ABSTRACTConcerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained anAzotobacter vinelandiimutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.

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16There is currently much interest in developing technology to use microlgae or 17 cyanobacteria for the production of bioenergy and biomaterials. Here we summarized 18 some remarkable achievements in strains improvement by traditional genetic 19 engineering and discussed common drawbacks for further progress. We present 20 general knowledge on natural microalgae-bacterial mutualistic interactions and discuss 21 the potential of recent developments in genetic engineering of multi-species microbial 22 cell-factories. This synthetic biology approach would rely on the assembly of complex 23 metabolic networks from optimized metabolic modules such as photosynthetic or 24 nitrogen-fixing parts. 25 26 3 Photosynthetic microbes for the production of bioenergy and biomaterials 27Increasing food and energy demand, global climate change and general environmental 28 decay are some of the main current challenges for Humankind. Biofuels, among other 29 alternative energy sources, have great potential to harmonize the food-energy-30 environment trilemma [1,2]. Photosynthetic organisms including plants, algae and 31 cyanobacteria capture solar energy and store it as chemical energy of their biomass 32 (bioenergy). Thus, agriculture might serve as a source of food and bioenergy. Since 33 bioenergy is mostly captured by photosynthetic CO 2 fixation, carbon-containing 34 biofuels have a varied tendency to be carbon neutral after combustion. The extent to 35 which this is accomplished largely depends on the nature of the feedstock, the 36 agricultural practice and the industrial process, and thus it might proportionally 37 contribute to climate change mitigation [3]. First-generation biofuels were based on 38 edible feedstocks. Consequently, several alternative feedstocks have been proposed, 39 mainly to alleviate food-bioenergy competition and land use change, leading to second 40 generation or more advanced biofuels [4]. 41The use of microbial cell factories for bioenergy or related purposes, although not 42 new, has regained attention as a result of increasing pressure for higher productivities, 43 novel bioproducts and environment protection. It is widely appreciated that the 44 microbial world contains by far the greatest fraction of biodiversity in the biosphere, 45and thus a corresponding innovation potential [5]. According to the advantages listed 46 in Box 1, there is currently much interest in developing the technology for the use of 47 photosynthetic microorganisms, such as eukaryotic microlgae or cyanobacteria [6,7]. 48Although some startup companies are already attempting to commercialize algal fuels 49[8], their actual potential is still a matter of debate [9]. 50 4 Identifying suitable microalgae strains is usually a starting point in the roadmap 51 towards microalgae-based technology development. The most appreciated traits for the 52 "ideal microalga" are listed in Box 2 [6,7]. Currently, there are no available strains 53 excelling in all these traits, what is not surprising considering that, conversely t...