Microalgae Biofilm Research Articles

Effects of environment-relevant concentrations of antibiotics on seawater Chlorella sp. biofilm in artificial mariculture effluent

Attached cultivation of microalgae using mariculture effluent could simultaneously realize cost-effective biomass production and effluent polishing, and the obtained microalgal biofilm could be readily applied to aquatic animals that feed directly on biofilms. However, the widespread existence of antibiotics in mariculture effluent may have inhibitory effects on microalgae. Existing studies regarding the influences of antibiotics on microalgae were overwhelmingly mostly conducted in suspended freshwater cultures, and the explored antibiotic concentrations were several orders of magnitudes higher than realist effluent, rendering limited guidance for practical applications. This study, therefore, studied three commonly observed antibiotics in mariculture effluent, i.e. sulfamethoxazole (SMX), tetracycline (TL), and clarithromycin (CLA), and explored their effects on attached seawater Chlorella sp. growth, nutrients removal, and antioxidative properties at environment-relevant concentrations (0.1–200 μg/L). Hormesis was shared among the three antibiotics, and highest biofilm density (14.6 g/m2) was observed under 0.1 μg/L SMX. SMX displayed strongest chlorophyll impairment, while SMX, TL, and CLA respectively favored lipid (up to 42.4 %), protein (up to 39.6 %), and polysaccharides (up to 65.4 %) accumulation. Highest correlation between biofilm density and extracellular polymeric substances (EPS) contents as well as highest total nitrogen (TN) removal were observed under TL exposure, while total phosphorus (TP) removal among all groups met the strictest local discharge standard for mariculture effluent (TP < 0.2 mg/L). Antioxidative responses revealed least stress towards attached seawater Chlorella sp. under TL treatment, while best acclimation under SMX exposure. Potential coping mechanisms of this microalgae under each antibiotic exposure were proposed as well. This study expands the knowledge of microalgae biofilm under environment-relevant concentrations of antibiotics.

A novel method to harvest microalgae biofilms by interfacial interaction

Microalgae biofilm-based culture has the advantages of less water requirement and higher biomass productivity. Generally, microalgae biofilms are collected by mechanical scraping, which easily leads to the damage of substrates and low harvest efficiency. In this work, a novel method to harvest microalgae biofilms by interfacial interactions was proposed and studied. The results indicated that N. oculata and Chlorella sp. biofilms could be peeled off from substrates when the biofilm was slowly immersed into liquids with surface tensions higher than 40 mJ·m−2. By developing a model, we found that when the detachment force of liquid applied onto cells was higher than ~60 × 10−6 mN, microalgae biofilms could be peeled off with efficiency of over 94 %. Although water was convenient to peel microalgae biofilms, the biofilms would break into loose pieces in the water within a short time after peeling. We found that saline was suitable to peel off microalgae biofilms and maintain biofilms with a stable structure. This work can provide valuable insights into developing new methods to harvest microalgae biofilms.

Temperature-controlled microalgae biofilm adsorption/desorption in a thermo-responsive light-guided 3D porous photo-bioreactor for CO2 fixation

Microalgae biofilm-based culture provides an efficient CO2 reduction and wastewater treatment method for its high photosynthetic efficiency and density. As supporting substrates for microalgae biofilm, porous materials have a big available adsorption area, but mutual shading makes it difficult to transmit external light to the internal surface for attached cells' photosynthesis. Thus, light-guided particles (SiO2) were introduced into photosensitive resin to fabricate a light-guided ordered porous photobioreactor (PBR) by 3D printing technology in this study. The space utilization of the PBR was significantly enhanced and the effective microalgae adsorption area was increased by 13.6 times. Further, a thermo-responsive hydrogel was grafted onto the surface of the substrate to form a smart temperature-controllable interface that could enhance microalgae adsorption and desorption in both directions. When the thermo-responsive layer received light, it would generate heat due to the hydrogel's photo-thermal effect. And the surface temperature would then raise to 33 °C, higher than the hydrogel phase transition point of 32 °C, making the surface shrinking and more hydrophobicity for microalgae cells attachment. The microalgae cells' adsorption capacity increased by 103%, resulting in a high microalgae growth rate of 3.572 g m−2 d−1. When turning off the light, the surface temperature would cool down to below 20 °C, the surface would shrink. And the biofilm shows a 564.7% increase in desorption ability, realizing temperature-controlled microalgae harvesting.

