Recent advances in sustainable hydrogen production from microalgae: Mechanisms, challenges, and future perspectives.

The depletion of fossil fuel reserves has resulted from their application in the industrial and energy sectors. As a result, substantial efforts have been dedicated to fostering the shift from fossil fuels to renewable energy sources via technological advancements in industrial processes. Microalgae can be used to produce biofuels such as biodiesel, hydrogen, and bioethanol. Microalgae are particularly suitable for hydrogen production due to their rapid growth rate, ability to thrive in diverse habitats, ability to resolve conflicts between fuel and food production, and capacity to capture and utilize atmospheric carbon dioxide. Therefore, microalgae-based biohydrogen production has attracted significant attention as a clean and sustainable fuel to achieve carbon neutrality and sustainability in nature. To this end, the review paper emphasizes recent information related to microalgae-based biohydrogen production, mechanisms of sustainable hydrogen production, factors affecting biohydrogen production by microalgae, bioreactor design and hydrogen production, advanced strategies to improve efficiency of biohydrogen production by microalgae, along with bottlenecks and perspectives to overcome the challenges. This review aims to collate advances and new knowledge emerged in recent years for microalgae-based biohydrogen production and promote the adoption of biohydrogen as an alternative to conventional hydrocarbon biofuels, thereby expediting the carbon neutrality target that is most advantageous to the environment.

A critical review on plastic waste life cycle assessment and management: Challenges, research gaps, and future perspectives.

The global production and consumption of plastics, as well as their deposition in the environment, are experiencing exponential growth. In addition, mismanaged plastic waste (PW) losses into drainage channels are a growing source of microplastic (MP) pollution concern. However, the complete understanding of their environmental implications throughout their life cycle is yet to be fully understood. Determining the potential extent to which MPs contribute to overall ecotoxicity is possible through the monitoring of PW release and MP removal during remediation. Life cycle assessments (LCAs) have been extensively utilized in many comparative analyses, such as comparing petroleum-based plastics with biomass and single-use plastics with multi-use alternatives. These assessments typically yield unexpected or paradoxical results. Nevertheless, there is still a paucity of reliable data and tools for conducting LCAs on plastics. On the other hand, the release and impact of MP have so far not been considered in LCA studies. This is due to the absence of inventory-related data regarding MP releases and the characterization factors necessary to quantify the effects of MP. Therefore, this review paper conducts a comprehensive literature review in order to assess the current state of knowledge and data regarding the environmental impacts that occur throughout the life cycle of plastics, along with strategies for plastic management through LCA.

Drivers of microbial food-web structure along productivity gradients.

Ratios between viruses, heterotrophic prokaryotes and chlorophyll a are key indicators of microbial food structure and both virus-prokaryote and prokaryote-chlorophyll ratios have been proposed to decrease with system productivity. However, the mechanisms underlying these responses are still insufficiently resolved and their consistency across aquatic ecosystem types requires critical evaluation. We assessed microbial community ratios in highly productive African soda-lakes and used our data from naturally hypereutrophic systems which are largely underrepresented in literature, to complement earlier across-system meta-analyses. In contrast to marine and freshwater systems, prokaryote-chlorophyll ratios in African soda-lakes did not decrease along productivity gradients. High-resolution time series from two soda-lakes indicated that this lack of response could be driven by a weakened top-down control of heterotrophic prokaryotes. Our analysis of virus-prokaryote relationships, revealed a reduction of virus-prokaryote ratios by high suspended particle concentrations in soda-lakes. This effect, likely driven by the adsorption of free-living viruses, was also found in three out of four additionally analysed marine datasets. However, the decrease of virus-prokaryote ratios previously reported in highly productive marine systems, was neither detectable in soda-lakes nor freshwaters. Hence, our study demonstrates that system-specific analyses can reveal the diversity of mechanisms that structure microbial food-webs and shape their response to productivity increases.

Short-term impacts of a large cultural event on the microbial pollution status of a pre-alpine river.

