OPTIMIZATION OF MICROALGAL IMMOBILIZATION FOR CULTIVATION IN AQUACULTURE WASTEWATER
Terhi Porkka MSc Thesis Environmental Science University of Eastern Finland, Department of Environmental and Biological Sciences May 2021
Terhi Porkka: Optimization of Microalgal Immobilization for Cultivation in Aquaculture Wastewater
MSc Thesis 86 pages
Supervisors: Ph.D. Amit Bhatnagar, Ph.D. Ehsan Daneshvar and Ph.D. Eila Torvinen May 2021
keywords: alginate beads, aquaculture wastewater, bead concentration, immobilization, microalgae, nutrient removal, Scenedesmus quadricauda
ABSTRACT
Pollution of the natural water bodies is a worldwide problem caused by inadequately treated wastewaters that are released to the environment. One of the industries causing water pollution is aquaculture which generates large amounts of nutrient-rich wastewaters. The use of microalgae to remove the excess nutrients from the wastewater may be a solution for the growing demand of efficient and affordable wastewater treatment techniques. Microalgae can be cultivated immobilized into spherical alginate beads to enhance the wastewater treatment process and ease the separation of microalgae from the treated effluent at the end of the wastewater treatment. With optimized immobilized conditions the best results can be achieved.
This work aimed at optimization of the immobilization of microalga Scenedesmus quadricauda for cultivation in aquaculture wastewater. The effect of bead concentration, bead size, initial algal concentration, and the addition of magnesium on the algal growth and nutrient removal efficiency were examined. In four separate experiments microalgae were immobilized into alginate beads with variation in the investigated parameter. The beads were cultivated in aquaculture wastewater and after 4.5 days of cultivation the nutrient concentrations of the wastewater (phosphate, nitrate, nitrite and ammonia) were analyzed spectrophotometrically.
The algal concentrations were measured after 6 days of cultivation by counting the cells with hemocytometer. Based on the experiments, immobilization enhanced nutrient removal from aquaculture wastewater. The optimal immobilized conditions for nutrient removal seem to be initial algal concentration of 5.1 105 cells/bead, beads to culture medium ratio of 1:10.7 and bead size of 3.4 mm. However also other mechanisms including assimilation by bacteria of the wastewater and adsorption to alginate contributed to the nutrient removal. Algal growth was not enhanced by the immobilization. Nevertheless, the optimal immobilized conditions for algal growth seem to be initial algal concentration of 1.2 105 or 2.3 105 cells/bead, beads to culture medium ratio of 1:5.4 and bead size of 4.7 mm. The addition of magnesium into the beads benefited neither nutrient removal nor algal growth. However, the results obtained in the experiments are only directional estimates. Due to issues in the experimental set-up and some of the measurements the results are not completely accurate and could not be analyzed statistically.
Terhi Porkka: Mikrolevän immobilisoinnin optimointi kalanviljelyn jätevedessä kasvatusta varten
Pro Gradu -tutkielma 86 sivua
Tutkielman ohjaajat: FT Amit Bhatnagar, FT Ehsan Daneshvar ja FT Eila Torvinen Toukokuu 2021
avainsanat: alginaattihelmet, immobilisointi, kalanviljelyn jätevesi, mikrolevä, ravinteiden poisto, Scenedesmus quadricauda
TIIVISTELMÄ
Käsittelemättömän jäteveden leviäminen ympäristöön aiheuttaa vesistöjen saastumista ympäri maailman. Yksi saastumista aiheuttavista teollisuudenaloista on kalanviljely, joka tuottaa suuria määriä ravinnepitoista jätevettä. Mikrolevien käyttö jätevesien ravinteiden poistossa voi auttaa täyttämään kasvavan tarpeen tehokkaille ja edullisille jätevedenkäsittelymenetelmille.
