Implications of a Catastrophic Refrigeration Failure on the Viability of Cryogenically Stored Samples.

Cryopreservation, the use of very low temperatures to preserve structurally intact living cells and tissues, is a key underpinning technology for life science research and medicine. It is employed to ensure the stability of critical biological resources including viruses, bacteria, protists, animal cell cultures, plants, reproductive materials and embryos. Fundamental to ensuring this stability is assuring stability of cryogenic storage temperatures. Here we report the occurrence of a failure in refrigeration in a cryostat holding > 600 strains of cyanobacteria and eukaryotic microalgae. A strategic approach was adopted to assess viability across a cross-section of the biodiversity held, both immediately after the potentially damaging temperature shift and 10 years later, on subsequent cryostorage in liquid-phase nitrogen (∼-196 °C). Furthermore, the event was replicated experimentally and the effects on the viability of cryo-tolerant and cryo-sensitive strains monitored. Our results have significant implications to all users of this storage method and parallels have been drawn with the ongoing development in other fields and in particular, human cell therapy. Based on our practical experience we have made a series of generic recommendations for emergency, remedial and ongoing preventative actions.

Enabling large-scale production of algal oil in continuous output mode.

Large-scale algal oil production requires continuous outputs and a trade-off between growth and oil content. Two unrelated marine algae (Nannochloropsis oceanica [CCAP 849/10] and Chlorella vulgaris [CCAP 211/21A]) that showed high oil production under batch culture were studied under controlled semicontinuous cultivation conditions. Three essential attributes maximized oil productivity: (i) downregulation of cell size to maximize light absorption under N limitation; (ii) low nutrient-depletion thresholds to trigger oil induction; (iii) a means of carbohydrate suppression in favor of oil. N. oceanica responded better to input N/P variations and is more suited to continuous oil production. A low N/P ratio was effective in both suppressing carbohydrate and reducing cell size concomitant with oil production. In C. vulgaris, nutrient starvation thresholds for oil were higher and carbohydrate was preferentially induced, which impeded stress-level optimization for oil. These differences, which impact continuous oil production at scale, are driven by species adaptation to specific marine habitats.

Removal of metals from aqueous solutions using dried Cladophora parriaudii of varying biochemical composition.

Macroalgal biosorption has shown promise for the removal of metal ions from wastewaters, whose presence can pose a threat to the aquatic environment. There is a wealth of literature published on macroalgal biosorption, the common thread being that the biosorbent material was collected from the field, under undefined conditions. These studies offer little insight into the impact of prior cultivation or biomass production practices upon the biosorbent material, its adsorptive physico-chemical properties and its subsequent capacity for metal removal. The present study sought to investigate the influence of changes in macroalgal cultivation, specifically nutrient regime, upon biomass properties and the resultant adsorption performance. The macroalga Cladophora parriaudii was cultivated under six different nutrient regimes; 2:1 and 12:1 N:P molar ratios, with nitrogen supplied either as ammonium (NH4+), nitrate (NO3-), or urea (CO(NH2)2). These nutrient regimes were designed to produce biomass of varying biochemical and cell surface profiles. After cultivation, the biomass was rinsed, dried, biochemically analysed and then used for the removal of four individual metals from solution. Metal removal varied considerably between treatments and across initial metal concentrations, with removal values of 46-85%, 9-80%, 8-71%, and 49-94% achieved for Al, Cu, Mn, and Pb, respectively, with initial metal concentrations varying between 0 and 150 mg L-1. The observed variation in metal removal can only be attributed to differences in biochemistry and cell surface properties of the biosorbent induced by nutrient regime, as all other variables were constant. This study demonstrates that prior cultivation conditions influence the biochemistry of a biosorbent material, namely macroalgae Cladophora parriaudii, which has an impact upon metal removal. This aspect should be given due consideration for future biosorption research and when reviewing already published literature.

Effects of cryopreservation on viability and functional stability of an industrially relevant alga.

