Microalgal Biofilms Research Articles

Effects of nutrient loading on Anabaena flos-aquae biofilm: biofilm growth and nutrient removals.

Effects of three different nutrient loadings (low nutrient loading, medium nutrient loading and high nutrient loading, denoted as LNS, MNS and HNS, respectively) on the structure and functions of algal biofilm using Anabaena flos-aquae were investigated using synthetic wastewater. Nutrients removal efficiencies, biofilm thickness, microalgae dehydrogenase activity (DHA) and exopolysaccharide (EPS) productions were examined. Results showed that the changes of nutrient concentration were insignificant after 4 days of experiment for the case of HNS condition; 9 days for the case of MNS condition, and 6 days for the case of LNS condition, respectively. The biofilm thickness, nutrient removal efficiencies, algae DHA and EPS productions increased with the increase of nutrient loadings in synthetic wastewater. For the case of HNS condition, the microalgal biofilm exhibited the best performance in terms of C, N and P removal efficiencies, reaching the removal rates of 68.45, 3.56 and 1.61 mg·L(-1)·d(-1) for C, N, P, respectively. This was likely because, fact with the high nutrient loading, the high biological activity could be achieved, thus resulting in high nutrient removals. The thickness of the biofilm in HNS condition was 75 μm, which was closely related to EPS production. DHA and EPS concentrations were 7.24 and 1.8 × 10(-2) mg·mm(-2), respectively. It was also shown that apart from the nutrient loading, the structure and functions of microalgal biofilm were also influenced by other factors, such as illumination and temperature.

Continuous removal of zinc from wastewater and mine dump leachate by a microalgal biofilm PSBR

Bio-removal of heavy metals from wastewater by microalgae has been investigated for decades. However, technical and economical limitations of cultivation systems for microalgae still impair progress toward application. Recently, a novel type of bioreactor for (immobilized) biofilm cultivation, the Porous Substrate Bioreactor (PSBR), has been shown to optimize biomass feedstock production and harvest, offering novel possibilities for application in the treatment of wastewater. We used two types of laboratory-scale Twin-Layer PSBRs to remove zinc (2–3mgZnL−1) from synthetic wastewater and real mine dump leachate in a continuous and batch process. The selection and use of a biofilm of a Zn-resistant strain of the green alga Stichococcus bacillaris (EC50 of 28.9mgZnL−1 based on Pulse-amplitude modulated (PAM) chlorophyll fluorescence analysis) led to a high zinc absorption capacity of 15–19mgZng−1 algal dry matter. The removal capacity for zinc correlated positively with biomass production and was thus, light dependent. Bio-removal properties observed here combined with biomass productivities of PSBR systems compare favorably with other algal-based bio-sorption technologies.

Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe.

Although estuarine microphytobenthos (MPB) is frequently exposed to excessive light and temperature conditions, little is known on their interactive effects on MPB primary productivity. Laboratory and in situ experiments were combined to investigate the short-term joint effects of high light (HL) and high temperature (37 °C versus 27 °C) on the operating efficiency of photoprotective processes [vertical migration versus non-photochemical quenching (NPQ)] exhibited by natural benthic diatom communities from two intertidal flats in France (FR) and Portugal (PT). A clear latitudinal pattern was observed, with PT biofilms being more resistant to HL stress, regardless the effect of temperature, and displaying a lower relative contribution of vertical migration to photoprotection and a stronger NPQ in situ. However, higher temperature leads to comparable effects, with photoinhibition increasing to about three times (i.e. from 3% to 10% and from 8% to 22% in PT and FR sites respectively). By using a number of methodological novelties in MPB research (lipid peroxidation quantification, Lhcx proteins immunodetection), this study brings a physiological basis to the previously reported depression of MPB photosynthetic productivity in summer. They emphasize the joint role of temperature and light in limiting, at least transiently (i.e. during emersion), MPB photosynthetic activity in situ.