Rivers are impacted by microbial faecal pollution from various sources. We report on a short-term faecal pollution event at the pre-alpine Austrian river Traisen caused by the large cultural event FM4 Frequency music festival, with around 200,000 visitors over 4 days. We observed a massive increase of the faecal indicator bacteria (FIB) intestinal enterococci during the event, while Escherichia coli concentrations were only slightly elevated. This increase poses a significant potential health threat to visitors and people recreating downstream of the festival area. A plausible explanation for the uncoupling of the two FIBs may have been a differential persistence caused by a combination of factors including water temperature, solar radiation, and the excessive presence of personal care products (PCPs) in the river water. However, a potential impact of PCPs on FIB assay performance cannot be ruled out. Our observations are relevant for other intensively used bathing sites; detailed investigations on persistence and assay performance of the FIB in response to different ingredients of PCPs are highly recommended. We conclude that for future festivals at this river or other festivals taking place under similar settings, a more effective management is necessary to reduce deterioration in water quality and minimise health risks.

Decolorization of reactive azo dye using novel halotolerant yeast consortium HYC and proposed degradation pathway.

The presence of high salinity levels in textile wastewater poses a significant obstacle to the process of decolorizing azo dyes. The present study involved the construction of a yeast consortium HYC, which is halotolerant and was recently isolated from wood-feeding termites. The consortium HYC was mainly comprised of Sterigmatomyces halophilus SSA-1575 and Meyerozyma guilliermondii SSA-1547. The developed consortium demonstrated a decolourization efficiency of 96.1% when exposed to a concentration of 50 mg/l of Reactive Black 5 (RB5). The HYC consortium significantly decolorized RB5 up to concentrations of 400 mg/l and in the presence of NaCl up to 50 g/l. The effects of physicochemical factors and the degradation pathway were systematically investigated. The optimal pH, salinity, temperature, and initial dye concentration were 7.0, 3%, 35 °C and 50 mg/l, respectively. The co-carbon source was found to be essential, and the addition of glucose resulted in a 93% decolorization of 50 mg/l RB5. The enzymatic activity of various oxido-reductases was assessed, revealing that NADH-DCIP reductase and azo reductase exhibited greater activity in comparison to other enzymes. UV-Visible (UV-vis) spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) were utilized to identify the metabolites generated during the degradation of RB5. Subsequently, a metabolic pathway was proposed. The confirmation of degradation was established through alterations in the functional groups and modifications in molecular weight. The findings indicate that this halotolerant yeast consortium exhibits promising potential of degrading dye compounds. The results of this study offer significant theoretical basis and crucial perspectives for the implementation of halotolerant yeast consortia in the bioremediation of textile and hypersaline wastewater. This approach is particularly noteworthy as it does not produce aromatic amines.

High Anthropogenic Organic Matter Inputs during a Festival Increase River Heterotrophy and Refractory Carbon Load.

Streams and rivers metabolize dissolved organic matter (DOM). Although most DOM compounds originate from natural sources, recreational use of rivers increasingly introduces chemically distinct anthropogenic DOM. So far, the ecological impact of this DOM source is not well understood. Here, we show that a large music festival held adjacent to the Traisen River in Austria increased the river's dissolved organic carbon (DOC) concentration from 1.6 to 2.1 mg L-1 and stream ecosystem respiration from -3.2 to -4.5 mg L-1. The DOC increase was not detected by sensors continuously logging absorbance spectra, thereby challenging their applicability for monitoring. However, the fluorescence intensity doubled during the festival. Using parallel factor analysis, we were able to assign the increase in fluorescence intensity to the chemically stable UV-B filter phenylbenzimidazole sulfonic acid, indicating organic compounds in sunscreen and other personal care products as sources of elevated DOC. This observation was confirmed by liquid chromatography coupled with mass spectrometry. The elevated respiration is probably fueled by anthropogenic DOM contained in beer and/or urine. We conclude that intense recreational use of running waters transiently increases the anthropogenic DOM load into stream ecosystems and alters the fluvial metabolism. We further propose that chemically distinct, manmade DOM extends the natural range of DOM decomposition rates in fluvial ecosystems.

