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# Production of polyhydroxybutyrate by the cyanobacterium cf. Anabaena sp. - ScienceDirect

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International Journal of Biological Macromolecules
Volume 191, 30 November 2021, Pages 92-99
Production of polyhydroxybutyrate by the cyanobacterium cf. Anabaena sp.
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Mara Simonazzi a
, 
Laura Pezzolesi a b
, Paola Galletti b c, Chiara Gualandi b d, Rossella Pistocchi a b, 
Nicole De Marco a
, 
Zoe Paganelli a
, Chiara Samorì b c
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https://doi.org/10.1016/j.ijbiomac.2021.09.054
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Highlights
•
Anabaena sp. accumulates 40% polyhydroxybutyrate (PHB) under mixotrophic conditions.
•
Phosphorus lack in the growth medium doubles the yield of PHB than nitrogen lack.
•
The molecular weight of the PHB extracted from Anabaena sp. was above 3 MDa.
Abstract
Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 gPHB gbiomass−1 d−1) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L−1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L−1, and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions.
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Introduction
Polyhydroxyalkanoates (PHAs) are a family of bio-based polyesters produced as intracellular inclusions in a wide variety of photosynthetic and heterotrophic organisms (e.g. the gram-negative bacterium Cupriavidus necator). The interest towards these biopolymers comes from their bio-based, biodegradable, compostable, and biocompatible nature, as well as on the possibility of tuning their chemo-physical characteristics by changing the carbon source used to feed the PHA-producers (the thermoplastic properties of PHAs can be tuned according to their co-monomer combination, that, in turns, is determined by the carbon source). These unique features make PHAs promising candidates for the replacement of petroleum-based non-biodegradable plastics. However, the current share of PHAs in the biopolymer market is limited to around 2% of all available biopolymers [1], and almost the entire global production is presently achieved by exploiting cultures of the gram-negative bacterium C. necator. Among the gram-negative bacteria known to produce PHAs, some species of Cyanobacteria have been described as capable of accumulating PHAs as secondary metabolites. Cyanobacteria are unicellular organisms constituting a phylogenetically coherent group of long evolutionary history prokaryotes, able to carry out oxygenic photosynthesis through chlorophyll a, as the major photosynthetic pigment, and phycobiliproteins that serve as light-harvesting pigments [2]. They are widely distributed throughout different aquatic and terrestrial environments, in which they play a key role in carbon and nitrogen fixation, thus impacting biogeochemical cycles in aquatic ecosystems as well as improving soil fertility [3]. Cyanobacteria are photoautotrophic and grow using CO2 as the sole source of carbon, although some strains have displayed an heterotrophic behavior especially when adapting to dark or semi-dark conditions, thus using other C-feedstocks like sugars under mixotrophic or heterotrophic regimes [4]. Cyanobacteria play significant roles both in the global primary production of the oceans (with a net primary production exceeding 1 Gt of wet biomass [2]), and in the biotechnological market [5], producing a variety of primary and secondary metabolites like proteins, phycobiliproteins (i.e. phycoerythrin and phycocyanin), and PHAs. PHAs can be produced by some Cyanobacteria strains via photosynthetic CO2-fixation (photoautotrophic conditions) [6], and their PHAs content is boosted when a mixotrophic metabolism is induced by the addition of organic carbon compounds (e.g. acetate) under specific growth conditions (e.g. lack of nitrogen) [7]. The accumulation of PHAs by Cyanobacteria seems to be species-specific, even if a general low PHA-productivity is usually observed: the best PHA-cyanobacterial producers (e.g. Nostoc muscorum, Synechococcus sp., or Synechocystis sp.) are capable of accumulating comparable amounts of PHAs than heterotrophic bacteria (e.g. Cupriavidus necator) (40–50 vs 60–70%) but their PHA-productivity is 150–200-folds lower (in the order of g L−1 d−1 for Cyanobacteria vs g L−1 h−1 for heterotrophic bacteria, respectively) [7]. The relatively lower PHB amount and productivity observed for Cyanobacteria have been ascribed to their larger cellular