Booster biocides and microfouling.

Antifouling (AF) paints are used to prevent the attachment of living organisms to the submerged surfaces of ships, boats and aquatic structures, usually by the release of biocides. Apart from copper, organic booster biocides are the main active components in AF paints, but their use can have a negative impact on the marine environment. The direct effects of biocides on marine bacteria are poorly known. This work investigates the impact of two biocides, viz. diuron and tolylfluanid, on the growth and the viability of marine microorganisms and on their ability to form biofilms. The biocides in solution were found to inhibit growth of two strains of marine bacteria, viz. Pseudoalteromonas and Vibrio vulnificus, at a high concentration (1000 microg ml(-1)), but only a small effect on viability was observed. Confocal laser scanning microscopy (CLSM) showed that the booster biocides decreased biofilm formation by both bacteria. At a concentration of 10 microg ml(-1), the biocides inhibited cell attachment and reduced biofilm thickness on glass surfaces. The percentage of live cells in the biofilms was also reduced. The effect of the biocides on two diatoms, Fragilaria pinnata and Cylindrotheca closterium, was also evaluated in terms of growth rate, biomass, chlorophyll a content and attachment to glass. The results demonstrate that diuron and tolylfluanid are more active against diatoms than bacteria.

Mycosporine-like amino acid and aromatic amino acid transcriptome response to UV and far-red light in the cyanobacterium Chlorogloeopsis fritschii PCC 6912.

The "UV sunscreen" compounds, the mycosporine-like amino acids (MAAs) are widely reported in cyanobacteria and are known to be induced under ultra-violet (UV) light. However, the impact of far red (FR) light on MAA biosynthesis has not been studied. We report results from two experiments measuring transcriptional regulation of MAA and aromatic amino acid pathways in the filamentous cyanobacterium Chlorogloeopsis fritschii PCC 6912. The first experiment, comparing UV with white light, shows the expected upregulation of the characteristic MAA mys gene cluster. The second experiment, comparing FR with white light, shows that three genes of the four mys gene cluster encoding up to mycosporine-glycine are also upregulated under FR light. This is a new discovery. We observed corresponding increases in MAAs under FR light using HPLC analysis. The tryptophan pathway was upregulated under UV, with no change under FR. The tyrosine and phenylalanine pathways were unaltered under both conditions. However, nitrate ABC transporter genes were upregulated under UV and FR light indicating increased nitrogen requirement under both light conditions. The discovery that MAAs are upregulated under FR light supports MAAs playing a role in photon dissipation and thermoregulation with a possible role in contributing to Earth surface temperature regulation.

Valorising nutrient-rich digestate: Dilution, settlement and membrane filtration processing for optimisation as a waste-based media for microalgal cultivation.

Digestate produced from the anaerobic digestion of food and farm waste is primarily returned to land as a biofertiliser for crops, with its potential to generate value through alternative processing methods at present under explored. In this work, valorisation of a digestate resulting from the treatment of kitchen and food waste was investigated, using dilution, settlement and membrane processing technology. Processed digestate was subsequently tested as a nutrient source for the cultivation of Chlorella vulgaris, up to pilot-scale (800L). Dilution of digestate down to 2.5% increased settlement rate and induced release of valuable compounds for fertiliser usage such as nitrogen and phosphorus. Settlement, as a partial processing of digestate offered a physical separation of liquid and solid fractions at a low cost. Membrane filtration demonstrated efficient segregation of nutrients, with micro-filtration recovering 92.38% of phosphorus and the combination of micro-filtration, ultra-filtration, and nano-filtration recovering a total of 94.35% of nitrogen from digestate. Nano-filtered and micro-filtered digestates at low concentrations were suitable substrates to support growth of Chlorella vulgaris. At pilot-scale, the microalgae grew successfully for 28 days with a maximum growth rate of 0.62 day-1 and dry weight of 0.86  g⋅L-1. Decline in culture growth beyond 28 days was presumably linked to ammonium and heavy metal accumulation in the cultivation medium. Processed digestate provided a suitable nutrient source for successful microalgal cultivation at pilot-scale, evidencing potential to convert excess nutrients into biomass, generating value from excess digestate and providing additional markets to the anaerobic digestion sector.

Far-Red Light Acclimation for Improved Mass Cultivation of Cyanobacteria.

Improving mass cultivation of cyanobacteria is a goal for industrial biotechnology. In this study, the mass cultivation of the thermophilic cyanobacterium Chlorogloeopsis fritschii was assessed for biomass production under light-emitting diode white light (LEDWL), far-red light (FRL), and combined white light and far-red light (WLFRL) adaptation. The induction of chl f was confirmed at 24 h after the transfer of culture from LEDWL to FRL. Using combined light (WLFRL), chl f, a, and d, maintained the same level of concentration in comparison to FRL conditions. However, phycocyanin and xanthophylls (echinone, caloxanthin, myxoxanthin, nostoxanthin) concentration increased 2.7-4.7 times compared to LEDWL conditions. The productivity of culture was double under WLFRL compared with LEDWL conditions. No significant changes in lipid, protein, and carbohydrate concentrations were found in the two different light conditions. The results are important for informing on optimum biomass cultivation of this species for biomass production and bioactive product development.

