Poly-ß-hydroxybutyrate production by Synechocystis MT_a24 in a raceway pond using urban wastewater.

Poly-ß-hydroxybutyrate (PHB) is a potential source of biodegradable plastics that are environmentally friendly due to their complete degradation to water and carbon dioxide. This study aimed to investigate PHB production in the cyanobacterium Synechocystis sp. PCC6714 MT_a24 in an outdoor bioreactor using urban wastewater as a sole nutrient source. The culture was grown in a thin-layer raceway pond with a working volume of 100 L, reaching a biomass density of up to 3.5 g L-1 of cell dry weight (CDW). The maximum PHB content was found under nutrient-limiting conditions in the late stationary phase, reaching 23.7 ± 2.2% PHB per CDW. These data are one of the highest reported for photosynthetic production of PHB by cyanobacteria, moreover using urban wastewater in pilot-scale cultivation which multiplies the potential of sustainable cultivation approaches. Contamination by grazers (Poterioochromonas malhamensis) was managed by culturing Synechocystis in a highly alkaline environment (pH about 10.5) which did not significantly affect the culture growth. Furthermore, the strain MT_a24 showed significant wastewater nutrient remediation removing about 72% of nitrogen and 67% of phosphorus. These trials demonstrate that the photosynthetic production of PHB by Synechocystis sp. PCC6714 MT_a24 in the outdoor thin-layer bioreactor using urban wastewater and ambient carbon dioxide. It shows a promising approach for the cost-effective and sustainable production of biodegradable carbon-negative plastics. KEY POINTS: • High PHB production by cyanobacteria in outdoor raceway pond • Urban wastewater used as a sole source of nutrients for phototrophic growth • Potential for cost-effective and sustainable production of biodegradable plastics.

Hibberdia magna (Chrysophyceae): a promising freshwater fucoxanthin and polyunsaturated fatty acid producer.

BACKGROUND: Algae are prominent producers of carotenoids and polyunsaturated fatty acids which are greatly prized in the food and pharmaceutic industry. Fucoxanthin represents a notable high-value carotenoid produced exclusively by algae. Its benefits range far beyond just antioxidant activity and include cancer prevention, anti-diabetes, anti-obesity, and many other positive effects. Accordingly, large-scale microalgae cultivation to produce fucoxanthin and polyunsaturated fatty acids is still under intensive development in the commercial and academic sectors. Industrially exploitable strains are predominantly derived from marine species while comparable freshwater fucoxanthin producers have yet to be explored. RESULTS: In this study, we searched for freshwater fucoxanthin producers among photoautotrophic flagellates including members of the class Chrysophyceae. The initial screening turned our attention to the chrysophyte alga Hibberdia magna. We performed a comprehensive cultivation experiments using a temperature × light cross-gradient to assess the impact of these conditions on the target compounds productivity. Here we present the observations that H. magna simultaneously produces fucoxanthin (max. 1.2% dry biomass) and polyunsaturated fatty acids (max. ~ 9.9% dry biomass) and is accessible to routine cultivation in lab-scale conditions. The highest biomass yields were 3.73 g L-1 accompanied by maximal volumetric productivity of 0.54 g L-1 d-1 which are comparable values to marine microalgae fucoxanthin producers in phototrophic mode. H. magna demonstrated different optimal conditions for biomass, fucoxanthin, and fatty acid accumulation. While maximal fucoxanthin productivities were obtained in dim light and moderate temperatures (23 °C× 80 µmol m-2 s-1), the highest PUFA and overall biomass productivities were found in low temperature and high light (17-20 °C × 320-480 µmol m-2 s-1). Thus, a smart biotechnology setup should be designed to fully utilize H. magna biotechnological potential. CONCLUSIONS: Our research brings pioneer insight into the biotechnology potential of freshwater autotrophic flagellates and highlights their ability to produce high-value compounds. Freshwater fucoxanthin-producing species are of special importance as the use of sea-water-based media may increase cultivation costs and prohibits inland microalgae production.

Photosynthetic performance of Chlamydopodium (Chlorophyta) cultures grown in outdoor bioreactors.