High light and carbon dioxide optimize surface productivity in a Twin-Layer biofilm photobioreactor

Immobilized cultivation of microalgae recently received increasing attention avoiding some of the major drawbacks of suspension cultivation. Twin-Layer photobioreactors (TL-PBRs), a type of porous substrate bioreactor (PSBR), have proven to be an efficient cultivation system for microalgal biofilms. However, the TL-PBR had not yet been optimized with respect to light and CO2. The effect of light intensity up to 1486μmolm−2s−1 as well as supplementary CO2 (0.12–5%) was investigated using the green alga Halochlorella rubescens as a model. With a combination of 1023μmolphotonsm−2s−1 and a CO2 level of 3% a surface productivity of 31.2gdry matter m−2d−1 was obtained. Along with a standing crop of 205gm−2 growth area (1011μmolphotonsm−2s−1 at atmospheric CO2 level) these values represent the highest biomass productivity and total biomass yield yet reported for a PSBR. This work, furthermore, demonstrates the successful transfer of laboratory scale data to a small prototype-scale greenhouse photobioreactor. With respect to an optimal Twin-Layer photobioreactor design, results suggest that the highest footprint productivities (~50gm−2d−1 without supplementary CO2) can be obtained at high light dilution rates. In contrast, a scenario using high irradiances and supplementary CO2 might be favourable from an economic point of view.

Phosphorus removal using a microalgal biofilm in a new biofilm photobioreactor for tertiary wastewater treatment

Eutrophication of surface water has been an important environmental issue for nearly half a century. High concentrations of phosphorus contribute to the process of eutrophication, resulting in the demand for effective and economic methods of phosphorus removal from treated water. The aim of this study was to evaluate the capacity for phosphorus removal of a microalgal biofilm during different light regimes. The photobioreactor was operated for nine months each year over a two-year period without interruption and without any need of re-inoculation. The algal biofilm was able to remove 97 ± 1% of total phosphorus from wastewater during 24 h of continuous artificial illumination. The average TP uptake rate in our experiments was 0.16 ± 0.008 g m−2 d−1. Phosphorus removal values ranged from 36 to 41% when the algal biofilm was illuminated by natural light (12 h sunlight–12 h night). The biomass production rate was 12.21 ± 10 g dry weight m−2 d−1 in experiments with continuous artificial light and 5.6 ± 1 g dry weight (DW) m−2 d−1 in experiments with natural light. These results indicate the great potential of microalgal biofilms in the tertiary treatment of wastewater.

Dissolved inorganic carbon enhanced growth, nutrient uptake, and lipid accumulation in wastewater grown microalgal biofilms

Microalgal biofilms grown to evaluate potential nutrient removal options for wastewaters and feedstock for biofuels production were studied to determine the influence of bicarbonate amendment on their growth, nutrient uptake capacity, and lipid accumulation after nitrogen starvation. No significant differences in growth rates, nutrient removal, or lipid accumulation were observed in the algal biofilms with or without bicarbonate amendment. The biofilms possibly did not experience carbon-limited conditions because of the large reservoir of dissolved inorganic carbon in the medium. However, an increase in photosynthetic rates was observed in algal biofilms amended with bicarbonate. The influence of bicarbonate on photosynthetic and respiration rates was especially noticeable in biofilms that experienced nitrogen stress. Medium nitrogen depletion was not a suitable stimulant for lipid production in the algal biofilms and as such, focus should be directed toward optimizing growth and biomass productivities to compensate for the low lipid yields and increase nutrient uptake.

Microalgal biofilms for biomass production

Microalgae are promising candidates for recycling of carbon dioxide (CO2) into renewable bioproducts. However, the low biomass concentration of current suspension culture systems leads to high water requirements, inefficient harvesting and high liquid transportation costs. Despite ongoing research, these still propose a challenge to the economic viability of microalgal cultivation. Microalgal biofilms provide an alternative approach to biomass production that could resolve these challenges by growing the cells attached to a surface, surrounded by a self-produced matrix of polymers. Microalgal biofilms have much higher biomass concentrations than suspension cultures, and the attached cells are easy to separate from the cultivation medium. However, cultivating microalgal biofilms requires the development of a purposefully designed cultivation system, especially due to interactions between cells and surface, persistent gradients in the biomass and the effects of flow, which play a critical role for biofilm productivity. A range of systems has been employed for the cultivation of microalgal biofilms, with biomass productivities of up to 60 grammes dry weight (g(DW)) m−2 day−1 (dry weight per ground area) outdoors and up to 80 g(DW) m−2 day−1 under laboratory conditions, respectively. However, there is considerable variation of reported results along with experimental conditions, which limits the capability for quantitative comparisons with other systems and hinders the identification of the drivers and variables that dictate microalgal biomass formation. Development of standard conditions and representative species would be required for closing this gap and for realising the full potential of microalgal biofilm cultivation as a viable process for industrial biomass production.