Adaptive responses of mature giant chloroplasts in the deep-shade lycopod Selaginella erythropus to prolonged light and dark periods.

Deep-shade plants have adapted to low-light conditions by varying morphology and physiology of cells and chloroplasts, but it still remains unclear, if prolonged periods of high-light or darkness induce additional modifications in chloroplasts' anatomy and pigment patterns. We studied giant chloroplasts (bizonoplasts) of the deep-shade lycopod Selaginella erythropus in epidermal cells of mature fully developed microphylls and subjected them to prolonged darkness and high-light conditions. Chloroplast size and ultrastructure were investigated by light and electron microscopy. Physiological traits were studied by pigment analyses, photosynthetic performance of photosystem II, and formation of reactive oxygen species. Results show that (a) thylakoid patterns and shape of mature bizonoplasts vary in response to light and dark conditions. (b) Prolonged darkness induces transitory formation of prolamellar bodies, which so far have not been described in mature chloroplasts. (c) Photosynthetic activity is linked to structural responses of chloroplasts. (d) Photosystem II is less active in the upper zone of bizonoplasts and more efficient in the grana region. (e) Formation of reactive oxygen species reflects the stress level caused by high-light. We conclude that during prolonged darkness, chlorophyll persists and even increases; prolamellar bodies form de novo in mature chloroplasts; bizonoplasts have spatial heterogeneity of photosynthetic performance.

Benthic-pelagic coupling drives non-seasonal zooplankton blooms and restructures energy flows in shallow tropical lakes.

Zooplankton blooms are a frequent phenomenon in tropical systems. However, drivers of bloom formation and the contribution of emerging resting eggs are largely unexplored. We investigated the dynamics and the triggers of rotifer blooms in African soda-lakes and assessed their impact on other trophic levels. A meta-analysis of rotifer peak densities including abundances of up to 6 × 105 individuals L-1 demonstrated that rotifer bloom formation was uncoupled from the food environment and the seasonality of climatic conditions. A time series with weekly sampling intervals from Lake Nakuru (Kenya) revealed that intrinsic growth factors (food quality and the physicochemical environment) significantly affected rotifer population fluctuations, but were of minor importance for bloom formation. Instead, rotifer bloom formation was linked to sediment resuspension, a prerequisite for hatching of resting-eggs. Population growth rates exceed pelagic birth rates and simulations of rotifer dynamics confirmed the quantitative importance of rotifer emergence from the sediment egg-bank and signifying a decoupling of bloom formation from pelagic reproduction. Rotifer blooms led to a top-down control of small-sized algae and facilitated a switch to more grazing-resistant, filamentous cyanobacteria. This shift in phytoplankton composition cascaded up the food chain and triggered the return of filter-feeding flamingos. Calculations of consequent changes in the lake's energy budget and export of aquatic primary production to terrestrial ecosystems demonstrated the large potential impact of nonseasonal disturbances on the functioning of shallow tropical lakes.

Seasonal succession of the travertine-forming desmid Oocardium stratum.

The calcifying Conjugatophyte Oocardium stratum occurs exclusively in spring-associated limestones (SAL) with active meteogene limestone deposition. The macroscopic colonies of Oocardium stratum form hemispherical, pinhead-like structures with a diameter of 0.5-2.0 mm. As its autecology is still poorly understood, we focused on the seasonal development of Oocardium stratum and linked environmental factors to its abundance. The study was conducted in a rivulet in Lunz/See (Austria) for 16 months on a weekly (growing season) to monthly (winter season) basis. Oocardium colonies were found throughout the whole year, with maximum abundance during the mid-summer months July and August. Repeated macro-mapping of three SAL sites measuring 750 cm(2) each showed a maximum Oocardium cover of around 30% in August; two smaller peaks developed in early summer and late autumn with ~10% cover. Diatom mats dominated by Cymbella excisiformis occurred in spring, autumn and winter, with more than 75% cover. The seasonal change between Oocardium and diatoms in limestone-precipitating springs causes a typical sequence pattern of limestone layers. Redundancy analysis revealed water temperature and bicarbonate content as the main structuring factors; these control the occurrence and growth of Oocardium, reflecting season as a background variable. Optimum growth conditions for Oocardium were an alkalinity around 4.7 meq · L(-1) and a water temperature around 13°C. Site openness, nitrate and dissolved carbon dioxide were inversely related to Oocardium biomass, the opposite for diatoms. Other environmental factors such as total ions or soluble reactive phosphorus had no significant influence on Oocardium stratum abundance.