The microalga Chlamydopodium fusiforme MACC-430 was cultured in two types of outdoor pilot cultivation units-a thin-layer cascade (TLC) and a raceway pond (RWP) placed in a greenhouse. This case study aimed to test their potential suitability for cultivation scale-up to produce biomass for agriculture purposes (e.g., as biofertilizer or biostimulant). The culture response to the alteration of environmental conditions was evaluated in "exemplary" situations of good and bad weather conditions using several photosynthesis measuring techniques, namely oxygen production, and chlorophyll (Chl) fluorescence. Validation of their suitability for online monitoring in large-scale plants has been one of the objectives of the trials. Both techniques were found fast and robust reliable to monitor microalgae activity in large-scale cultivation units. In both bioreactors, Chlamydopodium cultures grew well in the semi-continuous regime using daily dilution (0.20-0.25 day-1). The biomass productivity calculated per volume was significantly (about 5 times) higher in the RWPs compared to the TLCs. The measured photosynthesis variables showed that the build-up of dissolved oxygen concentration in the TLC was higher, up to 125-150% of saturation (%sat) as compared to the RWP (102-104%sat). As only ambient CO2 was available, its shortage was indicated by a pH increase due to photosynthetic activity in the thin-layer bioreactor at higher irradiance intensities. In this setup, the RWP was considered more suitable for scale-up due to higher areal productivity, lower construction and maintenance costs, the smaller land area required to maintain large culture volumes, as well as lower carbon depletion and dissolved oxygen build-up. KEY POINTS: • Chlamydopodium was grown in both raceways and thin-layer cascades in pilot-scale. • Various photosynthesis techniques were validated for growth monitoring. • In general, raceway ponds were evaluated as more suitable for cultivation scale-up.

Impact of photobioreactor design on microalgae-bacteria communities grown on wastewater: Differences between thin-layer cascade and thin-layer raceway ponds.

Thin-layer (TL) photobioreactors (PBRs) are characterised by high productivity. However, their use is limited to lab/pilot-scale, and a deeper level of characterisation is needed to reach industrial scale and test the resistance of multiple microalgae. Here, the performance and composition of eight microalgal communities cultivated in the two main TLs design (thin-layer cascade (TLC) and thin-layer raceway pond (RW)) were investigated through Illumina sequencing. Chlorella vulgaris showed robustness in both designs and often acted as an "invasive" species. Inoculum and reactor type brought variability. Eukaryotic microalgae inocula led to a more robust and stable community (higher similarity), however, RWs were characterised by a higher variability and did not favour the eukaryotic microalgae. The only cyanobacterial inoculum, Nostoc piscinale, was maintained, however the community was variable between designs. The reactor design had an effect on the N cycle with the TLC and RW configurations, enhancing nitrification and denitrification respectively.

Fractionation of the water insoluble part of the heterotrophic mutant green microalga Parachlorella kessleri HY1 (Chlorellaceae) biomass: Identification and structure of polysaccharides.

The water-insoluble part of Parachlorella kessleri HY1 biomass was subjected to the extraction of cell-wall polysaccharides using polar aprotic solvents (DMSO, LiCl/DMSO) and aqueous alkaline solutions (0.1, 1 and 4 mol·l-1 of NaOH). Proteins predominated in all the crude extracts and in the insoluble residues were partially removed by treatment with proteolytic enzymes (pepsin and pronase), and in some cases with the HCl/H2O2 reagent, yielding purified polysaccharide-enriched fractions. These treatments led to the solubilisation of some products in water. The composition and structure of isolated polysaccharides were characterised based on monosaccharide composition, glycosidic linkage and spectroscopic analyses. The DMSO extract contained mainly proteins, and polysaccharides were not detected. The water-soluble parts isolated from the LiCl/DMSO extract contained α-l-rhamnan, α-d-glucan and ß-d-glucogalactan; the water-insoluble part contained (1 â†’ 4)-ß-d-xylan, first isolated from the biomass of green microalgae. The alkali extracts contained polysaccharides of similar structure, and also water-insoluble (1 â†’ 4)-ß-d-mannan. The insoluble part after all extractions contained α-chitin as the main polysaccharide, which was confirmed by spectroscopic methods. All these polysaccharides can play a certain role in the cell wall structure of this microalga.

Impact of glgA1, glgA2 or glgC overexpression on growth and glycogen production in Synechocystis sp. PCC 6803.