Insight into the comprehensive effect of carbon dioxide, light intensity and glucose on heterotrophic-assisted phototrophic microalgae biofilm growth: A multifactorial kinetic model

Heterotrophic-assisted photoautotrophic microalgae biofilm cultivation was an alternative way to realize CO2 reduction and wastewater treatment. Growth kinetics supplied a channel to better understand how the cultivation conditions affect microalgal growth and CO2 reduction. However, the two growth modes (heterotroph and photoautotroph) have different needs for organic and inorganic nutrients. Thus, combining the threshold theory and multiplication theory, an integral multifactorial kinetic model that looking insight into the comprehensive effect of glucose, CO2, light intensity, and nitrate was developed for heterotrophic-assisted photoautotrophic microalgae biofilm growth in this study. R2 between model and experiment was 0.99. It predicted the maximum specific growth rate and maximum CO2 consumption rate of heterotrophic-assisted photoautotrophic microalgae biofilm was 1.868 h−1 and 1.02 h−1, respectively. This model fully explained the influence of the main factors on microalgae biofilm growth and reasonably predicted the growth rate of microalgae biofilm under different growth conditions.

Recycling banana pseudostem waste as a substrate for microalgae biofilm and their potential in arsenic removal

Arsenic (As) is the most toxic metalloid and class I carcinogen present in the drinking water affecting around 200 M people worldwide. Among various strategies microalgal biofilm based As removal is considered as most suitable option. However, sustainable technologies for As removal from water is scanty. In the present study, banana pseudostem waste was used as a substrate for growing microalgae biofilm (BSB) and its As removal capacity was compared with nylon mesh grown microalgae biofilm (NMB). Response surface methodology optimize initial As concentration as the most important parameter (p < 0.0001) for As removal as compared to substrate area and initial phosphate concentration in both BSB and NMB treatments. BSB treatment removed 97.6% of added arsenite [As(III)] and converted into less toxic forms like arsenate and dimethylarsenic acid within 5 d. The As removal efficiency of BSB treatment is 20% higher than NMB treatment due to the high As accumulation by the banana pseudostem. In both BSB and NMB treatments, As exposure led to change in microalgae community composition towards more tolerant species that produced more proteins and lipids facilitating binding of As and amelioration of induced stress, respectively. The present study signifies the use of banana pseudostem to grow tolerant microalgae for efficient removal of As from water. The study also paves way for proving the concept “waste into wealth” by utilizing the agri-wastes for the sustainable and more economical water treatment process.

The architecture and metabolic traits of monospecific photosynthetic biofilms studied in a custom flow-through system.

Microalgae biofilms have great ecological importance and high biotechnological potential. Nevertheless, an in-depth and combined structural (i.e., the architecture of the biofilm) and physiological characterization of microalgae biofilms is still missing. An approach able to provide the same time physiological and structural information during biofilm growth would be of paramount importance to understand these complex biological systems and to optimize their productivity. In this study, monospecific biofilms of a diatom and a green alga were grown under dynamic conditions in custom flow cells represented by UV/Vis spectroscopic cuvettes. Such flow cells were conceived to characterize the biofilms by several techniques mostly in situ and in a nondestructive way. Physiological traits were obtained by measuring variable chlorophyll a fluorescence by pulse amplitude modulated fluorometry and by scanning the biofilms in a spectrometer to obtain in vivo pigments spectral signatures. The architectural features were obtained by imaging the biofilms with a confocal laser scanning microscopy and an optical coherence tomography. Overall, this experimental setup allowed us to follow the growth of two biofilm-forming microalgae showing that cell physiology is more affected in complex biofilms likely as a consequence of alterations in local environmental conditions.

Understanding photosynthetic biofilm productivity and structure through 2D simulation.

We present a spatial model describing the growth of a photosynthetic microalgae biofilm. In this 2D-model we consider photosynthesis, cell carbon accumulation, extracellular matrix excretion, and mortality. The rate of each of these mechanisms is given by kinetic laws regulated by light, nitrate, oxygen and inorganic carbon. The model is based on mixture theory and the behaviour of each component is defined on one hand by mass conservation, which takes into account biological features of the system, and on the other hand by conservation of momentum, which expresses the physical properties of the components. The model simulates the biofilm structural dynamics following an initial colonization phase. It shows that a 75 μm thick active region drives the biofilm development. We then determine the optimal harvesting period and biofilm height which maximize productivity. Finally, different harvesting patterns are tested and their effect on biofilm structure are discussed. The optimal strategy differs whether the objective is to recover the total biofilm or just the algal biomass.