Mikrolevien kasvatus immobilisoituina pyöreisiin alginaattihelmiin tehostaa vedenkäsittelyprosessia ja helpottaa levän erottamista käsitellystä jätevedestä käsittelyn lopussa. Parhaisiin käsittelytuloksiin päästään optimoimalla immobilisointi. Tämän työn tavoitteena oli optimoida Scenedesmus quadricauda -mikrolevän immobilisointi kalanviljelyn jätevedessä kasvatusta varten. Helmien pitoisuuden, helmien koon, levän pitoisuuden ja magnesiumin lisäämisen vaikutusta levän kasvuun ja ravinteiden poistoon tutkittiin. Neljässä eri kokeessa levää immobilisoitiin alginaattihelmiin siten, että tutkittava muuttuja vaihteli eri käsittelyissä. Helmiä kasvatettiin kalanviljelyn jätevedessä, ja jäteveden ravinnepitoisuudet (fosfaatti, nitraatti, nitriitti, ammoniakki) mitattiin spektrofotometrillä 4,5 päivän kasvatuksen jälkeen. Leväpitoisuudet mitattiin kuuden päivän kasvatuksen jälkeen laskemalla solut hemosytometrin avulla. Tulosten perusteella immobilisointi paransi ravinteiden poistoa jätevedestä. Leväpitoisuus 5,1 105 solua/helmi, levän ja kasvatusalustan suhde 1:10,7 ja helmien koko 3,4 mm näyttivät muodostavan optimaalisen immobilisoinnin ravinteiden poistoa varten. Ravinteita poistui jätevedestä kuitenkin myös muiden reittien kuten jätevedessä esiintyvien bakteerien toiminnan ja alginaattiin sitoutumisen kautta. Immobilisointi ei parantanut levän kasvua. Leväpitoisuus 1,2 105 tai 2,3 105 solua/helmi, levän ja kasvatusalustan suhde 1:5,4 ja helmien koko 4,7 mm näyttivät muodostavan optimaalisen immobilisoinnin levän kasvua varten. Magnesiumin lisääminen helmiin ei parantunut ravinteiden poistoa tai levän kasvua. Kokeista saadut tulokset ovat kuitenkin vain suuntaa- antavia arvioita. Tulokset eivät ole täysin tarkkoja, eikä niitä voitu testata tilastollisesti koeasetelmassa ja osassa mittauksia tehtyjen virheiden takia.
The purpose of this work was to find the optimized immobilized conditions for the cultivation of microalga Scenedesmus quadricauda in aquaculture wastewater. The work was started in Water Chemistry Research Group at University of Eastern Finland (UEF) on January 2020 and the laboratory experiments were supposed to be performed in the course of the spring. However, when coronavirus pandemic arrived in Finland on March and shut down most of the society, including UEF, the experiments had to be interrupted and delayed. Since working in the laboratory was not possible, the months of the spring were used for working on the literature review instead. When UEF started to slowly open up again on June, the supervisors of the thesis informed that they would soon leave UEF. Due to the tight schedule caused by this change, the topic of the thesis had to be drastically changed. New experiments were performed during an intense one-month laboratory work period on the summer. The results were calculated during the remaining of the summer and the thesis was written on autumn.
I want to thank Amit Bhatnagar and Ehsan Daneshvar for being the supervisors of this work.
Thanks to also Eila Torvinen for agreeing to be the third supervisor when an extra supervisor was needed after the other supervisors had left UEF. I want to thank Eila Torvinen also for supervising my Bachelor’s thesis since everything I learned from her about writing a literature review during that time was a great help also in this project. Thanks to reviewers for reviewing the thesis. Thanks to all the members of the research group for kindly helping me to get around in the lab when I was a bit lost at first. I want to thank my family and friends for supporting me during this project. Thanks to especially Juri for all the much-needed support, I really appreciate that. Finally, I want to dedicate my thesis to the aquarium fish that died in the lab during the coronavirus shutdown and wish that the ones that survived will live long happy lives.
BOD C.
CaCl2
COD CO2
Biochemical oxygen demand Chlorella
Calcium chloride
Chemical oxygen demand Carbon dioxide
DON FAO
Dissolved organic nitrogen
Food and Agriculture Organization of the United Nations IMTA
MgSO4 7 H2O
Integrated multi-trophic aquaculture Magnesium sulfate
Na2CO3
NH3
NH4+
NO2-
NO3-
Sodium carbonate Ammonia
Ammonium Nitrite Nitrate PO43-
RAS S.