Microalgal biofilms on common yew needles in relation to anthropogenic air pollution in urban Prague, Czech Republic

Excessive occurrence of microalgae on needles of gymnosperms was reported for the first time in the 1980s from the Scandinavian countries. Since then, it has been repeatedly encountered on needles from various European forest habitats. The abundance of these biofilms has been related to the climatic conditions, such as temperature and precipitation, as well as to the air pollution by nitrogen and sulfur oxides. Urban areas typically have relatively homogenous climates and profound variation in levels of air pollution. Therefore, variation in the occurrence of biofilms in localities within an urban area may be related to local anthropogenic air pollution. We investigated the abundance of biofilms occurring on needles of the common yew (Taxus baccata) in the city of Prague, Czech Republic. The biofilms were composed of algae, fungi and particulate matter. The cover area of the biofilms was marginally explained by a positive influence of short-term maximum atmospheric levels of nitrogen dioxide (NO2). The amounts of the microalgae were also positively influenced by short-term maximum NO2 levels. In addition, high atmospheric levels of particulate matter (PM10) were related to low abundance of algae. The microbial biofilms growing on widely cultivated conifers, such as the common yew, form one of the few commonly occurring natural communities in highly urbanized central areas of temperate European cities. Consequently, we propose that microscopic analysis of biofilms may be used as a rapid and cheap method to collect ecological data. Such data may be used in biomonitoring schemes illustrating the effects of anthropogenic air pollution on natural microcommunities in urban areas.

Biofilm formation and lipid accumulation of attached culture of Botryococcus braunii.

The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that forms their immediate environment. The objective of this paper was to investigate the relationships between culture conditions, EPS, microalgal biofilms growth and lipid accumulation. Fresh water alga Botryococcus braunii was attached culture in multi-layers photobioreactors with different culture media and substrates. The results indicated that the production of EPS was affected by culture period, nutrient, and substrate. Increasing the production of EPS may enhance the biofilm growth. However, the EPS components, namely proteins and polysaccharide had a more profound effect on biofilm formation compared to total EPS, with protein being more significant than polysaccharide. Nitrogen-free and EPS strategies were conducted to increase the lipid content of B. braunii biofilm from 11.6 to 42.3% and 51.3%, respectively. Compared to suspended culture, the lipid quality was enhanced. The dominant component of hexadecanoic acid (16:0) was enhanced from 21.78 to 48.17% and 55.44%, respectively.

Biofilm Formation by Chlorella vulgaris is Affected by Light Quality

Formation of biofilm on surfaces is a common feature in aquatic environments. Major groups of inhabitants in conditions where light is present are photoautotrophic microorganisms, such as cyanobacteria and microalgae. This study examined the effect of light quality on growth and biofilm formation of the microalgal species Chlorella vulgaris. Dense biofilm formation and aggregated growth of cells were observed in treatments exposed to blue, purple and white light. Less dense biofilm formation and solitary growth of cells were observed in treatments exposed to red, yellow or green light. Microalgal biofilms are of high importance in many respects, not least from an economic perspective. One example is the intense efforts undertaken to control biofilm formation on technical surfaces such as ship hulls. The present study suggests that light quality plays a role in biofilm formation and that blue-light receptors may be involved.