Phytoplankton patterns along a series of small man-made reservoirs in Kenya.

We studied nine small man-made reservoirs located in different climate regions of Kenya to get an insight into the relationship between phytoplankton community structure and its environment. The investigated ponds form three groups of three reservoirs each found in the rural areas of Machakos district, Mount Kenya region, and Lake Victoria area with varied climatic characteristics. The ponds were sampled in monthly intervals between May 2007 and June 2008 for physicochemical variables including water chemistry, phytoplankton community composition, zooplankton abundance, and bacterial numbers. All ponds were classified as hypertrophic. Seasonal changes were reflected in the phytoplankton pattern, as all ponds showed a community shift after the short dry season in February. Due to high nutrient loads and increased turbidity, Cyanobacteria, which were initially thought to be predominating in all investigated water bodies, were found to play only a minor role except for the Bomet reservoir in Lake Victoria region. Instead, Chloro- and Streptophyta, Dinophyta, and Euglenophyta were abundant in the pelagial. A principal component analysis explained around 85 % of the data variance with four principal components (PCs) interpreted as "location", "ions", "zooplankton", and "particulate matter". A clear separation of ponds with and without cattle access based on algal species community data was found indicating the need for a sustainable use and regular monitoring program as the local population is largely dependent on these sensitive small-scale ecosystems.

Impact of heavy metals (Cu, Fe, Pb, Zn) on carbon and nitrogen uptake of the diatom-bearing benthic foraminifera Heterosteginadepressa.

Foraminifera are protists primarily living in benthic marine and estuarine environments. We studied uptake of inorganic carbon (C) and nitrogen (N) of the photosymbiont-bearing benthic coral reef foraminifera Heterostegina depressa in the presence of heavy metals. Incubation experiments were accomplished with artificial seawater enriched with copper, iron, lead and zinc at two different concentration levels (10 and 100 fold enriched in contrast to the usual culture medium). Additionally, isotopically labelled 13C-sodium bicarbonate and 15N-ammonium chloride were added to trace their assimilation over time (1 d, 3 d, 5 d, 7 d). Pulse-amplified modulated fluorescence measurements were performed to measure the potential impacts of heavy metals on chlorophyll fluorescence of the photosymbiont. Increased levels of copper (430.5 µg Cu/l) exhibited the greatest toxicity, while for low levels no effect on the overall metabolism of the foraminifera and the fluorescence activity of the photosymbiont could be detected. Iron (III) increased the symbiont activity, independent of concentration applied (44.5 and 513.3 µg Fe/l), which indicates Fe-limitation of the algal symbiont. Lead enrichment showed no detectable effect even at high concentration. Low concentrations of zinc (35.1 µg Zn/l) promoted the metabolism of the foraminifera, while high concentrations (598.4 µg Zn/l) were toxic. At low levels, two metals (Fe and Zn) promoted symbiont activity, at high levels, iron still boosted photosynthesis, but Zn and Cu had a negative impact on the obligatory photosynthetic symbionts.

Effect of micro-plastic particles on coral reef foraminifera.

