Miranda et al. Biotechnol Biofuels (2017) 10:120 DOI 10.1186/s13068-017-0798-9 Biotechnology for Biofuels RESEARCH Open Access Applications of microalgal bioflms for wastewater treatment and bioenergy production Ana F. Miranda1, Narasimhan Ramkumar2, Constandino Andriotis1, Thorben Höltkemeier3, Aneela Yasmin4, Simone Rochfort5, Donald Wlodkowic1, Paul Morrison1, Felicity Roddick6, German Spangenberg5, Banwari Lal2, Sanjukta Subudhi2 and Aidyn Mouradov1* Abstract Background: Microalgae have shown clear advantages for the production of biofuels compared with energy crops. Apart from their high growth rates and substantial lipid/triacylglycerol yields, microalgae can grow in wastewaters (animal, municipal and mining wastewaters) efciently removing their primary nutrients (C, N, and P), heavy met- als and micropollutants, and they do not compete with crops for arable lands. However, fundamental barriers to the industrial application of microalgae for biofuel production still include high costs of removing the algae from the water and the water from the algae which can account for up to 30–40% of the total cost of biodiesel production. Algal bioflms are becoming increasingly popular as a strategy for the concentration of microalgae, making harvest- ing/dewatering easier and cheaper. Results: We have isolated and characterized a number of natural microalgal bioflms from freshwater, saline lakes and marine habitats. Structurally, these bioflms represent complex consortia of unicellular and multicellular, photosyn- thetic and heterotrophic inhabitants, such as cyanobacteria, microalgae, diatoms, bacteria, and fungi. Bioflm #52 was used as feedstock for bioenergy production. Dark fermentation of its biomass by Enterobacter cloacae DT-1 led to the production of 2.4 mol of ­H2/mol of reduced sugar. The levels and compositions of saturated, monosaturated and poly- unsaturated fatty acids in Bioflm #52 were target-wise modifed through the promotion of the growth of selected individual photosynthetic inhabitants. Photosynthetic components isolated from diferent bioflms were used for tailoring of novel bioflms designed for (i) treatment of specifc types of wastewaters, such as reverse osmosis concen- trate, (ii) compositions of total fatty acids with a new degree of unsaturation and (iii) bio-focculation and concentra- tion of commercial microalgal cells. Treatment of diferent types of wastewaters with bioflms showed a reduction in the concentrations of key nutrients, such as phosphates, ammonia, nitrates, selenium and heavy metals. Conclusions: This multidisciplinary study showed the new potential of natural bioflms, their individual photosyn- thetic inhabitants and assembled new algal/cyanobacterial bioflms as the next generation of bioenergy feedstocks which can grow using wastewaters as a cheap source of key nutrients. Keywords: Bioflms, Bio-hydrogen, Biofuel, Cyanobacteria, Microalgae, Wastewater treatment Background search for the next generation of bioenergy feedstocks Extensive use of arable lands and high consumption of which will meet key selection criteria: (i) high growth freshwater by energy crops have triggered an intensive rates/biomass production; (ii) high content of bioenergy- producing molecules; (iii) high harvesting index and short rotation period; (iv) ability to grow on marginal *Correspondence: [email protected] lands and lack of competition with agricultural crops for 1 School of Sciences, RMIT University, Bundoora, VIC, Australia Full list of author information is available at the end of the article arable lands; (v) low freshwater usage; (vi) low costs for © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Miranda et al. Biotechnol Biofuels (2017) 10:120 Page 2 of 23 growth and harvest; and (vii) production of high-value In natural ecosystems, algal bioflms represent three- co-products [1]. Te use of wastewater as a source of dimensional, multilayered and multispecies structures key nutrients would signifcantly improve the econom- which involve consortia of heterotrophic and photoau- ics of biofuel production and reduce its energy require- totrophic prokaryotic and eukaryotic organisms [21–27]. ments. Tis has shifted attention from the application Photosynthetic organisms include flamentous and uni- of terrestrial energy crops towards the use of microal- cellular macro- and microalgae, and cyanobacterial spe- gae and aquatic plants. Microalgae have shown clear cies. Tese organisms are characterized by their typical advantages