Direct measurement and characterization of active photosynthesis zones inside wastewater remediating and biofuel producing microalgal biofilms

Microalgal biofilm based technologies are of keen interest due to their high biomass concentrations and ability to utilize light and CO2. While photoautotrophic biofilms have long been used for wastewater remediation, biofuel production represents a relatively new and under-represented focus area. However, the direct measurement and characterization of fundamental parameters required for industrial control are challenging due to biofilm heterogeneity. This study evaluated oxygenic photosynthesis and respiration on two distinct microalgal biofilms cultured using a novel rotating algal biofilm reactor operated at field- and laboratory-scales. Clear differences in oxygenic photosynthesis and respiration were observed based on different culturing conditions, microalgal composition, light intensity and nitrogen availability. The cultures were also evaluated as potential biofuel synthesis strategies. Nitrogen depletion was not found to have the same effect on lipid accumulation compared to traditional planktonic microalgal studies. Physiological characterizations of these microalgal biofilms identify fundamental parameters needed to understand and control process optimization.

Distribution patterns of subaerial corticolous microalgae in two European regions

Subaerial micro-algal biofilms growing on tree bark are omnipresent in a variety of ecosystems. However, terrestrial microhabitats have traditionally attracted far less attention from phycologists than marine and freshwater habitats, so their diversity and community structure is still very poorly known (Freystein & Reisser 2010, Rindi et al. 2010). Because of the lack of suitable discriminating characters and because species concepts are poor, species-level microscopic identification of most subaerial algae and Cyanobacteria is complicated (Ettl & Gartner 1995). In comparison to that of planktic and benthic communities, the morphological diversity of terrestrial microalgae is strikingly low; most taxa have coccoid spherical to oval cells, or simple filamentous thalli (Hoffmann 1989, Ettl & Gartner 1995). Consequently, most microscopic studies of subaerial assemblages have been based on either limited datasets acquired from approximate morphological identification of cultured strains (e.g. Nakano et al. 1991, Neustupa & Skaloud 2008), or direct observations of natural samples. Research on natural samples has typically been concentrated on several conspicuous groups, such as Trentepohliales, Klebsormidium, and Prasiola (e.g. Rindi & Guiry 2004, Hedenas et al. 2007). Recent molecular studies of subaerial microalgae have mostly been focused on phylogenetics and taxonomy of new and little-known microalgal and cyanobacterial lineages thriving in these microhabitats (Rindi et al. 2006, Zhang et al. 2008, Neustupa et al. 2013). They illustrated that the real phylogenetic diversity of subaerial microalgae was probably grossly underestimated by traditional morphological taxonomy. Molecular data revealed that the micro-algal communities of these habitats include numerous as yet unknown phylogenetic taxa. Some have been described as new micro-algal genera, such as Spongiochrysis, Heveochlorella, Hylodesmus, and Leptochlorella (Rindi et al. 2006, Zhang et al. 2008, Elias et al. 2010, Neustupa et al. 2013). Despite methodological limitations, microscopy still provides the very core of current knowledge on ecology and distribution patterns of subaerial algae, including those in corticolous biofilms. Tropical corticolous biofilms are often

Influence of Surface Zeta Potential on Adhesion of <i>Chlorella sp. to Substratum Surfaces</i>

Microalgae, in wet conditions, tend to grow on surfaces and form biofilms. The adhesion of microalgae to surfaces is very important for algal mass culture. The formation and development of microalgal biofims are in large denpend on the properties of cell surface, substratum surface and gowth medium. In this paper, the influence of substratum surface zeta potential on the microalgal biofilms was particularly investigated. We focused on a widely-used microalgal strain, the freshwater autotrophicChlorella sp..The adhesion phenomena ofChlorella sp.to surfaces with different zeta potential were observed microscopically. It was found thatChlorella sp.adhered easily to the surface with a positive zeta potential and difficultly to the surface with a negative zeta potential. We concluded that the surface zeta potential had a greater influence on the adhesion of microalgal cells to substratum surfaces. Our findings have important implications for microalgae mass culture and harvesting.