Foraminifera are single-celled protists which are important mediators of the marine carbon cycle. In our study, we explored the potential impact of polystyrene (PS) microplastic particles on two symbiont-bearing large benthic foraminifera species-Heterostegina depressa and Amphistegina lobifera-over a period of three weeks, employing three different approaches: investigating (1) stable isotope (SI) incorporation-via 13C- and 15N-labelled substrates-of the foraminifera to assess their metabolic activity, (2) photosynthetic efficiency of the symbiotic diatoms using imaging PAM fluorometry, and (3) microscopic enumeration of accumulation of PS microplastic particles inside the foraminiferal test. The active feeder A. lobifera incorporated significantly more PS particles inside the cytoplasm than the non-feeding H. depressa, the latter accumulating the beads on the test surface. Photosynthetic area of the symbionts tended to decrease in the presence of microplastic particles in both species, suggesting that the foraminiferal host cells started to digest their diatom symbionts. Compared to the control, the presence of microplastic particles lead to reduced SI uptake in A. lobifera, which indicates inhibition of inorganic carbon and nitrogen assimilation. Competition for particulate food uptake was demonstrated between algae and microplastic particles of similar size. Based on our results, both species seem to be sensitive to microplastic pollution, with non-feeding H. depressa being more strongly affected.

Impact of pesticides on marine coral reef foraminifera.

Our laboratory study looked into how pesticides affect the foraminifera species Heterostegina depressa and their obligatory algal endosymbionts. We incubated the foraminifera separately with different types of pesticides at varying concentrations (1 %, 0.01 % and 0.0001 %); we included the insecticide Confidor© (active substance: imidacloprid), the fungicide Pronto©Plus (tebuconazole), and the herbicide Roundup© (glyphosate). Our evaluation focused on the symbiont's photosynthetically active area (PA), and the uptake of dissolved inorganic carbon (DIC) and nitrogen (nitrate) to determine the vitality of the foraminifera. Our findings showed that even the lowest doses of the fungicide and herbicide caused irreparable damage to the foraminifera and their symbionts. While the insecticide only deactivated the symbionts (PA = 0) at the highest concentration (1 %), the fungicide, and herbicide caused complete deactivation even at the lowest levels provided (0.0001 %). The fungicide had the strongest toxic effect on the foraminiferal host regarding reduced isotope uptake. In conclusion, all pesticides had a negative impact on the holosymbiont, with the host showing varying degrees of sensitivity towards different types of pesticides.

Cytotoxicity and antibacterial susceptibility assessment of a newly developed pectin-chitosan polyelectrolyte composite for dental implants.

Biopolymers such as chitosan and pectin are currently attracting significant attention because of their unique properties, which are valuable in the food industry and pharmaceutical applications. These properties include non-toxicity, compatibility with biological systems, natural decomposition ability, and structural adaptability. The objective of this study was to assess the performance of two different ratios of pectin-chitosan polyelectrolyte composite (PCPC) after applying them as a coating to commercially pure titanium (CpTi) substrates using electrospraying. The PCPC was studied in ratios of 1:2 and 1:3, while the control group consisted of CpTi substrates without any coating. The pull-off adhesion strength, cytotoxicity, and antibacterial susceptibility tests were utilized to evaluate the PCPC coatings. In order to determine whether the composite coating was the result of physical blending or chemical bonding, the topographic surface parameters were studied using Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). PCPC (1:3) had the highest average cell viability of 93.42, 89.88, and 86.85% after 24, 48, and 72 h, respectively, as determined by the cytotoxicity assay, when compared to the other groups. According to the Kirby-Bauer disk diffusion method for testing antibacterial susceptibility, PCPC (1:3) showed the highest average diameter of the zone of inhibition, measuring 14.88, 14.43, and 11.03 mm after 24, 48, and 72 h of incubation, respectively. This difference was highly significant compared to Group 3 at all three time periods. PCPC (1:3) exhibited a significantly higher mean pull-off adhesion strength (521.6 psi) compared to PCPC (1:2), which revealed 419.5 psi. PCPC (1:3) coated substrates exhibited better surface roughness parameters compared to other groups based on the findings of the AFM. The FTIR measurement indicated that both PCPC groups exhibited a purely physical blending in the composite coating. Based on the extent of these successful in vitro experiments, PCPC (1:3) demonstrates its potential as an effective coating layer. Therefore, the findings of this study pave the way for using newly developed PCPC after electrospraying coating on CpTi for dental implants.