As algal biotechnology develops, there is an increasing requirement to conserve cultures without the cost, time and genetic stability implications of conventional serial transfers, including issues regarding potential loss by failure to regrow, contamination on transfer, mix up and/or errors in the documentation on transfer. Furthermore, it is crucial to ensure both viability and functionality are retained by stored stock-cultures. Low temperature storage, ranging from the use of domestic freezers to storage under liquid nitrogen, is widely being used, but the implication to stability and function rarely investigated. We report for the first time, retention of functionality in the maintenance of master stock-cultures of an industrially relevant, lipid-producing alga, under a variety of cryopreservation regimes. Storage in domestic (-15 °C), or conventional -80 °C freezers was suboptimal, with a rapid reduction in viability observed for samples at -15 °C and a >50% loss of viability, within one month, for samples stored at -80 °C. No reduction in viability occurred at -196 °C. Post-thaw culture functional performance was also influenced by the cryopreservation approach employed. Only samples held at -196 °C responded to nitrogen limitation in terms of growth characteristics and biochemical profiles (lipid production and chlorophyll a) comparable to the untreated control, cultured prior to cryopreservation. These results have important implications in microbial biotechnology, especially for those responsible for the conservation of genetic resources.

Cryopreservation studies of an artificial co-culture between the cobalamin-requiring green alga Lobomonas rostrata and the bacterium Mesorhizobium loti.

Algal-bacterial co-cultures, rather than cultures of algae alone, are regarded as having the potential to enhance productivity and stability in industrial algal cultivation. As with other inocula in biotechnology, to avoid loss of production strains, it is important to develop preservation methods for the long-term storage of these cultures, and one of the most commonly used approaches is cryopreservation. However, whilst there are many reports of cryopreserved xenic algal cultures, little work has been reported on the intentional preservation of both algae and beneficial bacteria in xenic cultures. Instead, studies have focused on the development of methods to conserve the algal strain(s) present, or to avoid overgrowth of bacteria in xenic isolates during the post-thaw recovery phase. Here, we have established a co-cryopreservation method for the long-term storage of both partners in a unialgal-bacterial co-culture. This is an artificial model mutualism between the alga Lobomonas rostrata and the bacterium Mesorhizobium loti, which provides vitamin B12 (cobalamin) to the alga in return for photosynthate. Using a Planer Kryo 360 controlled-rate cooler, post-thaw viability (PTV) values of 72% were obtained for the co-culture, compared to 91% for the axenic alga. The cultures were successfully revived after 6 months storage in liquid nitrogen, and continued to exhibit mutualism. Furthermore, the alga could be cryopreserved with non-symbiotic bacteria, without bacterial overgrowth occurring. It was also possible to use less controllable passive freezer chambers to cryopreserve the co-cultures, although the PTV was lower. Finally, we demonstrated that an optimised cryopreservation method may be used to prevent the overgrowth potential of non-symbiotic, adventitious bacteria in both axenic and co-cultures of L. rostrata after thawing.

A comparison of methods for the non-destructive fresh weight determination of filamentous algae for growth rate analysis and dry weight estimation.

The determination of rates of macroalgal growth and productivity via temporal fresh weight (FW) measurements is attractive, as it does not necessitate the sacrifice of biomass. However, there is no standardised method for FW analysis; this may lead to potential discrepancies when determining growth rates or productivity and make literature comparison problematic. This study systematically assessed a variety of lab-scale methods for macroalgal FW measurement for growth rate determination. Method efficacy was assessed over a 14-day period as impact upon algal physiology, growth rate on basis of FW and dry weight (DW), nitrate removal, and maintenance of structural integrity. The choice of method is critical to both accuracy and inter-study comparability of the data generated. In this study, it was observed that the choice of protocol had an impact upon the DW yield (P values = 0.036-0.51). For instance, those involving regular mechanical pressing resulted in a >25% reduction in the final DW in two of the three species studied when compared to algae not subjected to any treatment. This study proposes a standardised FW determination method employing a reticulated spinner that is rapid, reliable, and non-destructive and provides an accurate growth estimation.