Low production rates are still one limiting factor for the industrial climate-neutral production of biovaluable compounds in cyanobacteria. Next to optimized cultivation conditions, new production strategies are required. Hence, the use of established molecular tools could lead to increased product yields in the cyanobacterial model organism Synechocystis sp. PCC6803. Its main storage compound glycogen was chosen to be increased by the use of these tools. In this study, the three genes glgC, glgA1 and glgA2, which are part of the glycogen synthesis pathway, were combined with the Pcpc560 promoter and the neutral cloning site NSC1. The complete genome integration, protein formation, biomass production and glycogen accumulation were determined to select the most productive transformants. The overexpression of glgA2 did not increase the biomass or glycogen production in short-term trials compared to the other two genes but caused transformants death in long-term trials. The transformants glgA1_11 and glgC_2 showed significantly increased biomass (1.6-fold - 1.7-fold) and glycogen production (3.5-fold - 4-fold) compared to the wild type after 96 h making them a promising energy source for further applications. Those could include for example a two-stage production process, with first energy production (glycogen) and second increased product formation (e.g. ethanol).

Comparison of various approaches to detect algal culture contamination: a case study of Chlorella sp. contamination in a Phaeodactylum tricornutum culture.

Microalgal contamination in algal culture is a serious problem hampering the cultivation process, which can result in considerable economic and time losses. With the field of microalgal biotechnology on the rise, development of new tools for monitoring the cultures is of high importance. Here we present a case study of the detection of fast-growing green algae Chlorella vulgaris (as contaminant) in a diatom Phaeodactylum tricornutum culture using various approaches. We prepared mixed cultures of C. vulgaris and P. tricornutum in different cell-to-cell ratios in the range from 1:103 to 1:107. We compared the sensitivity among microscopy, cultivation-based technique, PCR, and qPCR. The detection of C. vulgaris contamination using light microscopy failed in samples containing cell ratios <1:105. Our results confirmed PCR/qPCR to provide the most reliable and sensitive results, with detection sensitivity close to 75 cells/mL. The method was similarly sensitive in a pure C. vulgaris culture as well as in a mixed culture containing 107-times more P. tricornutum cells. A next-generation sequencing analysis revealed a positive discrimination of C. vulgaris during DNA extraction. The method of cultivation media exchange from sea water to fresh water, preferred by the Chlorella contaminant, demonstrated a presence of the contaminant with a sensitivity comparable to PCR approaches, albeit with a much longer detection time. The results suggest that a qPCR/PCR-based approach is the best choice for an early warning in the commercial culturing of microalgae. This method can be conveniently complemented with the substitution-cultivation method to test the proliferating potential of the contaminant. KEY POINTS: • PCR-based protocol developed for detection of Chlorella cells. • Synergy of various approaches shows deeper insight into a presence of contaminants. • Positive/negative discrimination occurs during DNA extraction in mixed cultures. • Newly developed assays ready to use as in diagnostics of contamination.

Selenium Incorporation to Amino Acids in Chlorella Cultures Grown in Phototrophic and Heterotrophic Regimes.

Microalgae accumulate bioavailable selenium-containing amino acids (Se-AAs), and these are useful as a food supplement. While this accumulation has been studied in phototrophic algal cultures, little data exists for heterotrophic cultures. We have determined the Se-AAs content, selenium/sulfur (Se/S) substitution rates, and overall Se accumulation balance in photo- and heterotrophic Chlorella cultures. Laboratory trials revealed that heterotrophic cultures tolerate Se doses ∼8-fold higher compared to phototrophic cultures, resulting in a ∼2-3-fold higher Se-AAs content. In large-scale experiments, both cultivation regimes provided comparable Se-AAs content. Outdoor phototrophic cultures accumulated up to 400 µg g-1 of total Se-AAs and exhibited a high level of Se/S substitution (5-10%) with 30-60% organic/total Se embedded in the biomass. A slightly higher content of Se-AAs and ratio of Se/S substitution was obtained for a heterotrophic culture in pilot-scale fermentors. The data presented here shows that heterotrophic Chlorella cultures provide an alternative for Se-enriched biomass production and provides information on Se-AAs content and speciation in different cultivation regimes.

Rapid screening test to estimate temperature optima for microalgae growth using photosynthesis activity measurements.