Major and trace element geochemistry of Lake Bogoria and Lake Nakuru, Kenya, during extreme draught.

The physico-chemical properties of water samples from the two athalassic endorheic lakes Bogoria and Nakuru in Kenya were analysed. Surface water samples were taken between July 2008 and October 2009 in weekly intervals from each lake. The following parameters were determined: pH, salinity, electric conductivity, dissolved organic carbon (DOC), the major cations (FAAS and ICP-OES) and the major anions (IC), as well as certain trace elements (ICP-OES). Samples of superficial sediments were taken in October 2009 and examined using Instrumental Neutron Activation Analysis (INAA) for their major and trace element content including rare earth elements (REE). Both lakes are highly alkaline with a dominance of Na > K > Si > Ca in cations and HCO3 > CO3 > Cl > F > SO4 in anions. Both lakes also exhibited high concentrations of Mo, As and fluoride. Due to an extreme draught from March to October 2009, the water level of Lake Nakuru dropped significantly. This created drastic evapoconcentration, with the total salinity rising from about 20‰ up to 63‰. Most parameters (DOC, Na, K, Ca, F, Mo and As) increased with falling water levels. A clear change in the quality of DOC was observed, followed by an almost complete depletion of dissolved Fe from the water phase. In Lake Bogoria the evapoconcentration effects were less pronounced (total salinity changed from about 40‰ to 48‰). The distributions of REE in the superficial sediments of Lake Nakuru and Lake Bogoria are presented here for the first time. The results show a high abundance of the REE and a very distinct Eu depletion of Eu/Eu* = 0.33-0.45.

The change in metabolic activity of a large benthic foraminifera as a function of light supply.

We studied metabolic activity of the symbiont-bearing large benthic foraminifer Heterostegina depressa under different light conditions. Besides the overall photosynthetic performance of the photosymbionts estimated by means of variable fluorescence, the isotope uptake (13C and 15N) of the specimens (= holobionts) was measured. Heterostegina depressa was either incubated in darkness over a period of 15 days or exposed to an 16:8 h light:dark cycle mimicking natural light conditions. We found photosynthetic performance to be highly related to light supply. The photosymbionts, however, survived prolonged darkness and could be reactivated after 15 days of darkness. The same pattern was found in the isotope uptake of the holobionts. Based on these results, we propose that 13C-carbonate and 15N-nitrate assimilation is mainly controlled by the photosymbionts, whereas 15N-ammonium and 13C-glucose utilization is regulated by both, the symbiont and the host cells.

Microalgae-based wastewater treatment: Mechanisms, challenges, recent advances, and future prospects.

The rapid expansion of both the global economy and the human population has led to a shortage of water resources suitable for direct human consumption. As a result, water remediation will inexorably become the primary focus on a global scale. Microalgae can be grown in various types of wastewaters (WW). They have a high potential to remove contaminants from the effluents of industries and urban areas. This review focuses on recent advances on WW remediation through microalgae cultivation. Attention has already been paid to microalgae-based wastewater treatment (WWT) due to its low energy requirements, the strong ability of microalgae to thrive under diverse environmental conditions, and the potential to transform WW nutrients into high-value compounds. It turned out that microalgae-based WWT is an economical and sustainable solution. Moreover, different types of toxins are removed by microalgae through biosorption, bioaccumulation, and biodegradation processes. Examples are toxins from agricultural runoffs and textile and pharmaceutical industrial effluents. Microalgae have the potential to mitigate carbon dioxide and make use of the micronutrients that are present in the effluents. This review paper highlights the application of microalgae in WW remediation and the remediation of diverse types of pollutants commonly present in WW through different mechanisms, simultaneous resource recovery, and efficient microalgae-based co-culturing systems along with bottlenecks and prospects.

Hidden genetic diversity in the green alga Spirogyra (Zygnematophyceae, Streptophyta).