Different types of bubbly flows in a confined channel with the aim of limiting microalgae biofilm development – Part II: Study of the development and removal of microalgae biofilm

This study aims at investigating the role of bubbles passage to prevent biofouling in thin-gap bubble column photobioreactors (PBRs) where the biomass concentration is important and the reactor thickness is low. Different types of gas sparging are tested in a square channel with a cross-section of 4x4 mm2 mimicking a thin-gap bubble column PBR. Firstly, it is shown that it is very difficult to detach the biofilm once it has adhered to the wall: only the bubbling conditions with high wall shear rate fluctuations are able to detach part of the superficial layers of the biofilm, but no effect is observed on the detachment of the conditioning layer. Secondly, bubbling generating either high shear rate fluctuations, or high mean shear stress are tested to assess their ability to delay biofilm development: both accentuate biofilm adhesion, showing that the biofilm may be compacted by highly confined bubbles. Thus, small bubbles whose diameter (1.4 mm) is smaller than the side of the square channel are investigated, allowing bubbles oscillations and a 3D hydrodynamics around the bubble. This bubbling, which creates the most important shear rate fluctuations, is the most adapted to limit the development of biofilm in thin-gap PBR.

Microalgae Biofilm and Bacteria Symbiosis in Nutrient Removal and Carbon Fixation from Wastewater: a Review

Microalgae Biofilm and Bacteria Symbiosis in Nutrient Removal and Carbon Fixation from Wastewater: a Review

A synchronous photoautotrophic-heterotrophic biofilm cultivation mode for Chlorella vulgaris biomass and lipid simultaneous accumulation

Microalgae biofilm was a porous structure formed by the accumulation of microalgae cells. The cells in upper layer of biofilm could receive light for photosynthetic growth and meanwhile removed the inorganic nitrogen and phosphorus etc. From wastewater to cleaner biomass. While with the increase of biofilm thickness, light could not reach the underlayer, forming a natural dark area in the bottom of the biofilm for microalgae heterotrophic growth. The naturally formed synchronous photoautotrophic-heterotrophic microalgae growth environment realized the synchronization of biofilm growth and lipid enrichment. The transmission of metabolic gases made the biofilm loose and porous (120.18% increase on the biofilm porosity), which provided more channels for the transmission of nutrients. Due to the synchronous heterotrophic enrichment in the bottom layer of biofilm, the maximum growth rate of biomass cultivated under synchronous photoautotrophic-heterotrophic biofilm was 3.04 times higher than that of photo-autotrophic biofilm and lipid yield increased by 2.22 times. The proportion of photosynthetic autotrophy of cells in the biofilm increased with the increase of light intensity which made an increase of 33.45% in the growth rate of biofilm but with a price of 43.88% reducing in the lipid content. While, the increase of heterotrophic proportion in the biofilm realized the simultaneous enhancement of biomass density and lipid content. The biomass productivity increased to 55.81 g m−2 d−1 and the lipid content increased to 46.79% at the same time. Thus, it solved the contradictory between the growth of microalgae and the accumulation of lipid, and provided a new idea for the application of changing wastewater resources to clean biofuel. It would have an important significant to the development of low-carbon renewable and clean energy.

Temperature-Controlled Microalgae Biofilm Adsorption/Desorption in a Thermo-Responsive Light-Guided 3d Porous Photo-Bioreactor for Co2 Fixation

Temperature-Controlled Microalgae Biofilm Adsorption/Desorption in a Thermo-Responsive Light-Guided 3d Porous Photo-Bioreactor for Co2 Fixation

The mechanism study of attached microalgae cultivation based on reverse osmosis concentrated water (WROC)

Reverse osmosis membrane is widely applied in advanced wastewater treatment. Along with the production of clean water, 30%∼50% reverse osmosis concentrated water (WROC), containing massive nitrogen and phosphorus, is also produced. WROC would bring ecological risk if discharged into the natural water body. Hence, attached microalgae cultivation based on WROC was proposed to recovery the nutrients from water in this study. The results showed that the initial concentrations of nitrogen and phosphorus in WROC (TN≈50 mg/L, TP≈10 mg/L) were not too high to inhibit attached microalgae growth. Among the complex chemicals in WROC, non-oxidizing bacteriostatic agent had the worst impact on the growth of microalgae with an 8-day inhibition ratio of 25%. Compared with the microalgae cultured by nutrient medium, the lipid and protein contents in attached microalgae cultured by WROC were relatively high and stable during the whole cultivation period. Seen from the micro level, there was no obvious resistance to the transfer of TN and TP in the microalgae biofilm. However, the photosynthetic rate of microalgae in the biofilm decreases along with the depth. In conclusion, uneven light distribution in biofilm and the non-oxidizing bacteriostatic agent in WROC were the main resistance to the growth of attached cultured microalgae, which should be optimized or per-treated.