We have worked out a rapid 1-day test based on photosynthesis measurements to estimate suitable growth temperature of microalgae cultures. To verify the proposed procedure, several microalgae-Chlorella, Nostoc, Synechocystis, Scenedesmus, and Cylindrospermum-were cultured under controlled laboratory conditions (irradiance, temperature, mixing, CO2, and nutrient supply) to find the optima of photosynthetic activity using the range between 15 and 35 °C. These activities were recorded at each temperature step after 2 h of acclimation which should be sufficient as oxygen production and the PQ cycle are regulated by fast processes. Photosynthetic activity was measured using three techniques-oxygen production/respiration, saturating pulse analysis of fluorescence quenching, and fast fluorescence induction kinetics-to estimate the temperature optima which should correspond to high growth rate. We measured all variables that might have been directly related to growth-photosynthetic oxygen evolution, maximum photochemical yield of PSII, Fv/Fm, relative electron transport rate rETRmax, and the transients Vj and Vi determined by fast fluorescence induction curves. When the temperature optima for photosynthetic activity were verified in growth tests, we found good correlation. For most of tested microalgae strains, temperature around 30 °C was found to be the most suitable at this setting. We concluded that the developed test can be used as a rapid 1-day pre-screening to estimate a suitable growth temperature of microalgae strains before they are cultured in a pilot scale.

Bioethanol production from microalgae polysaccharides.

The worldwide growing demand for energy permanently increases the pressure on industrial and scientific community to introduce new alternative biofuels on the global energy market. Besides the leading role of biodiesel and biogas, bioethanol receives more and more attention as first- and second-generation biofuel in the sustainable energy industry. Lately, microalgae (green algae and cyanobacteria) biomass has also remarkable potential as a feedstock for the third-generation biofuel production due to their high lipid and carbohydrate content. The third-generation bioethanol production technology can be divided into three major processing ways: (i) fermentation of pre-treated microalgae biomass, (ii) dark fermentation of reserved carbohydrates and (iii) direct "photo-fermentation" from carbon dioxide to bioethanol using light energy. All three technologies provide possible solutions, but from a practical point of view, traditional fermentation technology from microalgae biomass receives currently the most attention. This study mainly focusses on the latest advances in traditional fermentation processes including the steps of enhanced carbohydrate accumulation, biomass pre-treatment, starch and glycogen downstream processing and various fermentation approaches.

Development of thin-layer cascades for microalgae cultivation: milestones (review).

In this work, the key moments of the development of the so-called thin-layer cascades (TLC) for microalgae production are described. Development started at the end of the 1950s when the first generation of TLCs was set-up in former Czechoslovakia. Since, similar units for microalgae culturing, which are relatively simple, low-cost and highly productive, have been installed in a number of other countries worldwide. The TLCs are characterized by microalgae growth at a low depth (< 50 mm) and fast flow (0.4-0.5 m/s) of culture compared to mixed ponds or raceways. It guarantees a high ratio of exposed surface to total culture volume (> 100 1/m) and rapid light/dark cycling frequencies of cells which result in high biomass productivity (> 30 g/m2/day) and operating at high biomass density, > 10 g/L of dry mass (DW). In TLCs, microalgae culture is grown in the system of inclined platforms that combine the advantages of open systems-direct sun irradiance, easy heat derivation, simple cleaning and maintenance, and efficient degassing-with positive features of closed systems-operation at high biomass densities achieving high volumetric productivity. Among significant advantages of thin layer cascades compared to raceway ponds are the operation at much higher cell densities, very high daylight productivities, and the possibility to store the culture in retention tanks at night, or in unfavourable weather conditions. Concerning the limitations of TLCs, one has to consider contaminations by other microalgae that limit cultivation to robust, fast-growing strains, or those cultured in selective environments.

Microbial communities responsible for the degradation of poly(lactic acid)/poly(3-hydroxybutyrate) blend mulches in soil burial respirometric tests.

The microbial communities responsible for the degradation of poly(lactic acid)/poly(3-hydroxybutyrate) (PLA/PHB) blend foils were investigated in 1 year long laboratory soil burial experiments. Different PLA/PHB foils were tested: (a) PLA/PHB original transparent foil, (b) PLA/PHB carbon black filled foil and (c) PLA/PHB black foil previously exposed for 90 days to sun light. The microbiome diversity of these three types of foil was compared with that identified from soil/perlite sample at the beginning of experiment and that developed on a cellulose mat. Culture-dependent and culture-independent (DGGE-cloning) approaches together with PLA, PHB and PLA/PHB degradation plate assays were employed. The cultivation strategy combined with degradation tests permitted the isolation and evaluation of several PLA/PHB blend degrading microorganisms such as members of the genera Bacillus, Paenibacillus, Streptomyces, Rhodococcus, Saccharothrix, Arthrobacter, Aureobasidium, Mortierella, Absidia, Actinomucor, Bjerkandera, Fusarium, Trichoderma and Penicillium. The DGGE-cloning investigation increased the information about the microbial communities occurring during bioplastic degradation detecting several bacterial and fungal taxa and some of them (members of the orders Anaerolineales, Selenomonadales, Thelephorales and of the genera Pseudogymnoascus and Pseudeurotium) were revealed here for the first time. This survey showed the microbiome colonizing PLA/PHB blend foils and permitted the isolation of several microorganisms able to degrade the tested polymeric blends.

Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.

Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.

Water-related environments: a multistep procedure to assess the diversity and enzymatic properties of cultivable bacteria.

Studying the culturable portion of environmental bacterial populations is valuable for understanding the ecology, for discovering taxonomically interesting isolates and for exploiting their enzymatic abilities. In this study, diverse water-related samples, iced water (3 °C) from river, the sediment (29 °C) and water (55 °C) of a hot-spring, were investigated by two cultivation strategies, Dry and novel Wet approach. The isolates were clustered by fluorescent internal transcribed spacer PCR and identified by 16S rRNA sequencing. Several bacterial groups were also sub-typed through the application of Random Amplified Microsatellite Polymorphisms method. A broad enzymatic screening of all bacterial isolates was performed in order to assess the proteolytic, cellulolytic, lipolytic, esterolytic, amylolytic properties, as well as catalase and peroxidase activities. The Wet cultivation demonstrated to be suitable for the isolation of potential new species belonging to genera Massilia, Algoriphagus, Rheinheimera and Pandoraea. Valuable microbial resources with extensive enzymatic activities were recognized among the psychrophilic (Pantoea brenneri and Serratia sp.), mesophilic (Pandoraea, Massilia, Pseudomonas, Stenotrophomonas, Bacillus and Aeromonas) and thermophilic bacteria (Aeribacullus pallidus and Geobacillus kaustophilus). The experimental strategy developed in this study includes simple investigation tools able to reveal the genetic and enzymatic peculiarities of isolated microorganisms. It can be applied to different kinds of aquatic samples and extreme environments similar to those described in this study.

Biodeterioration of epoxy resin: a microbial survey through culture-independent and culture-dependent approaches.

During the 20th century, synthetic polymers were greatly used in the field of art. In particular, the epoxy resins were used for both conservation and for creating sculptures. The biodeterioration of these polymers has not been adequately studied. The aim of this investigation was to examine the microflora responsible for the deterioration of an epoxy statue exposed to outdoor conditions. Fungal and bacterial microflora were isolated from the art object, clustered by fluorescence-ITS (internal transcribed spacer), identified by ITS and 16S rRNA sequencing and tested for their lipolytic abilities by three agar assays. Different algal, bacterial, cyanobacterial and fungal clone libraries were constructed. The surrounding airborne microflora was analyzed using culture-dependent and culture-independent approaches. The results indicated the presence, on the statue surface, of an interesting and differentiate microbial community composed of rock-inhabiting members, algal photobionts (Trebouxia spp., Chloroidium ellipsoideum and Chlorella angustoellipsoidea), Cyanobacteria (Leptolyngbya sp., Phormidium sp., Cylindrospermum stagnale, Hassallia byssoidea and Geitlerinema sp.), black yeasts related to the species Friedmanniomyces endolithicus, Pseudotaeniolina globosa, Phaeococcomyces catenatus and Catenulostroma germanicum and several plant-associated fungi. This investigation provides new information on the potential microfloral inhabitants of epoxy resin discovering a new ecological niche, occupied mainly by several members of rock-colonizing microbial species.

Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids.

High transformation competency of Escherichia coli is one of the critical factors in the bacterial artificial chromosome (BAC)-based DNA library construction. Many electroporation protocols have been published until now, but the majority of them was optimized for transformation of small plasmids. Large plasmids with a size above 50 kbp display reduced transformation efficiency and thereby require specific conditions in the preparation and electroporation of electrocompetent cells. In the present work, we have optimized the parameters critical to the application of BAC DNA electrotransformation into E. coli. Systematic evaluation of electroporation variables has revealed several key factors like temperature of growth, media supplements, washing buffer, and cell concentration. Improvements made in the transformation protocol have led to electrocompetent cells with transformation efficiency up to 7 × 10(8) transformants per microgram of 120 kbp BAC plasmid DNA. We have successfully used in-house prepared competent cells, the quality of which is comparable with those produced by different companies, in the construction of metagenomic libraries from the soil. Our protocol can also be beneficial for other application with limited DNA source.