BACKGROUND: The unbranched filamentous green alga Spirogyra (Streptophyta, Zygnemataceae) is easily recognizable based on its vegetative morphology, which shows one to several spiral chloroplasts. This simple structure falsely points to a low genetic diversity: Spirogyra is commonly excluded from phylogenetic analyses because the genus is known as a long-branch taxon caused by a high evolutionary rate. RESULTS: We focused on this genetic diversity and sequenced 130 Spirogyra small subunit nuclear ribosomal DNA (SSU rDNA) strands of different origin. The resulting SSU rDNA sequences were used for phylogenetic analyses using complex evolutionary models (posterior probability, maximum likelihood, neighbor joining, and maximum parsimony methods). The sequences were between 1672 and 1779 nucleotides long. Sequence comparisons revealed 53 individual clones, but our results still support monophyly of the genus. Our data set did not contain a single slow-evolving taxon that would have been placed on a shorter branch compared to the remaining sequences. Out of 130 accessions analyzed, 72 showed a secondary loss of the 1506 group I intron, which formed a long-branched group within the genus. The phylogenetic relationship to the genus Spirotaenia was not resolved satisfactorily. The genetic distance within the genus Spirogyra exceeded the distances measured within any other genus of the remaining Zygnemataceae included in this study. CONCLUSION: Overall, we define eight distinct clades of Spirogyra, one of them including the genus Sirogonium. A large number of non-homoplasious synapomorphies (NHS; 114 NHS in total) was found for Spirogyra (41 NHS) and for each clade (totaling 73 NHS). This emphasizes the high genetic diversity of this genus and the distance to the remaining Zygnematophyceae.

Estimating Biomass and Vitality of Microalgae for Monitoring Cultures: A Roadmap for Reliable Measurements.

Estimating algal biomass is a prerequisite for monitoring growth of microalgae. Especially for large-scale production sites, the measurements must be robust, reliable, fast and easy to obtain. We compare the relevant parameters, discuss potential hurdles and provide recommendations to tackle these issues. The focus is on optical density and in vivo autofluorescence of chlorophyll, which have proven to be ideal candidates for monitoring purposes. Beyond biomass, cell vitality is also crucial for maintaining cultures. While maximizing biomass yield is often the primary consideration, some applications require adverse growth conditions for the synthesis of high-quality compounds. The non-invasive technique of pulse-amplified modulated (PAM) fluorescence measurements provides an ideal tool and is increasingly being employed due to ever more affordable devices. We compared three devices and studied the robustness of the dark fluorescence yield of photosystem II (Fv/Fm) at various cell densities. Although the so-called inner filter effects influence the fluorescence signal, the resulting Fv/Fm remain stable and robust over a wide range of cell densities due to mutual effects.

How to Verify Non-Presence-The Challenge of Axenic Algae Cultivation.

Many phycological applications require the growth and maintenance of pure algae cultures. In some research areas, such as biochemistry and physiology, axenic growth is essential to avoid misinterpretations caused by contaminants. Nonetheless, axenicity-defined as the state of only a single strain being present, free of any other organism-needs to be verified. We compare the available methods to assess axenicity. We first purified unialgal Limnospira fusiformis cultures with an established series of axenicity treatments, and by including two additional treatment steps. The presumable axenic cultures were then tested for their axenic state by applying conventional tests on LB (lysogeny broth) agar-plates, 16S rRNA gene amplicon sequencing, flow-cytometry and epifluorescence microscopy. Only the plate tests indicated axenic conditions. We found a linear relationship between total cell counts of contaminants achieved by flow cytometry and epifluorescence microscopy, with flow cytometry counts being consistently higher. In addition, 16S rRNA gene amplicon sequencing demonstrated its superiority by not only being an efficient tool for axenicity testing, but also for identification of persistent contaminants. Although classic plate tests are still commonly used to verify axenicity, we found the LB-agar-plate technique to be inappropriate. Cultivation-independent methods are highly recommended to test for axenic conditions. A combination of flow-cytometry and 16S rRNA gene amplicon sequencing complement each other and will yield the most reliable result.