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# Pilot-scale production of poly-β-hydroxybutyrate with the cyanobacterium Synechocytis sp. CCALA192 in a non-sterile tubular photobioreactor - ScienceDirect

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Algal Research
Volume 34, September 2018, Pages 116-125
Pilot-scale production of poly-β-hydroxybutyrate with the cyanobacterium Synechocytis sp. CCALA192 in a non-sterile tubular photobioreactor
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Clemens Troschl a
, 
Katharina Meixner a
, 
Ines Fritz a
, 
Klaus Leitner a
, 
Alejandra Palacios Romero a
, 
Adriana Kovalcik b
, Petr Sedlacek b, 
Bernhard Drosg a
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https://doi.org/10.1016/j.algal.2018.07.011
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Highlights
•
The biopolymer poly-β-hydroxybutyrate (PHB) was produced in four production cycles over a period of 75 days
•
A special cultivation strategy with partially anoxic conditions was successfully applied
•
Maximum biomass concentration was 1.0 g/L and maximum PHB concentration was 12.5% of cell dry weight
•
A three-stage cultivation mode with a ripening stage is proposed
Abstract
The biopolymer poly-β-hydroxybutyrate (PHB) can be used as a promising bioplastic. It has a broad range of applications and is degraded relatively rapidly by soil organisms. Like many prokaryotes, the cyanobacterium Synechocystis sp. CCALA192 produces this biopolymer as a storage compound, especially under nutrient limitation.
In a 200-L tubular photobioreactor, we cultivated Synechocystis sp. CCALA192 semi-continuously over a period of 75 days with CO2 as sole carbon source. A two-stage cultivation strategy was performed, where after 5–7 days nitrogen was depleted and the culture started to produce PHB and gradually turned from blue-green to yellow. After 16–20 days, 90% of the culture were harvested and the residual 10% were used as inoculum for the following cycle. The harvested culture had an average biomass concentration of 1.0 g/L with an average PHB content of 12.5% of cell dry weight. After restarting with fresh nutrients, the yellow culture turned blue-green again and degraded the PHB within 24–48 h. When nitrogen of the medium was consumed, PHB was produced again and the cycle continued. In the late stage of each production cycle, a ripening process was observed, where no CO2 was consumed but the PHB concentration was still rising at the expense of the existing glycogen rich biomass.
Establishing a stable Synechocystis sp. CCALA192 culture under non-sterile conditions turned out to be difficult, as this small unicellular organism is very sensitive and easily grazed by protozoa. Therefore, a special cultivation strategy with partially anoxic conditions was necessary.
Introduction
According to the Intergovernmental Panel on Climate Change (IPCC), scientific evidence for anthropogenic carbon dioxide (CO2) as the main driver for climate change is unequivocal [1]. Therefore, more and more effort has been made to utilize CO2 as raw material and transform it into different products [2]. Apart from the chemical possibilities, there are also biotechnological ways using CO2 as a substrate. Cultivating microalgae with CO2 as carbon source is one promising approach [3, 4] and there is a wide variety of microalgal products, ranging from fuels like biodiesel [[5], [6], [7], [8]] and bioethanol [9, 10] over platform chemicals like isoprene [11] to high-value products like polyunsaturated fatty acids or astaxanthin [12, 13].
Another interesting product synthesized by cyanobacteria, formerly known as blue-green algae, is the bioplastic poly-β-hydroxybutyrate (PHB). PHB is the most common polymer in the group of polyhydroxyalkanoates (PHAs) and the only PHA produced, when using CO2 as sole carbon source. It has favorable mechanical properties similar to polypropylene and can be processed by thermoplastic methods, including fiber spinning or injection molding. But in opposite to petroleum-based polypropylene, it is compostable and degraded rapidly by soil organisms [14]. This could avoid serious environmental problems caused by spillage and litter from persistent plastics. For example, in the Austrian Danube river, plastic litter had temporarily outnumbered fish larvae. The small plastic particles are ingested by a wide range of organisms with yet unknown consequences [15].
Cyanobacteria produce PHB as an intracellular energy and carbon storage compound. The most important trigger for PHB production is nutrient deprivation, especially nitrogen limitation. The role of PHB in cyanobacteria is still not clear; besides its storage function, it is supposed that it serves as an electron sink and helps to restore NADP+ and maybe supports the cell's stress resistance [16]. Generally, PHB concentrations in nutrient-deprived, photoautotrophic cyanobacteria range between 5 and 20% of cell dry weight (cdw), which is still quite low, compared to heterotrophic PHB producers with concentrations above 70% of cdw. However, in contrast to heterotrophic bacteria, cyanobacteria do not consume sugars, that are responsible for an estimated 50% of the total production costs [17]. Furthermore, cyanobacteria do not depend on agricultural crops, what makes them even more attractive as sustainable biomass producers.