Recognition of key factors on attached microalgae growth from the internal sight of biofilm

Microalgae cultivation with wastewater could realize the advanced water treatment and pollutant conversion to biomass. Attached microalgae cultivation mode, that can avoid the high-cost and energy-extensive consumption process of biomass recovery from water in suspended cultivation mode, is getting increasing attention. During the attached cultivation, light and nutrient concentration in the internal biofilm, play a direct and crucial role in regulating the growth of microalgae. Hence, the distribution of light and nutrients at different depths of biofilm were first explored in this study together with the change rules of its internal distribution under different external nutrient levels. It demonstrated that the gross growth rate was enhanced by increasing the external nutrient level. Seen from the internal sight of biofilm, the internal nutrient level had a positive response to the external nutrient change. Nutrients (especially nitrogen) distributed homogeneously through the biofilm, and no serious nutrient starvation occurred at the surface layer of biofilm. Photosynthesis rate linearly decreased along the depth of microalgae biofilm (10–120 μm). In conclusion, light, rather than nutrient, would be the key influencing factor on attached microalgae growth. How to optimize the internal light distribution would determine the wastewater purification efficiency based on attached microalgae cultivation.

Enhanced Transpiration by Attached Microalgae-Simulated Plants for Zero-Discharge of Reverse Osmosis Concentrated Water (WROC)

Inspired by plant transpiration, an attached microalgae−simulated plant system was designed to enhance the transpiration of reverse osmosis concentrated water (i.e., WROC) and realize the conversion of pollutants to microalgae biomass. The results showed that the production rate of clean water could reach as high as 14.84 L·m−2·day−1, which was significantly influenced by the humidity of the air and the growth status of the attached microalgae. Moreover, the enhancement of water evaporation by microalgae was more obvious under relatively low humidity. Pollutants, transported along with the water, could transform into microalgae biofilm or crystallize at the top of the microfiber. TN and TP transformation into biomass resources were maximized in 40% diluted WROC, with efficiencies of 60.91% and 38.49%, respectively. Of note, the accumulation of phosphorus in the micro-environment of attached microalgae may inhibit microalgal growth in the later stages of cultivation, owing to the relatively low movability. Hence, this system could be applied for high-efficiency wastewater purification, especially under high humidity. Wastewater dilution and periodic microalgae harvest could guarantee the attached microalgae growth and increase the pollutant-bioresource conversion rate.

The efficiency of microalgae biofilm in the phycoremediation of water from River Kaduna

This study is aimed at investigating the efficiency of microalgae biofilm in the phycoremediation of water from a polluted river. Freshwater microalgae biofilm inherent in a contaminated petrochemical stream was employed to remediate water from the River Kaduna, which is the largest river in Kaduna town, Kaduna State, Nigeria, and serves as the primary water source in Kaduna town. The results indicate high reduction efficiency of some physicochemical parameters and pollutants (turbidity (71%), conductivity (9.8%), sulfate (37.5%), alkalinity (62.5%), chloride (11.5%), TDS (9.9%), TSS (66.7%), nitrate (42.9%), COD (24%), and BOD (33%), Cd (70.0%), Ni (74.0%) and Pb (71.0%)), indicating the effectiveness of microalgae biofilm in the phycoremediation of water from River Kaduna. According to scanning electron microscope (SEM) observation, the microalgae biofilm has rough surface morphology after the treatment of the river water, which implies that the biofilm was capable of removing the pollutants in water via biosorption. Other characterizations such as XRF, XRD, and FTIR also buttressed that biosorption was the primary removal mechanism of pollutants by microalgae biofilm. Besides, the results also show the production of ROS during the treatment of water from the River Kaduna by the microalgae biofilm. This high concentration of ROS produced during the treatment correlates significantly with pollutant degradation. The GC-MS analysis of the microalgae biofilm shows the involvement of some phytochemicals in the process of pollutant degradation. As a result, microalgae biofilm is a simple and cost-effective method of polluted water phycoremediation with promising applications and future prospects.