SS
Phosphate
Recirculating aquaculture system Scenedesmus
Suspended solids
TN Total nitrogen
TP TSS
Total phosphorus Total suspended solids
1. INTRODUCTION
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2. LITERATURE REVIEW
112.1 MICROALGAE 11
2.1.1 Microalgae in the Nature
2.1.2 Cultivation of Microalgae for Industrial and Commercial applications 2.1.3 Methods for Cultivating and Harvesting Microalgae
2.2 IMMOBILIZATION OF MICROALGAE
2.2.1 Advantages and Disadvantages of Immobilization of Microalgae 2.2.2 Immobilization Methods
2.2.2.1 Immobilization Matrices
2.2.2.2 Immobilization Process for the Production of Alginate Beads 2.2.3 Nutrient Removal Mechanisms of the Treatment with Microalgal Beads 2.2.4 Applications of Immobilized Microalgae
2.3 OPTIMIZATION OF TREATMENT WITH MICROALGAL BEADS 2.3.1 Optimization of the Initial Algal Concentration of the Beads 2.3.2 Optimization of the Bead Concentration of the Culture Medium 2.3.3 Optimization of the Bead Size
2.4 AQUACULTURE
2.4.1 Aquaculture Production
2.4.2 The Characteristics of Aquaculture Wastewater 2.4.3 Aquaculture Wastewater Treatment
2.4.4 Environmental Impacts of Aquaculture Wastewater
11 12 14 17 17 18 19 20 22 23 24 24 28 32 33 33 35 37 41
3. OBJECTIVES
444. MATERIALS AND METHODS
4.1 OVERVIEW OF THE EXPERIMENTS 4.2 MICROALGAL STRAIN
4.3 AQUACULTURE WASTEWATER 4.4 PREPARATION OF THE BEADS
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4.4.3 Preparation of the Beads for the Bead Size Experiment
4.4.4 Preparation of the Beads for the Algal Concentration Experiment 4.4.5 Preparation of the Beads for the Mg Addition Experiment
4.5 CULTIVATION CONDITIONS
4.6 SAMPLING AND SAMPLE PREPARATION
4.6.1 Sampling and Sample Preparation for Nutrient Concentration Analyses 4.6.2 Sampling and Sample Preparation for Determination of Algal Concentration 4.7 NUTRIENT CONCENTRATION ANALYSES
4.7.1 Phosphate Concentration Analysis 4.7.2 Nitrate Concentration Analysis 4.7.3 Nitrite Concentration Analysis 4.7.4 Ammonia Concentration Analysis
4.8 DETERMINATION OF ALGAL CONCENTRATION 4.9 PROCESSING OF THE RESULTS
50 51 51 51 52 52 53 53 53 54 54 55 55 56
5. RESULTS
5.1 OBSERVED CELL LEAKAGE
5.2 THE RESULTS OF THE BEAD CONCENTRATION EXPERIMENT 5.2.1 The Effect of Bead Concentration on Nutrient Removal
5.2.2 The Effect of Bead Concentration on Algal Growth 5.3 THE RESULTS OF THE BEAD SIZE EXPERIMENT 5.3.1 The Effect of Bead Size on Nutrient Removal 5.3.2 The Effect of Bead Size on Algal Growth
5.4 THE RESULTS OF THE ALGAL CONCENTRATION EXPERIMENT 5.4.1 The Effect of Algal Concentration on Nutrient Removal
5.4.2 The Effect of Algal Concentration on Algal Growth 5.5 THE RESULTS OF THE Mg ADDITION EXPERIMENT 5.5.1 The Effect of Mg Addition on Nutrient Removal 5.5.2 The Effect of Mg Addition on Algal Growth
58 58 58 58 61 61 61 63 63 63 65 66 66 68
6.1 EVALUATION OF THE EXPERIMENTAL SET-UP AND THE SOURCES OF ERRORS
6.2 CELL LEAKAGE
6.3 NUTRIENT REMOVAL MECHANISMS 6.4 THE EFFECTS OF IMMOBILIZATION
6.5 THE EFFECTS OF INITIAL ALGAL CONCENTRATION 6.6 THE EFFECTS OF BEAD CONCENTRATION
6.7 THE EFFECTS OF BEAD SIZE 6.8 THE EFFECTS OF Mg ADDITION
6.9 OPTIMIZATION OF IMMOBILIZED ALGAL CULTIVATION IN AQUA- CULTURE WASTEWATER
7. CONCLUSIONS REFERENCES
70 72 74 75 76 77 79 80
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1. INTRODUCTION
The pollution of the natural water bodies is a major problem all around the world. One of the industries causing water pollution is aquaculture i.e. farming of fish and aquatic animals for human consumption. Aquaculture production generates large volumes of wastewaters that contain uneaten fish feed, feces of fish, antibiotics, and hormones. (Turcios and Papenbrock 2014). If not treated properly, nutrient-rich aquaculture wastewater can cause eutrophication and oxygen deficiency in the receiving waters. In the developing countries that practice aquaculture widely, simple and inefficient wastewater treatment techniques (sedimentation and constructed wetlands) are applied due to the lack of resources. This together with the insufficient regulation of aquaculture pollution worsens the environmental impacts of aquaculture in the developing countries. (Kathijotes et al. 2015).