size and mass with thicker cell walls than heterotrophic bacterial cells [8], confirming similar PHA-synthesizing ability [9]. Moreover, it has been estimated that the costs related to the carbon source (the amount of exogenous carbon is more than one order of magnitude lower if compared to that needed by heterotrophic bacteria [6]), the nutrient requirements (Cyanobacteria can be cultivated in wastewaters, using inorganic nitrogen and phosphorus for their metabolism), and the oxygen supply necessary in PHAs production processes could be lower when Cyanobacteria are used than when heterotrophic bacteria like C. necator are used, de facto lowering the final PHAs cost [8], [9], [10], [11]. The capability of fixing nitrogen seems not to be directly correlated to the capability of producing PHAs, since among the best PHA-cyanobacterial producers, there are both nitrogen-fixing (e.g. Nostoc muscorum) and non-fixing Cyanobacteria (e.g. Synechocystis sp. and Synechococcus sp.) [12]. Among the diazotrophic Cyanobacteria, A. cylindrica has been reported to accumulate PHAs; however, this characteristic is not common to the other species of the Anabaena genus [13]. Although A. cylindrica accumulates polyhydroxybutyrate (PHB, the homopolymer constituted by 3-hydroxybutyrate units) when grown with acetate as carbon source and under nitrogen starvation, nor A. variabilis neither A. torulosa were able to accumulate the polymer under similar conditions [13], [14], [15]; the maximum PHB content accumulated by Anabaena spp. was 2% when cultivated with acetate and without nitrogen, 10–20 times lower than what was observed for other nitrogen-fixing Cyanobacteria such as Nostoc muscorum [12], [13].
The present paper aims at deepening the knowledge on PHB production by a newly isolated strain of the genus Anabaena to better understand its potential as a PHB producer. The growth conditions were initially optimized to maximize PHB accumulation, and then the cultivation was scaled up (10 L) to study the whole biomass composition (in terms of PHB, lipids, fatty acids, polysaccharides, and proteins) and the PHB recovery in terms of extraction yields and PHB characteristics.
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Section snippets
Chemicals
All reagents, solvents and chemicals used in this work were of analytical grade and purchased from various commercial suppliers. Commercial polyhydroxybutyrate (PHB) was bought from Biomer (Germany).
Cf. Anabaena sp. isolation
The cyanobacterial strain was isolated from surface water collected in Reno river (Emilia-Romagna, Italy) in July 2018, and morphologically identified as cf. Anabaena sp. (hereafter referred to as Anabaena sp.) using an inverted light microscope (ZEISS Axiovert 100) at 320× magnification according
Anabaena sp. growth under balanced and mixotrophic conditions
Anabaena sp. was initially cultivated in a nitrogen-free (ØN) growth medium with sodium acetate (NaOAc, 5.0 g L−1); the culture was placed in a low-light environment for 7 and 11 days and compared with the control (balanced growth medium in a low-light environment) in terms of PHB content, dry weight, and photosynthetic efficiency (Table 1, entries 1–3). As expected, Anabaena sp. grown in a balanced medium with no additional source of NaOAc did not accumulate PHB (entry 1), in line with what
Conclusion
The mixotrophic production of PHB by Cyanobacteria is rarely studied in comparison to the heterotrophic one operated by non-photosynthetic microorganisms. However, some cyanobacterial strains have been reported to accumulate high PHB amounts, comparable to what occurs for heterotrophic bacteria. The unique potential of Cyanobacteria to produce PHB exploiting CO2 as the sole carbon source can be boosted when an exogenous carbon source is provided, nutrients are limited, or stress conditions are
CRediT authorship contribution statement
C.S. and M.S. have made substantial contributions to the conception and design of the study; C.G., M.S., N.D., and Z.P. have made substantial contributions to the acquisition, collection and assembly of data; C.S., M.S., L.P., and C.G. have made substantial contributions to the analysis and interpretation of data; C.S. has made substantial contributions to the drafting of the article; P.G., R.P. and L.P. have made substantial contributions to the critical revision of the article for important
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
We acknowledge Romagna Acque - Società delle Fonti S.p.A. sede di Ravenna (IT) for PhD funding.
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