Scenario Analysis of Nutrient Removal from Municipal Wastewater by Microalgal Biofilms

Microalgae can be used for the treatment of municipal wastewater. The application of microalgal biofilms in wastewater treatment systems seems attractive, being able to remove nitrogen, phosphorus and COD from wastewater at a short hydraulic retention time. This study therefore investigates the area requirement, achieved effluent concentrations and biomass production of a hypothetical large-scale microalgal biofilm system treating municipal wastewater. Three scenarios were defined: using microalgal biofilms: (1) as a post-treatment; (2) as a second stage of wastewater treatment, after a first stage in which COD is removed by activated sludge; and (3) in a symbiotic microalgal/heterotrophic system. The analysis showed that in the Netherlands, the area requirements for these three scenarios range from 0.32 to 2.1 m2 per person equivalent. Moreover, it was found that it was not possible to simultaneously remove all nitrogen and phosphorus from the wastewater, because of the nitrogen:phosphorus ratio in the wastewater. Phosphorus was limiting in the post-treatment scenario, while nitrogen was limiting in the two other scenarios. Furthermore, a substantial amount of microalgal biomass was produced, ranging from 13 to 59 g per person equivalent per day. These findings show that microalgal biofilm systems hold large potential as seasonal wastewater treatment systems and that it is worthwhile to investigate these systems further.

Nitrogen and phosphorus removal from municipal wastewater effluent using microalgal biofilms

Microalgal biofilms have so far received little attention as post-treatment for municipal wastewater treatment plants, with the result that the removal capacity of microalgal biofilms in post-treatment systems is unknown. This study investigates the capacity of microalgal biofilms as a post-treatment step for the effluent of municipal wastewater treatment plants. Microalgal biofilms were grown in flow cells with different nutrient loads under continuous lighting of 230 μmol/m 2/s (PAR photons, 400–700 nm). It was found that the maximum uptake capacity of the microalgal biofilm was reached at loading rates of 1.0 g/m 2/day nitrogen and 0.13 g/m 2/day phosphorus. These maximum uptake capacities were the highest loads at which the target effluent values of 2.2 mg/L nitrogen and 0.15 mg/L phosphorus were still achieved. Microalgal biomass analysis revealed an increasing nitrogen and phosphorus content with increasing loading rates until the maximum uptake capacities. The internal nitrogen to phosphorus ratio decreased from 23:1 to 11:1 when increasing the loading rate. This combination of findings demonstrates that microalgal biofilms can be used for removing both nitrogen and phosphorus from municipal wastewater effluent.

Effects of zinc on microalgal biofilms in intertidal and subtidal habitats

Microalgal biofilms are sensitive to environmental conditions. Impacts of contaminants on assemblages of marine biofilm are often investigated in laboratories or in mesocosms. Such experiments are rarely representative of the effects of contaminants on biofilms under natural conditions. Studies in field situations, with enough power to detect impacts, are necessary to develop a better understanding of the effects of contaminants on ecological processes. Metals are a common contaminant of marine systems and can cause disturbances to assemblages. Using a new technique to experimentally deliver contaminants to microalgal assemblages, hypotheses were tested regarding the effects of zinc on microalgal biofilms growing on settlement panels in subtidal and intertidal habitats. PAM fluorometry was used to assess the amount and physiological state of biofilms on panels. Control panels deployed for 1 month in each habitat had significantly greater amounts of biofilm than those exposed to zinc. After deployment for 3 months, the results varied with location. The observed effects on the biofilm did not, however, cause significant changes in the macro-invertebrate assemblages that developed on the panels.

Species and material considerations in the formation and development of microalgal biofilms

The development of microalgal biofilms has received very limited study despite its relevance in the design of photobioreactors where film growth may be advantageous for biomass separation or disadvantageous in fouling surfaces. Here, the effects of species selection, species control, and substrate properties on biofilms of Scenedesmus obliquus and Chlorella vulgaris were investigated. Experiments were conducted in batch culture and in continuous culture modes in a flow cell. Cell growth was monitored using confocal laser scanning microscopy and gravimetrically. Species selection and species control had significant effects on biofilm development. On non-sterile wastewater, C. vulgaris shifted from primarily planktonic (23.7% attachment) to primarily sessile (79.8% attachment) growth. The biofilms that developed in non-sterile conditions were thicker (52 ± 19 μm) than those grown in sterile conditions (7 ± 6 μm). By contrast, S. obliquus attained similar thicknesses (54 ± 31 and 53 ± 38 μm) in both sterile and non-sterile conditions. Neither species was able to dominate a non-sterile biofilm. The effect of substrate surface properties was minimal. Both species grew films of similar thickness (approximately 30 μm for S. obliquus, < 10 μm for C. vulgaris) on materials ranging from hydrophilic (glass) to hydrophobic (polytetrafluoroethylene). Surface roughness created by micropatterning the surface with 10 μm grooves did not translate into long-term increases in biofilm thickness. The results indicate that species selection and control are more important than surface properties in the development of microalgal biofilms.

