Porous Substrate Bioreactors: A Paradigm Shift in Microalgal Biotechnology?

Many of the demands in the production of microalgae at a technical scale cannot presently be met by state-of-the-art cultivation technologies based on suspensions. Immobilized cultivation using porous substrate bioreactors (PSBRs) is characterized by a reduction of liquid reaction volumes by several orders of magnitude and has solved several volume-related problems. Recently, PSBRs demonstrated potential for both established and novel applications in microalgal biotechnology, and first insights into biophysical processes have provided an understanding of the benefits of PSBR biofilm cultivation. Further efforts should primarily focus on scale-up and engineering challenges in this emerging field and, additionally, provide experience in the long-term operation of bioreactors. The results may contribute to assessing the technical and economic potential of PSBR cultivation.

Microscale profiling of photosynthesis-related variables in a highly productive biofilm photobioreactor.

In the present study depth profiles of light, oxygen, pH and photosynthetic performance in an artificial biofilm of the green alga Halochlorella rubescens in a porous substrate photobioreactor (PSBR) were recorded with microsensors. Biofilms were exposed to different light intensities (50-1,000 µmol photons m(-2) s(-1) ) and CO2 levels (0.04-5% v/v in air). The distribution of photosynthetically active radiation showed almost identical trends for different surface irradiances, namely: a relatively fast drop to a depth of about 250 µm, (to 5% of the incident), followed by a slower decrease. Light penetrated into the biofilm deeper than the Lambert-Beer Law predicted, which may be attributed to forward scattering of light, thus improving the overall light availability. Oxygen concentration profiles showed maxima at a depth between 50 and 150 µm, depending on the incident light intensity. A very fast gas exchange was observed at the biofilm surface. The highest oxygen concentration of 3.2 mM was measured with 1,000 µmol photons m(-2) s(-1) and 5% supplementary CO2. Photosynthetic productivity increased with light intensity and/or CO2 concentration and was always highest at the biofilm surface; the stimulating effect of elevated CO2 concentration in the gas phase on photosynthesis was enhanced by higher light intensities. The dissolved inorganic carbon concentration profiles suggest that the availability of the dissolved free CO2 has the strongest impact on photosynthetic productivity. The results suggest that dark respiration could explain previously observed decrease in growth rate over cultivation time in this type of PSBR. Our results represent a basis for understanding the complex dynamics of environmental variables and metabolic processes in artificial phototrophic biofilms exposed to a gas phase and can be used to improve the design and operational parameters of PSBRs.

A method to determine photosynthetic activity from oxygen microsensor data in biofilms subjected to evaporation.

Phototrophic biofilms are widely distributed in nature and their ecological importance is well recognized. More recently, there has been a growing interest in using artificial phototrophic biofilms in innovative photobioreactors for production of microalgal biomass in biotechnological applications. To study physiological processes within these biofilms, microsensors have been applied in several studies. Here, the 'light-dark shift method' relies on measurement of photosynthetic activity in terms of light-induced oxygen production. However, when applied to non-submerged biofilms that can be found in numerous locations in nature, as well as in some types of photobioreactors, limitations of this approach are obvious due to rapid removal of gaseous species at the biofilm surface. Here, we introduce a mathematical correction to recover the distribution of the actual photosynthetic activity along the depth gradient in the biofilm, based on a numerical solution of the inversed diffusion equation of oxygen. This method considers changes in mass transport during the measurement period as can found on biofilms possessing a thin flow/mass transfer boundary layer (e. g., non-submerged biofilms). Using both simulated and real microsensor data, the proposed method was shown to be much more accurate than the classical method, which leads to underestimations of rates near the biofilm surface. All test profiles could be recovered with a high fit. According to our simulated microsensor measurements, a depth resolution of ≤20 µm is recommended near the surface. We conclude that our method strongly improves the quality of data acquired from light-dark measurements of photosynthetic activity in biofilms.

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 (2-3 mg Zn L(-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 28.9 mg Zn L(-1) based on Pulse-amplitude modulated (PAM) chlorophyll fluorescence analysis) led to a high zinc absorption capacity of 15-19 mg Z ng(-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.

Immobilized growth of the peridinin-producing marine dinoflagellate Symbiodinium in a simple biofilm photobioreactor.

Products from phototrophic dinoflagellates such as toxins or pigments are potentially important for applications in the biomedical sciences, especially in drug development. However, the technical cultivation of these organisms is often problematic due to their sensitivity to hydrodynamic (shear) stress that is a characteristic of suspension-based closed photobioreactors (PBRs). It is thus often thought that most species of dinoflagellates are non-cultivable at a technical scale. Recent advances in the development of biofilm PBRs that rely on immobilization of microalgae may hold potential to circumvent this major technical problem in dinoflagellate cultivation. In the present study, the dinoflagellate Symbiodinium voratum was grown immobilized on a Twin-Layer PBR for isolation of the carotenoid peridinin, an anti-cancerogenic compound. Biomass productivities ranged from 1.0 to 11.0 g m(-2) day(-1) dry matter per vertical growth surface and a maximal biomass yield of 114.5 g m(-2), depending on light intensity, supplementary CO2, and type of substrate (paper or polycarbonate membrane) used. Compared to a suspension culture, the performance of the Twin-Layer PBRs exhibited significantly higher growth rates and maximal biomass yield. In the Twin-Layer PBR a maximal peridinin productivity of 24 mg m(-2) day(-1) was determined at a light intensity of 74 µmol m(-2) s(-1), although the highest peridinin content per dry weight (1.7 % w/w) was attained at lower light intensities. The results demonstrate that a biofilm-based PBR that minimizes hydrodynamic shear forces is applicable to technical-scale cultivation of dinoflagellates and may foster biotechnological applications of these abundant marine protists.