Comparing Nutrient Removal from Membrane Filtered and Unfiltered Domestic Wastewater Using Chlorella vulgaris.

The nutrient removal efficiency of Chlorella vulgaris cultivated in domestic wastewater was investigated, along with the potential to use membrane filtration as a pre-treatment tool during the wastewater treatment process. Chlorella vulgaris was batch cultivated for 12 days in a bubble column system with two different wastewater treatments. Maximum uptake of 94.18% ammonium (NH4-N) and 97.69% ortho-phosphate (PO4-P) occurred in 0.2 µm membrane filtered primary wastewater. Membrane filtration enhanced the nutrient uptake performance of C. vulgaris by removing bacteria, protozoa, colloidal particles and suspended solids, thereby improving light availability for photosynthesis. The results of this study suggest that growing C. vulgaris in nutrient rich membrane filtered wastewater provides an option for domestic wastewater treatment to improve the quality of the final effluent.

Using microalgae in the circular economy to valorise anaerobic digestate: challenges and opportunities.

Managing organic waste streams is a major challenge for the agricultural industry. Anaerobic digestion (AD) of organicwastes is a preferred option in the waste management hierarchy, as this processcangenerate renewableenergy, reduce emissions from wastestorage, andproduce fertiliser material.However, Nitrate Vulnerable Zone legislation and seasonal restrictions can limit the use of digestate on agricultural land. In this paper we demonstrate the potential of cultivating microalgae on digestate as a feedstock, either directlyafter dilution, or indirectlyfromeffluent remaining after biofertiliser extraction. Resultant microalgal biomass can then be used to produce livestock feed, biofuel or for higher value bio-products. The approach could mitigate for possible regional excesses, and substitute conventional high-impactproducts with bio-resources, enhancing sustainability withinacircular economy. Recycling nutrients from digestate with algal technology is at an early stage. We present and discuss challenges and opportunities associated with developing this new technology.

Formulation and utilisation of spent anaerobic digestate fluids for the growth and product formation of single cell algal cultures in heterotrophic and autotrophic conditions.

Spent anaerobically digested effluents of agricultural origin were collected and treated using membrane filtration to achieve three-large particle free-nutrient streams of N:P ratios of 16.53, 3.78 and 14.22. Three algal species were grown on these streams, achieving good levels of bioremediation of digester fluids simultaneously with biomass and associated end product formation. Nannochloropsis oceanica and Scenedesmus quadricuada, where proven highly effective in remediating the streams achieving ammonia and phosphate reduction over 60% while for Schizochytrium limacinum SR21 these serve as an ideal production medium for lipids and biomass reaching 16.70w/w% and 1.42gL-1 correspondingly. These processes thus provide treatment of sludge, avoiding the disposal problems by land spreading. The solid components are nutrient depleted but rich in organic matter as a soil enhancer, while the fluids rich in nutrients can be efficiently utilised for growth to generate high value materials of microalgae facilitating water reclamation.

Bioremediation efficacy-comparison of nutrient removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation.

An algal consortium was isolated from an integrated steelmaking site at TATA Steel Strip Products Ltd. in Port Talbot, UK, and its bioremediation capacity tested. Excellent "bioremediation" was observed when the mixed culture was "applied" to diluted effluent from an enhanced anaerobic digestion plant at Dwr Cymru Welsh Water at Port Talbot, UK. After 5 days of cultivation in a 600-L photobioreactor, 99% of the total nitrogen (initial level, 4500 µmol L-1) and total phosphorus (initial level, 690.4 µmol L-1) were removed from the waste stream. The consortium was deposited in the Culture Collection of Algae and Protozoa (CCAP), an international depository authority for microalgal patents, as CCAP 293/1. This material has been successfully cryopreserved using a two-step cryopreservation protocol with dimethyl sulphoxide (5% v/v) used as a cryoprotectant. On recovery of samples after 3 months storage at -196 °C, the specific bioremediation activity of the revived consortium was tested. The capacity of the revived culture to bioremediate effluent was not significantly different (p < 0.05) from a non-cryopreserved control, with 99% of total nitrogen and phosphorus remediated by day 4. Although non-axenic algal cultures have previously been cryopreserved, this is the first report of the successful cryopreservation of mixed algal consortium, with validation of its ability to bioremediate after thawing comparing non-cryopreserved cultures with a revived post-thaw algal consortium. The study also highlights the need to ensure the long-term security and the requirement to validate the functionality of conserved inocula with biotechnological/bioremediation potential.

Utilising light-emitting diodes of specific narrow wavelengths for the optimization and co-production of multiple high-value compounds in Porphyridium purpureum.