Media Screening for Obtaining Haematococcus pluvialis Red Motile Macrozooids Rich in Astaxanthin and Fatty Acids.

Astaxanthin from Haematococcus pluvialis is commercially produced in a two-stage process, involving green vegetative (macrozooid) and red aplanospore stages. This approach has been scaled up to an industrial process but constraints limit its commercial success and profitability, including: contamination issues, high pigment extraction costs, requirements for high light levels and photo-bleaching in the red stage. However, in addition to the aplanospore stage, this alga can produce astaxanthin in vegetative palmelloid and motile macrozooid cells. In this study, a two-stage process utilising different media in the green stage, with subsequent re-suspension in medium without nitrate was employed to optimise the formation of red motile macrozooids. Optimal growth in the green phase was obtained on cultivation under mixotrophic conditions in EG:JM media followed by re-suspension in medium without nitrate resulting in red motile macrozooids with an astaxanthin content of 2.74% (78.4% of total carotenoids) and a lipid content of 35.3% (rich in unsaturated fatty acids. It is envisaged that the red motile macrozooids could be harvested and fed as a whole-cell product directly in the animal feed and aquaculture sectors, or used as a blend of carotenoids and polyunsaturated fatty acids (PUFAs) in nutraceutical products.

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.

Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Developing an ecologically relevant heterogeneous biofilm model for dental-unit waterlines.

This study monitored the biodiversity of microbes cultured from a heterogeneous biofilm which had formed on the lumen of a section of dental waterline tubing over a period of 910 days. By day 2 bacterial counts on the outlet-water showed that contamination of the system had occurred. After 14 days, a biofilm comparable to that of clinical waterlines, consisting of bacteria, fungi and amoebae had formed. This showed that the proprietary silver coating applied to the luminal surface of the commercial waterline tubing failed to prevent biofilm formation. Molecular barcoding of isolated culturable microorganisms showed some degree of the diversity of taxa in the biofilm, including the opportunistic pathogen Legionella pneumophila. Whilst the system used for isolation and identification of contaminating microorganisms may underestimate the diversity of organisms in the biofilm, their similarity to those found in the clinical environment makes this a promising test-bed for future biocide testing.

Challenges for the Maintenance and Cryopreservation of Multiple Isolates of Model Microorganisms: An Example Using the Marine Diatom Skeletonema marinoi.

Modern genomic and metabolomic tools have provided the possibility of generating and interrogating large datasets that can provide answers to previously imponderable taxonomic, evolutionary, ecological, and physiological questions. However, the curatorial tools needed to provide and maintain the relevant biological resources on which new knowledge can be built have not kept pace with this meteoric rise in scientific capacity, its associated activity, or the huge increase in published science. The availability of biological material of guaranteed identity and quality in Biological Resource Centers is fundamental for scientific research, but it crucially depends on there being adequate preservation/maintenance methods that are capable of ensuring phenotypic, genotypic, and functional security of the biological material(s). This article highlights the challenges to the long-term maintenance of genetic resources in general, focusing specifically on the issues associated with the maintenance of a large collection of strains of the ecologically significant diatom Skeletonema marinoi. This research collection, held at the Department of Marine Sciences, University of Gothenburg, has been systematically tested for its capacity to survive cryopreservation. A method, involving incubation in the dark for 20-24 hours before cryopreservation, followed by cryoprotection employing 10% dimethysulphoxide (DMSO) and conventional cooling in a passive cooler, before plunging into liquid nitrogen was successfully applied to ∼80% of the strains tested. In addition, the growth characteristics of exemplar strains were confirmed after storage.

Biofouling community composition across a range of environmental conditions and geographical locations suitable for floating marine renewable energy generation.