Application of a prototype-scale Twin-Layer photobioreactor for effective N and P removal from different process stages of municipal wastewater by immobilized microalgae.

In the view of limited phosphorous resources and tightened discharge regulations, the recovery of phosphate and nitrate from wastewater is of great interest. Here, the integration of microalgae into wastewater treatment processes is a promising approach. A prototype-scale Twin-Layer photobioreactor immobilizing the green alga Halochlorella rubescens on vertical sheet-like surfaces was constructed and operated using primary and secondary municipal wastewater. The process was not impaired by suspended solids, bacteria or loss of algal biomass by leaching. The average areal microalgal growth was 6.3 gm(-2) d(-1). After treatment, P and N concentrations in the effluents could efficiently be reduced by 70-99%, depending on element and type of wastewater. Mean effluent values of ⩽ 1.0mg L(-1)P and 1.3 mg L(-1)N met the legal discharge limits of the European Water Framework Directive and show a potential to comply with upcoming, more stringent legislation.

Genetic Programming as a tool for identification of analyte-specificity from complex response patterns using a non-specific whole-cell biosensor.

Whole-cell biosensors are mostly non-specific with respect to their detection capabilities for toxicants, and therefore offering an interesting perspective in environmental monitoring. However, to fully employ this feature, a robust classification method needs to be implemented into these sensor systems to allow further identification of detected substances. Substance-specific information can be extracted from signals derived from biosensors harbouring one or multiple biological components. Here, a major task is the identification of substance-specific information among considerable amounts of biosensor data. For this purpose, several approaches make use of statistical methods or machine learning algorithms. Genetic Programming (GP), a heuristic machine learning technique offers several advantages compared to other machine learning approaches and consequently may be a promising tool for biosensor data classification. In the present study, we have evaluated the use of GP for the classification of herbicides and herbicide classes (chemical classes) by analysis of substance-specific patterns derived from a whole-cell multi-species biosensor. We re-analysed data from a previously described array-based biosensor system employing diverse microalgae (Podola and Melkonian, 2005), aiming on the identification of five individual herbicides as well as two herbicide classes. GP analyses were performed using the commercially available GP software 'Discipulus', resulting in classifiers (computer programs) for the binary classification of each individual herbicide or herbicide class. GP-generated classifiers both for individual herbicides and herbicide classes were able to perform a statistically significant identification of herbicides or herbicide classes, respectively. The majority of classifiers were able to perform correct classifications (sensitivity) of about 80-95% of test data sets, whereas the false positive rate (specificity) was lower than 20% for most classifiers. Results suggest that a higher number of data sets may lead to a better classification performance. In the present paper, GP-based classification was combined with a biosensor for the first time. Our results demonstrate GP was able to identify substance-specific information within complex biosensor response patterns and furthermore use this information for successful toxicant classification in unknown samples. This suggests further research to assess perspectives and limitations of this approach in the field of biosensors.

The 96-well twin-layer system: a novel approach in the cultivation of microalgae.

A novel system for the growth and maintenance of microalgae has been developed that allows the cultivation of a large number of strains with little manual effort. The system is based on a 96-well microtiter plate in which a membrane filter constitutes the bottom of each well. Algal strains are immobilised on the membranes and provided with culture medium through contact with layers of glass fibre located beneath the membranes in a special cultivation chamber. The configuration effectively separates culture medium from algal cells which allows the simultaneous exchange of the culture medium for 96 strains within a few minutes without the need to transfer the algae. If necessary, algal strains can be transferred using multi-channel pipettes. We demonstrate that a large variety of microalgal strains including delicate flagellates can be reliably grown in the system under axenic conditions and without cross-contamination. As an array system, the 96-well twin-layer system using immobilised algae is also amenable to high-throughput and massively parallel applications increasingly sought after in algal bio- and environmental technology.

The use of multiple-strain algal sensor chips for the detection and identification of volatile organic compounds.

Although biosensors detecting a great variety of toxicants have been developed during the last decades, the simultaneous detection and identification of several targets by one biosensor is not possible in the majority of the biosensor systems. In our study we proved the concept of the detection and identification of two different volatile toxic compounds with a non-selective biochip-based algal biosensor. For that purpose we produced array plate biochips to utilise three membrane-immobilised algal strains of genus Klebsormidium and Chlorella in one biosensor system. A novel IMAGING-PAM chlorophyll fluorometer was applied to measure the impact of volatile organic compounds (VOC) on photosynthesis of chip-immobilized algae in terms of quantum efficiency of electron transport (DeltaF/F'm). Formaldehyde (FA) vapour was detectable with statistical significance in concentrations relevant to human health from 10 ppb to 10 ppm. The biosensor response recorded within minutes was concentration-dependent and reversible. Moreover, vapours of formaldehyde (0.05-1 ppm) and methanol (MeOH) (200-1000 ppm) were significantly identified by the compound-specific response rate as a quotient of the biosensor responses of the respective algal strains. Using the IMAGING-PAM chlorophyll fluorometer, data sampling proved to be highly efficient. Based on our results we conclude that the principle of the algal sensor chip (ASC) suggests further research on the detection and identification of VOCs and other toxic substances in gaseous environment with that biochip system.