Microalgae biofilm formation and antioxidant responses to stress induce by Lemna minor L., Chlorella vulgaris, and Aphanizomenon flos-aquae

The study shows how microalgae biofilm formation and antioxidant responses to the production of reactive oxygen species (ROS) is alter by the presences of Lemna minor L., Chlorella vulgaris, and Aphanizomenon flos-aquae. The study involves the cultivation of the biofilm of Chlorella vulgaris and Aphanizomenon flos-aquae in three bioreactors. The condition of growth for the biofilm formation was varied across the three bioreactors to enable the dominance Chlorella vulgaris and Aphanizomenon flos-aquae in one of the bioreactors. Lemna minor L. was also introduce into one of the bioreactors to determine its effect on the biofilm formation. The result obtained shows that C. vulgaris and A. flos-aquae dominate the biofilm, resulting in a high level of H2O2 and O2- (H2O2 was 0.122 ± 0.052 and 0.183 ± 0.108 mmol/L in C. vulgaris and A. flos-aquae, respectively, and O2- was 0.261 ± 0.039 and 0.251 ± 0.148 mmol/L in C. vulgaris and A. flos-aquae, respectively). The study also revealed that the presence of L. minor L. tend to reduce the oxidative stress to the biofilm leading to low production of ROS (H2O2 was 0.086 ± 0.027 and 0.089 ± 0.045 mmol/L in C. vulgaris and A. flos-aquae respectively, and O2- was 0.185 ± 0.044 and 0.161 ± 0.065 mmol/L in C. vulgaris and A. flos-aquae respectively). The variation in the ability of the biofilm of C. vulgaris and A. flos-aquae to respond via chlorophyll, carotenoid, flavonoid, anthocyanin, superoxide dismutase, peroxidase, catalase, glutathione reductase activities, antioxidant reducing power, phosphomolybdate activity, DPPH reduction activity, H2O2 scavenging activity, lipid content and organic carbon also supports the fact that the presence of biomass of microalgae and aquatic macrophytes tend to affect the process of microalgae biofilm formation and the ability of the biofilm to produce antioxidant. This high nutrient utilization leads to the production of biomass which can be used for biofuel production and other biotechnological products.

The self-adaption capability of microalgal biofilm under different light intensities: Photosynthetic parameters and biofilm microstructures

Microalgae biofilm-based culture systems have received considerable attention because their water requirements and harvesting costs are lower, and biomass productivity is higher, than in suspended culture systems. The photosynthetic properties and microstructure of microalgae biofilms have fundamental effects on biofilm growth. We investigated the photosynthetic parameters, microstructures, biomass accumulation, and CO2 fixation rate of Chlorella sp. biofilms cultured under a series of light intensities (20, 50, 100, 200, and 400 μmol·m−2·s−1). The results indicated that microalgae biofilms had higher photosynthetic potentials and more porous microstructures under low light intensities, and that both of these promoted the efficiency of photon use. In contrast, biofilms under high light intensities exhibited higher photo-protection capabilities and more compact microstructures, which protected microalgae against photo damage. The study revealed that microalgae biofilms have excellent self-adaption capabilities to cope with varying light intensities, and these have significant implications for selection of the optimal light intensity when designing more efficient biofilm-based microalgae culture systems.

Spawning, larval culture, settlement and juvenile production of the west Indian Sea egg, Tripneustes ventricosus (Lamarck, 1816), under hatchery conditions