Knowledge of biofouling typical of marine structures is essential for engineers to define appropriate loading criteria in addition to informing other stakeholders about the ecological implications of creating novel artificial environments. There is a lack of information regarding biofouling community composition (including weight and density characteristics) on floating structures associated with future marine renewable energy generation technologies. A network of navigation buoys were identified across a range of geographical areas, environmental conditions (tidal flow speed, temperature and salinity), and deployment durations suitable for future developments. Despite the perceived importance of environmental and temporal factors, geographical location explained the greatest proportion of the observed variation in community composition, emphasising the importance of considering geography when assessing the impact of biofouling on device functioning and associated ecology. The principal taxa associated with variation in biofouling community composition were mussels (Mytilus edulis), which were also important when determining loading criteria.

Unlocking nature's treasure-chest: screening for oleaginous algae.

Micro-algae synthesize high levels of lipids, carbohydrates and proteins photoautotrophically, thus attracting considerable interest for the biotechnological production of fuels, environmental remediation, functional foods and nutraceuticals. Currently, only a few micro-algae species are grown commercially at large-scale, primarily for "health-foods" and pigments. For a range of potential products (fuel to pharma), high lipid productivity strains are required to mitigate the economic costs of mass culture. Here we present a screen concentrating on marine micro-algal strains, which if suitable for scale-up would minimise competition with agriculture for water. Mass-Spectrophotometric analysis (MS) of nitrogen (N) and carbon (C) was subsequently validated by measurement of total fatty acids (TFA) by Gas-Chromatography (GC). This identified a rapid and accurate screening strategy based on elemental analysis. The screen identified Nannochloropsis oceanica CCAP 849/10 and a marine isolate of Chlorella vulgaris CCAP 211/21A as the best lipid producers. Analysis of C, N, protein, carbohydrate and Fatty Acid (FA) composition identified a suite of strains for further biotechnological applications e.g. Dunaliella polymorpha CCAP 19/14, significantly the most productive for carbohydrates, and Cyclotella cryptica CCAP 1070/2, with utility for EPA production and N-assimilation.

Proteomic-based biotyping reveals hidden diversity within a microalgae culture collection: An example using Dunaliella.

Accurate and defendable taxonomic identification of microalgae strains is vital for culture collections, industry and academia; particularly when addressing issues of intellectual property. We demonstrate the remarkable effectiveness of Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) biotyping to deliver rapid and accurate strain separation, even in situations where standard molecular tools prove ineffective. Highly distinctive MALDI spectra were obtained for thirty two biotechnologically interesting Dunaliella strains plus strains of Arthrospira, Chlorella, Isochrysis, Tetraselmis and a range of culturable co-occurring bacteria. Spectra were directly compared with genomic DNA sequences (internal transcribed spacer, ITS). Within individual Dunaliella isolates MALDI discriminated between strains with identical ITS sequences, thereby emphasising and enhancing knowledge of the diversity within microalgae culture collections. Further, MALDI spectra did not vary with culture age or growth stage during the course of the experiment; therefore MALDI presents stable and accurate strain-specific signature spectra. Bacterial contamination did not affect MALDI's discriminating power. Biotyping by MALDI-TOF-MS will prove effective in situations wherein precise strain identification is vital, for example in cases involving intellectual property disputes and in monitoring and safeguarding biosecurity. MALDI should be accepted as a biotyping tool to complement and enhance standard molecular taxonomy for microalgae.

Comparison of screening methods for high-throughput determination of oil yields in micro-algal biofuel strains.

The phenotypic and phylogenetic diversity of micro-algae capable of accumulating triacylglycerols provides a challenge for the accurate determination of biotechnological potential. High-yielding strains are needed to improve economic viability and their compositional information is required for optimizing biodiesel properties. To facilitate a high-throughput screening programme, a very rapid direct-derivatization procedure capable of extracting lyophilized material for GC analysis was compared with a scaled-down Folch-based method. This was carried out on ten micro-algal strains from 6 phyla where the more rapid direct-derivatization approach was found to provide a more reliable measure of yield. The modified Folch-based procedure was found to substantially underestimate oil yield in one Chlorella species (P < 0.01). In terms of fatty acid composition however, the Folch procedure proved to be slightly better in recovering polyunsaturated fatty acids, in six out of the ten strains. Therefore, direct-derivatization is recommended for rapid determination of yields in screening approaches but can provide slightly less compositional accuracy than solvent-based extraction methods.