The use of microalgae to remove the excess nutrients from wastewater can help to fulfill the high need for affordable and efficient wastewater treatment techniques. These simple photosynthetic microorganisms, that live on aquatic environments, have shown potential for the treatment of both municipal and industrial wastewaters. Due to their large surface to volume ratio, microalgae are effective in assimilating nutrients from their environment, which can be utilized in wastewater treatment. (Aishvarya et al. 2015). In addition to nutrients, microalgae can remove chemicals, antibiotics, and metals from wastewater. At the end of the wastewater treatment, valuable algal biomass is obtained in addition to purified effluent. Microalgal metabolism can produce many high-value molecules including lipids and pigments, and thus the applications for microalgal biomass are vast. The industries utilizing microalgal biomass range from human dietary supplements and pharmaceuticals to fertilizers and biofuels. (Delrue et al. 2016).
Even though microalgae have promising abilities for wastewater treatment, the separation of algal cells from the effluent still poses a problem. Traditionally microalgae are cultivated suspended in the culture medium in open ponds or closed photobioreactors. At the time of the harvesting dilute algal culture has to be processed to remove the majority of the culture medium.
Currently available methods for harvesting include sedimentation, flotation, filtration, and centrifugation. These methods are, however, either too slow or too expensive for cost-effective algal harvesting. (Aishvarya et al. 2015). To avoid the problematic harvesting step of the algal cultivation, the use of immobilized culture method has been suggested. In immobilized
cultivation, algae are entrapped inside the polymer matrix, which most often consists of alginate in the shape of a spherical bead. The immobilization matrix allows the nutrients of the culture medium to reach the algae, but keeps the algae separated from the culture medium during the cultivation. In addition to easing the harvesting process, immobilization can enhance both algal metabolism and nutrient removal efficiency, protect algae from the predators that graze on them, and improve the resistance of algae to toxic compounds that can be present in wastewater.
However, using wastewater as a culture medium for immobilized culture can lead to complications as the microorganisms and contaminants of the wastewater can deteriorate the immobilization matrix, causing algal cells to leak to the culture medium. Another downside of the immobilized cultivation is the high cost of the immobilization process and polymers. (de- Bashan and Bashan 2010).
It is important to optimize the immobilized conditions in a treatment with algal beads. The size of the beads, the initial algal concentration of the beads, and the bead concentration of the culture medium can affect the outcome of the treatment with algal beads. These parameters can be adjusted to achieve optimal results both in terms of nutrient removal and algal growth.
(Abdel Hameed 2007). This work investigates the optimization of treatment with microalgal beads for nutrient removal from aquaculture wastewater.
2. LITERATURE REVIEW
2.1 MICROALGAE
2.1.1 Microalgae in the Nature
Microalgae are microscopic photosynthetic organisms living primarily in different aquatic environments. The size of these simple uni- or multicellular organisms ranges between 2 and 200 μm. Microalgae are one of the oldest life forms on the earth. (Aishvarya et al. 2015).
Cyanobacteria, which are classified as microalgae, were responsible for the production of oxygen to the early atmosphere, and to this date approximately half of the photosynthetic productivity on the earth results from microalgal activity. Microalgae are not a monophyletic group and include eukaryotic algal species in addition to prokaryotic cyanobacteria. (Andersen 2013). Diatoms, green algae, blue-green algae, and golden algae are the most abundant groups of microalgae. The diversity of microalgal species is vast: the estimated number of species is between 200 000 and several millions, the majority of which remains undescribed. (Aishvarya et al. 2015).
As a result of the species diversity, the morphology of microalgae varies greatly. Coccoid algae are spherical in shape while flagellate algae can use their flagella to move around in water.
