embargo, este inconveniente puede convertirse en una ventaja cuando las bacterias productoras de H2 se cultivan conjuntamente con Chlamydomonas. En un tercer estudio, tres cepas de bacterias (Stenotrophomonas sp., Microbacterium sp. y Bacillus sp.) fueron aisladas e identificadas de una comunidad de bacterias silvestres. Se observó que Microbacterium es un socio bacteriano mutualista para Chlamydomonas en término de crecimiento. En medios de cultivo suplementados con azúcares y fuentes de nitrógeno inorgánico, Microbacterium no pudo crecer de forma aislada, sin embargo, sí lo pudo hacer cuando se co-cultivó con Chlamydomonas. Posiblemente el alga permite el crecimiento de Microbacterium al proporcionar nutrientes esenciales, probablemente fuentes de nitrógeno orgánico. Por otro lado, la producción de ácido acético por parte de Microbacterium, a partir de la fermentación de azúcares, favorece el crecimiento de Chlamydomonas y la fotoproducción de H2. Bajo esta cooperación, se produjo una cantidad considerable de H2, 313 ml/L de cultivo en cocultivos de Chlamydomonas-Microbacterium en medios ricos en azúcar. Finalmente, se logró un nivel aceptable de coordinación entre los resultados del modelado y los datos empíricos en términos de crecimiento, producción de H2 y absorción de ácido acético, tanto en co-cultivos de Chlamydomonas-Pseudomonas putida como en los monocultivos control. Este modelo de red metabólica basado en restricciones puede ser prometedor para predecir el comportamiento de organismos en sistemas de mono y co-cultivo.Hydrogen gas (H2) is considered a clean energy carrier with a very high energy content per mass. Photobiological production of H2 by green algae can potentially be a clean and renewable method for H2 generation. Chlamydomonas reinhardtii (Chlamydomonas) is a model unicelular green microalga capable of H2 photoproduction. In this work we studied the effect of light intensity and different carbon and nitrogen sources on H2 production in Chlamydomonas-bacteria co-cultures to gain a better knowledge on how alga-bacteria interactions can improve H2 production. First, we studied co-cultivation of Chlamydomonas with different bacteria, including Pseudomonas spp., Escherichia coli and Rhizobium etli cultured in acetate-containing nutrientreplete media at three different light intensities (12, 50 and 100 PPFD). Increasing from 0.87 to 18.2 ml H2/L culture, was the highest enhancement in H2 production which was obtained when Chlamydomonas was co-cultivated with Pseudomonas putida 12264 under 100 PPFD. Enhancement of H2 production in co-cultures was clearly related to the lower capacity of these co-cultures to consume the acetic acid from the media. The longer the acetic acid remained in the media, the longer the cultures were able to sustain hypoxia and support H2 production. Then, we found out that algal H2 photoproduction is possible in Chlamydomonas-bacteria co-cultures grow on sugars as the only carbon source when acetic acid is produced by the bacteria. These results suggested that acetic acid assimilation is linked to H2 production beside its ability to promote oxygen consumption. We also observed that co-culturing Chlamydomonas with Escherichia coli led to synergetic H2 production that 60% more H2 was produced in co-cultures compared with the sum of production in alga and bacterium monocultures. The accumulation of acetic acid is one of the main drawbacks of the dark fermentative H2 production. However, this drawback could be switched into an advantage when H2 producing bacteria are co-cultivated with Chlamydomonas. In the following, three bacteria strains including Stenotrophomonas sp., Microbacterium sp. and Bacillus sp. were isolated and identified from an unknown bacteria community. Microbacterium was found to be a mutualistic bacterial partner for Chlamydomonas in term of growth. In culture media supplemented with sugars and inorganic nitrogen source, Microbacterium alone was not able to grow, however it grew when co-cultivated with Chlamydomonas. It seemed that this alga allowed Microbacterium growth by providing essential key nutrients, probably organic nitrogen sources. On the other hand, Microbacterium was able to produce acetic acid through fermentation of sugars which favors Chlamydomonas growth and H2 production. Following this cooperative relationship, a considerable amount of H2, 313 ml/L culture was produced in Chlamydomonas- Microbacterium co-cultures in sugar-rich media. Finally, an acceptable level of coordination between the results of modeling and the empirical data in terms of growth, H2 production and acetic acid uptake in Chlamydomonas-Pseudomonas putida co-cultures and pure control cultures was achieved. Therefore, constraint-based metabolic network model can be a promising potential to predict and especially compare the behavior of organisms in mono- and co-culture systems

Algae-Bacteria Consortia as a Strategy to Enhance H2 Production

Biological hydrogen production by microalgae is a potential sustainable, renewable and clean source of energy. However, many barriers limiting photohydrogen production in these microorganisms remain unsolved. In order to explore this potential and make biohydrogen industrially affordable, the unicellular microalga Chlamydomonas reinhardtii is used as a model system to solve barriers and identify new approaches that can improve hydrogen production. Recently, Chlamydomonas–bacteria consortia have opened a new window to improve biohydrogen production. In this study, we review the different consortia that have been successfully employed and analyze the factors that could be behind the improved H2 production

Stenotrophomonas goyi sp. nov., a novel bacterium associated with the alga Chlamydomonas reinhardtii [version 2; peer review: 2 approved]

Background: A culture of the green algae Chlamydomonas reinhardtii was accidentally contaminated with three different bacteria in our laboratory facilities. This contaminated alga culture showed increased algal biohydrogen production. These three bacteria were independently isolated. Methods: The chromosomic DNA of one of the isolated bacteria was extracted and sequenced using PacBio technology. Tentative genome annotation (RAST server) and phylogenetic trees analysis (TYGS server) were conducted. Diverse growth tests were assayed for the bacterium and for the alga-bacterium consortium. Results: Phylogenetic analysis indicates that the bacterium is a novel member of the Stenotrophomonas genus that has been termed in this work as S. goyi sp. nov. A fully sequenced genome (4,487,389 base pairs) and its tentative annotation (4,147 genes) are provided. The genome information suggests that S. goyi sp. nov. is unable to use sulfate and nitrate as sulfur and nitrogen sources, respectively. Growth tests have confirmed the dependence on the sulfur-containing amino acids methionine and cysteine. S. goyi sp. nov. and Chlamydomonas reinhardtii can establish a mutualistic relationship when cocultured together. Conclusions: S. goyi sp. nov. could be of interest for the design of biotechnological approaches based on the use of artificial microalgae-bacteria multispecies consortia that take advantage of the complementary metabolic capacities of their different microorganisms