Bacterially mediated mineralisation processes lead to biodeterioration of artworks in Maltese catacombs

Mineral structures formed by bacterial and microalgal biofilms growing on the archaeological surface in Maltese hypogea were studied using Energy Dispersive X-Ray Spectroscopy (EDS) coupled to Environmental Scanning Electron Microscopy (ESEM), X-ray micro-diffraction (XRD) and X-ray fluorescence (XRF). These techniques have shown that mineral structures having different morphologies and chemical composition were associated with the microorganisms in the subaerophytic biofilm. Salt efflorescences and mineral deposits on the archaeological surface were often formed from gypsum (CaSO 4∙ 2H 2O), halite (NaCl) and calcite (CaCO 3). Biogenic carbonates produced by microbial activities were a common occurrence. These assumed different forms, such as the production of mineral coats around cyanobacterial sheaths and the occurrence of calcite fibres with different morphologies on the surface of the biofilms. Moreover, vaterite (CaCO 3) spherulites which appeared hollow in cross-section were observed. The presence of struvite was recorded from one catacomb site. These investigations have facilitated the study of the neoformation of metastable minerals by microbially mediated processes, which potentially contribute to a better understanding of the biodeterioration of artworks in Maltese palaeo-Christian catacombs.

Initial Attachment, Metamorphosis, Settlement, and Survival of Black‐footed Abalone,Haliotis iris, on Microalgal Biofilms Containing Different Amino Acid Compositions

Microalgal biofilms with different amino acid profiles were investigated for their ability to induce abalone,Haliotis iris, attachment, metamorphosis, shell stage, and survival. Twenty microalgal strains, isolated from rocky shores and sandy estuaries, were grown in the laboratory to produce young and matured monospecific biofilms. Abalone larvae were exposed to the different biofilm treatments and controls (no biofilms) for 7 (attachment and metamorphosis) and 14 (shell stage and survival) d. The larvae performed significantly better in biofilms compared with controls across microalgal strains, but attachment, metamorphosis, shell stage, and survival were generally less than about 50, 35, 25, and 25%, respectively. Some microalgal strains belonging to the same species but collected from different sites had different effects on the larvae, likely due to variations in biochemical composition and activity among strains. Although no clear relationship was found between the microalgal amino acid profiles and larval processes, percentage biofilm cover and total amino acids were positively correlated with some of these processes. In addition, abalone performed significantly better when exposed to mature compared with young biofilms. These results may be due to the greater amounts of microalgal cells and their extracellular polymers within older biofilms, which may cue larvae toward more nutritionally favorable environments.

Effects of Dual Microalgal Species Biofilms on New Zealand Black-Footed Abalone (Hailotis iris) Larval/Post-Larval Processes

Dual microalgal species biofilms with distinct amino acid characteristics were tested for their effects on abalone (Haliotis iris) initial attachment, metamorphosis, settlement, and survival. Nine microalgal species, isolated from around Auckland, New Zealand, were grown in the laboratory, and used to produce nine dual microalgal species biofilms. Abalone larvae were exposed to the different biofilm treatments and controls (no biofilms) for seven (attachment and metamorphosis) and 14 (settlement and survival) days. The larvae performed significantly better in the dual microalgal biofilms compared to controls, and some biofilms resulted in better attachment, metamorphosis, settlement, and survival than others. For example, the best-performing biofilms almost always contained cyanobacteria (Oscillatoria cf. erythrata, Anacystis cf. aeruginosa, and Schizothrix cf. calcicola), which are attributed with better nutritional values. However, the amino acid profiles did not produce a clear pattern with regard to their effects on the four larval processes. Instead, total amino acid (TAA) content was positively correlated with percentage of attachment and metamorphosis. Greater TAA contents are likely to reflect greater amounts of extra-cellular polymers within the biofilms, which are suggested to improve larval performance.