Resumen de Estudios experimentales sobre el metabolismo de la producción de hidrógeno en consorcios de algas-bacterias
1. introducción o motivación de la tesis Hydrogen gas (H2) is considered a clean energy carrier with a very high energy content per mass (Graetz, 2009). Photobiological production of H2 by green algae can potentially be a clean and renewable method for H2 generation. Chlamydomonas reinhardtii (Chlamydomonas) is a model unicellular green microalga capable of H2 photoproduction (Melis et al., 2000). However, photobiological H2 production is still far from commercialization due to low rate and yield of production. One of the main bottlenecks of this process is that oxygen (O2) which is inevitably produced through photosynthesis inhibits both expression and activity of hydrogenases which are in charge of H2 production in Chlamydomonas (Stiebritz and Reiher, 2012). To overcome this issue, co-culturing Chlamydomonas with bacteria, following the idea that bacterial respiration keeps the O2 level in the culture low enough for hydrogenase activity has attracted attention in recent decades (Lakatos et al., 2014; Xu et al., 2017; Ban et al., 2018; He et al., 2018). 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. Also, constraint-based metabolic network modeling was applied to investigate the potential of mathematical model to predict alga-bacteria interactions.
2.contenido de la investigación First, we studied co-cultivation of Chlamydomonas with different bacteria, including Pseudomonas putida 12264, Pseudomonas putida 291, Pseudomonas stutzeri, Escherichia coli and Rhizobium etli cultured in acetate-containing nutrient-replete media at three different light intensities (12, 50 and 100 PPFD). Compared with Escherichia coli and Rhizobium etli, Pseudomonas spp. showed stronger impact on H2 production in co-cultures. Co-cultivation with Pseudomonas spp. enhanced H2 production in Chlamydomonas at all light intensities. As reported before, at low light intensity (12PPFD) Chlamydomonas monocultures are able to reach hypoxia and produce considerable amount of H2 (Jurado-Oller et al., 2015). Therefore, at higher light intensities the influence of bacteria co-cultivation on improving H2 production was more evident. Improvement from 0.87 to 18.2 ml H2/L culture, was the highest enhancement in H2 production, 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. Results showed that the longer acetic acid remained in the media, the longer the cultures were able to sustain hypoxia and support H2 production.
Then, we found out that Chlamydomonas was able to produce H2 in sugar containing media as the sole carbon source, when it was co-cultured with Pseudomonas putida 12264 (40.8 ml H2/L culture), Escherichia coli (35.1 ml H2/L culture) and Rhizobium etli (16.1 ml H2/L culture). All the bacteria were able to produce acetic acid through fermentation of sugars which was a key for induction of hypoxia and H2 production. Also, results suggested that acetic acid assimilation is linked to H2 production beside its ability to promote oxygen consumption. Escherichia coli excreted more acetic acid to the medium compared to the two other bacteria which correlated well with high amount of H2 production in Chlamydomonas-Escherichia coli co-cultures on the first day. However, low amount of acetic acid produced by Pseudomonas putida and Rhizobium etli led to sustained and slow H2 production in the corresponding co-cultures. Escherichia coli produces high amount of organic acids through fermentation of glucose (Stokes, 1949), which reduced the pH of the medium to 4.5. Therefore, intense H2 production in Escherichia coli co-cultures lasted only for one day which was probably due to impairment of Chlamydomonas cells in low pH medium. 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 by bacteria (Oh et al., 2011; Ding et al., 2016). However, this drawback could be switched into an advantage when H2 producing bacteria are co-cultivated with Chlamydomonas since acetic acid uptake is in favor of H2 production by both alga and bacterium.
In the following, three bacteria strains including Stenotrophomonas sp., Microbacterium sp. and Bacillus sp. were isolated and identified from an unknown bacteria community. Out of three bacteria, Microbacterium had a considerable effect on H2 production in Chlamydomonas-Microbacterium co-cultures in sugar rich media. Microbacterium was able to grow only in presence of organic compounds like tryptone, yeast extract or one of the 20 amino acids. In culture media supplemented with sugars and inorganic nitrogen source, Microbacterium alone was not able to grow, however it grew when it was 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. Microbacterium was found to be a mutualistic bacterial partner for Chlamydomonas in term of growth. 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.
3.conclusión In summary, alga-bacteria co-cultures are promising candidates for biological H2 production. Presence of acetic acid in the culture media is a key parameter which can control hypoxia and extend H2 production. Therefore, acetic acid produced by bacteria through fermentation of sugars can lead to sustained H2 production in Chlamydomonas-bacteria co-cultures. In addition, co-cultivation of Chlamydomonas with anaerobic bacteria in sugar rich media supports synergetic H2 production since acetic acid uptake by Chlamydomonas is in favor of H2 production by both alga and bacterium. Furthermore, a mutualistic relationship between Chlamydomonas and Microbacterium in co-cultures supplemented with sugars leads to sustained and high yield of H2 production.
Algae-bacteria interactions are hard to understand, and they are time and energy consuming. In this study, constraint-based metabolic network modeling shows considerable potential to be an efficient tool to predict the behavior of Chlamydomonas-bacteria co-cultures.
4. bibliografía Ban S, Lin W, Wu F, Luo J (2018) Algal-bacterial cooperation improves algal photolysis-mediated hydrogen production. Bioresour Technol 251: 350–357 Ding C, Yang KL, He J (2016) Biological and fermentative production of hydrogen. Handb Biofuels Prod Process Technol Second Ed. doi: 10.1016/B978-0-08-100455-5.00011-4 Graetz J (2009) New approaches to hydrogen storage. Chem Soc Rev 38: 73–82 He J, Xi L, Sun X, Ge B, Liu D, Han Z, Pu X, Huang F (2018) Enhanced hydrogen production through co-cultivation of Chlamydomonas reinhardtii CC-503 and a facultative autotrophic sulfide-oxidizing bacterium under sulfurated conditions. Int J Hydrogen Energy 43: 15005–15013 Jurado-Oller JL, Dubini A, Galván A, Fernández E, González-Ballester D (2015) Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures. Biotechnol Biofuels 8: 149 Lakatos G, Deák Z, Vass I, Rétfalvi T, Rozgonyi S, Rákhely G, Ördög V, Kondorosi É, Maróti G (2014) Bacterial symbionts enhance photo-fermentative hydrogen evolution of Chlamydomonas algae. Green Chem 16: 4716–4727 Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M, Cournac L, Peltier G (2000) Sustained Photobiological Hydrogen Gas Production upon Reversible Inactivation of Oxygen Evolution in the Green Alga Chlamydomonas reinhardtii. Plant Physiol 122: 127–136 Oh YK, Raj SM, Jung GY, Park S (2011) Current status of the metabolic engineering of microorganisms for biohydrogen production. Bioresour Technol 102: 8357–8367 Stiebritz MT, Reiher M (2012) Hydrogenases and oxygen. Chem Sci 3: 1739–1751 Stokes JL (1949) Fermentation of glucose by suspensions of Escherichia coli. J Bacteriol 57: 147–58 Xu L, Cheng X, Wu S, Wang Q (2017) Co-cultivation of Chlamydomonas reinhardtii with Azotobacter chroococcum improved H2 production. Biotechnol Lett 39: 731–738