There are numerous reports about PHB production with cyanobacteria: Kamravamanesh et al. produced 16.4% PHB of cdw with Synechocystis sp. PCC6714 [18]. Panda et al. produced 11.2% PHB of cdw with Synechocystis sp. PCC6803 [19]. Khetkorn et al. produced 26% PHB of cdw with a genetically modified Synechocystis sp. PCC6803 [20]. An exceptional high PHB content of 55% of cdw was produced with the thermophilic Synechococcus sp. MA19 [21]. Generally higher PHB contents could be produced with adding carbon sources like acetate or glucose. With this strategy, Bhati et al. produced 78% PHB of cdw with Nostoc Muscorum Agardh [22]. However, the addition of organic carbon sources will change the metabolism from autotrophic growth to heterotrophic growth. For further examples we recommend the latest reviews about this topic [[23], [24], [25], [26]].
Almost all of the reported experiments were performed under sterile laboratory conditions and there are hardly any reports about pilot-scale production of PHB under non-sterile conditions. Apart from Arthrospira sp., which can be grown in a highly alkaline environment, cyanobacteria are not grown in an industrial scale. Arthrospira sp. produces comparable low amounts of PHB, therefore other strains like Synechocystis sp. or Synechococcus sp. are more promising. There are next to no reports about full-scale production plants with these small cyanobacteria. Indications are strong that the primary reason may be the difficulty of establishing a stable process – avoiding disturbances or overgrowth by other microorganisms. The main difference of large photobioreactors compared to lab-scale systems is the impossibility of sterilization. Therefore, large photobioreactors are susceptible to biological pollutants. Contamination problems due to protozoa are a major drawback when cultivating microalgae in an industrial scale [27].
This work investigates the pilot-scale production of PHB with Synechoystis sp. CCALA192 in a non-sterile photobioreactor with a volume of 200 L. Over a period of 75 days, four production cycles were performed with a special cultivation strategy of partially anoxic conditions. Several parameters such as biomass concentration, PHB concentration, glycogen concentration and CO2 consumption were measured. The culture was routinely examined under the microscope and occurring contaminants are described. Furthermore, a new operation mode is proposed for a more efficient PHB production with cyanobacteria.
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Section snippets
Organism and culture conditions for strain maintenance
Synechocystis sp. CCALA192 was ordered from the Culture Collection of Autotrophic Organisms, Institute of Botany, Trebon, Czech Republic. This strain was chosen as it proved to be the most promising PHB producer within a previously performed screening of roughly 30 strains. For strain maintenance in the laboratory, the strain was cultivated in BG11 medium [28] supplemented with 1 g/L sodium hydrogencarbonate (NaHCO3) with a resulting pH of 8.5. Shaking flasks containing 50 mL of medium were
Description of the culture and contamination monitoring
We previously described the necessary contamination control for the non-sterile cultivation of Synechocystis sp. [33]. Stable cultivation of this small cyanobacterium was only possible with partially anoxic conditions. Through degassing with pure nitrogen, the oxygen in the culture was removed during the night and inhibited the growth of the ciliate Colpoda steinii. This cultivation method was performed throughout all cultivations in this study. Furthermore, Synechocystis sp. CCALA192 was
Conclusion
This study demonstrates the long term, non-sterile cultivation of Synechocystis sp. CCALA192 in a tubular photobioreactor for PHB production. A special cultivation strategy is necessary to maintain a stable culture, as this small unicellular cyanobacterium is vulnerable to predators. However, even with selective conditions such as high pH values and partially anoxic conditions, the cyanobacterial culture appeared as a complex ecosystem and side organisms such as bacteria, ciliates, amoeba and
Acknowledgements
This research was thankfully financed by the Austrian climate and energy fund and FFG (Austrian Research Promotion Agency). Grant number is 848783. The authors further acknowledge the support by the project “Materials Research Centre – Sustainability and Development” Nr. LO1211 - Ministry of Education, Youth and Sports of the Czech Republic.
Declaration of author's contribution
Clemens Troschl designed and performed experiments, analyzed data and wrote the article. Katharina Meixner has done substantial work at the photobioreactor and revised the manuscript. Klaus Leitner and Alejandra Palacios Romero performed experiments. Adriana Kovalcik and Petr Sedlacek analyzed the purified PHB sample and revised the manuscript. Ines Fritz and Bernhard Drosg supervised work, analyzed data and revised the manuscript.
Conflict of interest statement
The authors declare no conflict of interest.
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights applicable.
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