Tripneustes ventricosus is a commercially important species of sea urchin native of the tropical Atlantic Ocean. With the purpose of providing a scientific basis for the development of its production by aquaculture, several experiments were performed under hatchery conditions, including spawning induction, larval culture, settlement and juvenile culture. The efficiency of different spawning stimuli was assessed, including the use of UV-irradiated seawater; thermal and electric shocks, and injections of potassium chloride (KCl) and acetylcholine (ACh). Growth and survival of larvae maintained at different culture conditions were compared, including culture densities (2, 6 and 12 zygotes mL−1; 0.5, 1 and 2 larvae mL−1), culture systems (9 L aquaria, 75 L and 500 L tanks), water renewal rates (total and partial), feed amount (15 and 30 cells μL−1 d−1), feeding regime (constant or increasing) and type of diet (Isochrysis galbana, and a mixture of I. galbana and Chaetoceros calcitrans). Different stimuli to induce the larval settlement were tested, including low temperature, KCl, l-glutamine and γ-aminobutyric acid (GABA), as well as different substrates (artificial and benthonic microalgae biofilms). Finally, the growth and survival of juveniles were assessed under indoor conditions. The highest percentage of spawning individuals and the shorter response times were obtained using ACh. The average fecundity was 1.5 × 106 oocytes female−1. This variable exhibited a positive correlation with the female body size. One of the most relevant results of the present study was the completion of the embryo and larval developmental stages of T. ventricosus. Higher levels of larval either survival or growth were found in the tanks with the highest volume (500 L), maintaining conditions of low culture densities and complete water renewal and a constant supply of microalgae during all the larval development stage (15 cells of I. galbana and C. calcitrans μL−1 day−1). The larval settlement was obtained using low temperatures in all the tested substrates, except the substrate with artificial biofilm containing Sargassum and Thalassia. After 9 months of culture in laboratory conditions, juveniles of 44-mm length were obtained, using a regime of complete water renewal, and feeding the larvae with artificial biofilm, benthic microalgae and macroalgae. Although the present study made possible to identify appropriate culture conditions for obtaining hatchery-reared juveniles of white sea urchin, the low survival rates observed during the larval culture and the settlement stage must be improved with further research on the feeding requirements and the optimal water quality conditions for this species.

Settlement and culture of post-larvae, spat and juveniles of the West Indian topshell, Cittarium pica (Linne, 1758)

The settlement, survival and growth rates of C. pica post-larvae, spat and juveniles were evaluated under different culture conditions. The factorial effect of six stimuli (low temperature, γ-aminobutyric acid, potassium chloride, hydrogen peroxide, dibromo methane, and coralline algae extract), five types of biofilm substrates (artificial, artificial with coralline algae, artificial with congener mucus, benthic microalgae, and microalgae with congeners mucus) and five stimulation times (6, 12, 24, 36 and 72 h) on the larval settlement of the topshell were assessed. In addition, growth and survival rates of post-larvae fed with artificial biofilm and benthic microalgae were compared. Similarly, growth and survival rates were assessed in spat cultured in different environments (outdoor and indoor), and juveniles grown in concrete tanks with different finished of the inner walls (gray rough finish, and white smooth walls with epoxy paint). A higher larval settlement was observed in substrates with artificial biofilm exposed to cold water (20 °C) for 24 h. Treatments with the stimulus of KCl and dibromo methane had toxic effects on larvae. There were not differences in the shell length of the settled larvae between stimuli, substrata or exposition time. Post-larvae fed with artificial biofilm had survival and growth rates similar to those maintained with benthic microalgae biofilm. The spat that were cultured in outdoor conditions showed a higher growth rate than those kept indoor, but the survival was similar in both cases. Finally, juveniles grown in tanks with rough, gray concrete inner walls had higher growth and survival rates, compared to those held in tanks with smooth white finish. The improvements obtained in growth and survival of post-larvae, spat and juveniles of C. pica suggest that there is a great biological potential for its wild populations restocking and for its aquaculture in sea-based systems.

A novel microalgal biofilm reactor using walnut shell as substratum for microalgae biofilm cultivation and lipid accumulation

Microalgae are a potential candidate for biofuels, but the high cost and low biomass yield hinder the scale-up application of microalgae. In this study, a novel walnut shell based self-permeating biofilm reactor (WSPBR) was proposed for cultivation of Chlorella vulgaris (C. vulgaris) and Scenedesmus obliquus (S. obliquus). Major influencing factors such as walnut shell size, light intensity and CO2 concentration were studied in detail. The results showed that CO2 can enhance the storage of metabolic products in microalgal cells. Thermodynamics analysis indicated that C. vulgaris and S. obliquus were likely to adhere on the surface of walnut shells. S. obliquus and C. vulgaris biomass reached the maximum values of 97.43 and 70.49 g m−2, respectively, corresponding to the walnut shell size of 0.8–1.2 mm, light intensity of 150 μmol m−2 s−1 and CO2 concentration of 2%. The lipid contents of these microalgal cells were 34.32% and 28.94%, respectively. These results indicated that WSPBR was an efficient system for microalgae cultivation as feedstock for biofuels.