Post-cryopreservation viability of the benthic freshwater diatom Planothidium frequentissimum depends on light levels.

Over recent years, several planktonic and benthic freshwater diatom taxa have been established as laboratory model strains. In common with most freshwater diatoms the pennate diatom Planothidium frequentissimum suffers irreversible cell shrinkage on prolonged maintenance by serial transfers, without induction of the sexual cycle. Therefore, alternative strategies are required for the long-term maintenance of this strain. Conventional colligative cryopreservation approaches have previously proven unsuccessful with no regrowth. However, in this study using 5% dimethyl sulfoxide (Me2SO), controlled cooling at 1 °C min(-1), automated ice seeding and cooling to -40 °C with a final plunge into liquid nitrogen, viability levels were enhanced from 0.3 ± 0.4% to 80 ± 3%, by incorporating a 48 h dark-recovery phase after rewarming. Omission, or reduction, of this recovery step resulted in obvious cell damage with photo-bleaching of pigments, indicative of oxidative-stress induced cell damage, with subsequent deterioration of cellular architecture.

Isolation of clonal cultures of endosymbiotic green algae from their ciliate hosts.

Using Paramecium bursaria as a model organism improved protocols have been developed to isolate clonal endosymbiotic algae. This involved micromanipulation of individual protists, rupturing to release endosymbionts followed by enrichment on complex media and a series of plating steps, under low light (PAR ~10µmol photons m(-2)s(-1)).

Effects of temperature and nutrient regimes on biomass and lipid production by six oleaginous microalgae in batch culture employing a two-phase cultivation strategy.

Commercial success of algal-based biofuels depends on growth characteristics and lipid metabolism of the production species. The oleaginous microalgae, Thalassiosira pseudonana, Odontella aurita, Nannochloropsis oculata, Isochrysis galbana, Chromulina ochromonoides, and Dunaliella tertiolecta, were cultivated under a matrix of two temperatures (10 and 20 °C) and two nutrient regimes (deplete and replete). For all species, a strong negative correlation between growth rate and lipid content was observed. Multiple stressors have no additive effect on lipid accumulation. Total oil content (fatty acid methyl esters, FAMEs, pg cell(-1)) was increased more by nutrient limitation than by temperature stress. In response to nutrient stress, N. oculata emerged as the most robust species with an increase in lipid accumulation of up to three to four-fold compared to the accumulation under nutrient sufficient conditions. Although stress conditions led to reduced fatty acid unsaturation in most taxa due to increased triacylglycerol (TAG) production, a high proportion of eicosapentaenoic acid (EPA) was maintained in O. aurita.

Early detection of protozoan grazers in algal biofuel cultures.

Future micro-algal biofuels will most likely be derived from open-pond production systems. These are by definition open to "invasion" by grazers, which could devastate micro-algal mass-cultures. There is an urgent requirement for methodologies capable of early detection and control of grazers in dense algal cultures. In this study a model system employing the marine alga Nannochloropsis oculata was challenged by grazers including ciliates, amoebae and a heterotrophic dinoflagellate. A FlowCAM flow-cytometer was used to detect all grazers investigated (size range <20->80 µm in length) in the presence of algae. Detection limits were <10 cells ml(-1) for both "large" and "small" model grazers, Euplotes vannus (80 × 45 µm) and an unidentified holotrichous ciliate (~18 × 8 µm) respectively. Furthermore, the system can distinguish the presence of ciliates in N. oculata cultures with biotechnologically relevant cell densities; i.e. >1.4 × 10(8) cells ml(-1) (>0.5 g l(-1) dry wt.).