for the production of biofuels compared with pigment content, which is used to capture solar energy energy crops. Apart from their high-growth rates and and to protect the cells from radiation [28]. Heterotrophic substantial lipid/triacylglycerol (TAG) yields, micro- organisms can include protozoa, fagellates, bacterial and algae can grow in wastewater efciently removing the fungal cells. In this highly heterogeneous structure, dif- primary nutrients, heavy metals, and micropollutants. ferent species usually colonize diferent zones within the And, microalgae do not compete with crops for arable bioflm structure that are most suitable for their growth land [2–4]. However, some fundamental barriers to the [21]. Heterotrophic and photoautotrophic organisms in industrial application of microalgae for biofuel produc- this complex and highly productive ecosystems can be tion still exist and include high costs for their growth, held together by a matrix of EPS, the functions of which harvesting and high freshwater requirements. Te main include adhesion, aggregation, retention of water and techniques used for harvesting and concentration of nutrients, difusion barrier for toxins and heavy met- microalgal cells (centrifugation, fltration, focculation, als, cell motility, protection barriers against grazers and gravity sedimentation and fotation) are still not eco- harmful chemicals or environmental conditions [24, 29– nomically viable for the large-scale microalgal industry 31]. EPS, which can account for 90% of dry bioflm mass, [5–13]. Bio-focculation methods are becoming increas- are typically composed of the high molecular weight het- ingly popular because of their high efciency and low eropolysaccharides containing linear or branched repeat- energy input [9, 14, 15]. Fungal-assisted bio-focculation ing units comprising 2–10 monosaccharides, such as as a new strategy for attachment and concentration of hexoses, pentoses, glucose, mannose, arabinose, uronic microalgal cells within fungal flaments, via hydrogen acids, and deoxy-sugars. A low contamination of intracel- bonds, electrostatic interactions and/or using a matrix lular polymers and proteins in EPS suggests low rates of of extracellular polymeric substance (EPS) secreted by cell rupture within bioflms [24]. fungal and algal cells, started attracting attention since Wastewater treatment is one of the favourable applica- it was shown to be highly efcient for the concentra- tions of algal bioflm systems because they ofer a sim- tion of microalgal cells, does not require added chemi- ple, energy-efcient technology for absorption of the key cals and has a low energy input requirement [6, 16–20]. nutrients, nitrogen and phosphorus followed by easy and Moreover, co-cultivation of microalgae and a flamen- robust separation of the algal biomass from the bulk of tous fungus showed an additive efect on total biomass the wastewater [32–37]. However, in spite of the obvious production, lipid yield, and wastewater treatment ef- advantages of low-cost nutrient removal and low energy ciency. Application of alternative sources of carbon from biomass production of bioflm-based technology, wide- lignocellulosic wastes, nitrogen, and phosphorus from spread application of algal bioflm-based treatment of wastewaters for fungal and algal growth improves the municipal, industrial, and agricultural waste streams has economics of biofuel production [19, 20]. Tis strategy, been limited so far [38]. however, is limited because of environmental concerns Reverse osmosis (RO) technology is being used world- over potential contamination of treated water with fun- wide for full-scale municipal wastewater reclamation to gal spores as a result of large-scale fungal production. produce high-quality recycled water to meet the increas- Moreover, the addition of carbon sources such as glu- ing water supply demand [39]. However, this technology cose for heterotrophic fungal growth under non-sterile generates highly saline RO concentrate (ROC) streams, conditions will trigger growth of microbial populations, which contain almost all of the contaminants and nutri- which will signifcantly limit the application of this ents derived from the secondary efuent at elevated technology under environmental, non-axenic condi- levels (4–6 times higher) [40]. With commercial scale tions. Tis concern has triggered a search for alterna- capacities up to 3 million m3/day of clean water, genera- tive environmentally friendly and low-cost strategies