The effect of specific narrow light-emitting diode (LED) wavelengths (red, green, blue) and a combination of LED wavelengths (red, green and blue - RGB) on biomass composition produced by Porphyridium purpureum is studied. Phycobiliprotein, fatty acids, exopolysaccharides, pigment content, and the main macromolecules composition were analysed to determine the effect of wavelength on multiple compounds of commercial interest. The results demonstrate that green light plays a significant role in the growth of rhodophyta, due to phycobiliproteins being able to harvest green wavelengths where chlorophyll pigments absorb poorly. However, under multi-chromatic LED wavelengths, P. purpureum biomass accumulated the highest yield of valuable products such as eicosapentaenoic acid (∼2.9% DW), zeaxanthin (∼586µgg-1DW), ß-carotene (397µgg-1DW), exopolysaccharides (2.05g/L-1), and phycobiliproteins (∼4.8% DW). This increased accumulation is likely to be the combination of both photo-adaption and photo-protection, under the combined specific wavelengths employed.

Cultivating Chlorella vulgaris and Scenedesmus quadricauda microalgae to degrade inorganic compounds and pesticides in water.

This work evaluates the possibility of cultivating Scenedesmus quadricauda and Chlorella vulgaris microalgae in wastewater from the hydroponic cultivation of tomatoes with the aim of purifying the water. S. quadricauda and C. vulgaris were also used in purification tests carried out on water contaminated by the following active ingredients: metalaxyl, pyrimethanil, fenhexamid, iprodione, and triclopyr. Fifty-six days after the inoculum was placed, a reduction was found in the concentration of nitric nitrogen, ammonia nitrogen, and soluble and total phosphorus. The decrease was 99, 83, 94, and 94 %, respectively, for C. vulgaris and 99, 5, 88, and 89 %, respectively, for S. quadricauda. When the microalgae were present, all the agrochemicals tested were removed more quickly from the water than from the sterile control (BG11). The increase in the rate of degradation was in the order metalaxyl > fenhexamid > iprodione > triclopyr > pyrimethanil. It was demonstrated that there was a real degradation of fenhexamid, metalaxyl, triclopyr, and iprodione, while in the case of pyrimethanil, the active ingredient removed from the substrate was absorbed onto the cells of the microalgae. It was also found that the agrochemicals used in the tests had no significant effect on the growth of the two microalgae. The experiment highlighted the possibility of using cultivations of C. vulgaris and S. quadricauda as purification systems for agricultural wastewater which contains eutrophic inorganic compounds such as nitrates and phosphates and also different types of pesticides.

Comparative efficiency of macroalgal extracts and booster biocides as antifouling agents to control growth of three diatom species.

The application of 'booster biocides' Diuron, Tolylfluanid and Copper thiocyanate inbantifouling paints, used to prevent development of biofouling, needs to be monitored before assessing their impacts on the environment. An alternative approach aims to propose eco-friendly and effective antifoulants isolated from marine organisms such as seaweeds. In this study, the effects of 'booster biocides' and the ethanol and dichloromethane extracts from a brown (Sargassum muticum) and a red alga (Ceramium botryocarpum) have been compared by algal growth inhibition tests of marine diatoms. The most efficient extracts were ethanol fraction of S. muticum and C. botryocarpum extracts with growth EC(50)=4.74 and 5.3µg mL(-1) respectively, with reversible diatom growth effect. The booster biocides are more efficient EC(50)=0.52µg mL(-1), but are highly toxic. Results validate the use of macroalgal extracts as non toxic antifouling compounds, and they represent valuable environmentally friendly alternatives in comparison with currently used biocides.

Antifouling activity of macroalgal extracts on Fragilaria pinnata (Bacillariophyceae): a comparison with Diuron.

The tributyltin-based products and organic biocides which are incorporated into antifouling paints have had a negative impact on the marine environment, and the ban on tributyltin-based antifouling products has urged the industry to find substitutes to prevent the development of fouling on ship hulls. Natural antifouling agents could be isolated from marine resources, providing an alternative option for the industry. The effects of different marine seaweed extracts from Sargassum muticum and Ceramium botryocarpum on the growth, pigment content and photosynthetic apparatus of the marine diatom Fragilaria pinnata were compared with those of Diuron, a biocide widely used in antifouling paints. The addition of the macroalgal extracts in the culture medium resulted in an inhibition of the growth of F. pinnata, but this inhibition was lower than that obtained with Diuron. After transfer to a biocide-free medium, F. pinnata cells previously exposed to the macroalgal extracts exhibited normal growth, in contrast to Diuron-treated cells, which died, demonstrating that the effects of the natural antifouling agents were reversible. Macroalgal extracts and Diuron-induced modifications in F. pinnata cellular pigment content. Chlorophyll a, fucoxanthin, and the xanthophyll pool, diadinoxanthin and diatoxanthin, were the most affected. Changes in the structure and function of the photosynthetic apparatus were studied by microspectrofluorimetry, and provided a comprehensive evaluation of the inhibition of the diatom Photosystem II (PSII) by the biocides. This study confirms that natural extracts from the macroalgae studied have the potential to be used as a substitute to commercial biocides in antifouling paints.