Among these forms are species living both independently as a single-celled organism and as a part of a colony. The colonies of sarcinoid algae are cubical while filamentous algae form ribbon-like chains. Palmelloid algae live within a gelatinous matrix, where the gel holds the cells together. Amoeboid algae are able to crawl on the surfaces of their habitat. (Andersen 2013).
Also the modes of nutrition differ between the microalgal species. Most microalgae are autotrophs and able to produce their nutrition through photosynthesis, but some microalgal species are not capable of that. These heterotrophic species take in and digest particles and bacteria from their environment. Mixotrophic algae can fulfil their nutrition demand using both of these modes. (Andersen 2013). In the nature microalgae live in various environments all around the world. The most common habitats for microalgae are fresh, brackish, and marine water environments. (Aishvarya et al. 2015). In addition to aquatic environments, microalgae grow on different surfaces including rocks, soil, snow, and plants. Some microalgal species have symbiotic relationships with other organisms e.g. in lichens and corals. (Andersen 2013).
2.1.2 Cultivation of Microalgae for Industrial and Commercial Applications
The diversity of microalgal species holds enormous potential for industrial and commercial applications. Microalgae produce many high-value molecules in their metabolism and the cultivation of microalgae can provide these compounds for several industries. (Delrue et al.
2016). The main constituents of microalgal biomass are proteins, lipids, and carbohydrates. The protein content of microalgae is usually high whereas the lipid content varies between the species, being 5-20% on average. Under optimal conditions lipid content can, however, reach up to 80-90% of the dry weight. Other compounds present in microalgal biomass include biopolymers and pigments like chlorophyll and carotenoids. Microalgae and valuable compounds originating from microalgae can be utilized in pharmaceutical and cosmetics industries or as human nutritional supplements, animal feed, and fertilizer. (Aishvarya et al.
2015). In addition, biochar for soil amendment purposes can be produced from microalgae through pyrolysis. Microalgae can also be used as a feedstock for biofuels. Algal lipids can be extracted and converted to biofuel through transesterification, or the biomass as such can be processed through anaerobic digestion or hydrothermal liquefaction to produce biofuels.
(Delrue et al. 2016). Compared to other energy crops, advantages of microalgae include higher photosynthetic efficiency, growth rate, and biomass production. For some microalgal species with high growth rates, the doubling of biomass concentration can occur within hours. In addition, cultivation of microalgae does not compete for arable land with food crops. (Pires et al. 2013).
Successful cultivation of microalgae for industrial and commercial applications requires optimal cultivation conditions for microalgae. Microalgae need nutrients, light, and carbon dioxide (CO2) for their growth. In addition, pH, temperature, and salinity of the culture medium need to be suitable for the cultured algal species, since both optimal and tolerated growth conditions are species specific. The most important nutrients for the growth of microalgae are phosphorus and nitrogen, which microalgae can take up as nitrate (NO3-), ammonia (NH3), and urea. Other essential nutrients include sulphur, potassium, sodium, iron, magnesium, calcium, and some trace elements. The culture medium must supply microalgae with the nutrients.
Several culture media (e.g. BBM, BG11, F/2) have been developed for the cultivation of microalgae with different nutrient requirements. However, large-scale cultivation of microalgae in these media is not feasible due to the high cost of the media. Microalgae need light for photosynthesis. Light intensity and spectral quality are the most important variables when illuminating microalgal culture, but for some species also photoperiod (e.g. alternation of light
and dark) plays an important role. Adjusting the light intensity according to the needs of the microalgae is important since low intensity limits photosynthesis and high intensity causes photo-inhibition. Microalgae use wavelengths between 400 and 700 nm for photosynthesis. As light from the red and blue parts of the spectrum is most actively used in the photosynthesis, illuminating the culture with fluorescent lights of these colors can be beneficial for the growth of microalgae. During the cultivation algae consume CO2 from the culture medium as it is also needed in photosynthesis. Decreased dissolved CO2 concentration can limit the algal growth if the supply of CO2 is not assured by aerating the culture with either ambient air or air enriched with CO2. In addition to providing dissolved CO2 to the culture, aeration keeps the culture moving and thus prevents the formation of algal sediment, and contributes to maintaining even conditions (temperature, pH, distribution of nutrients and light) within the culture. Optimal and tolerated pH and temperature ranges for most microalgal species are 8.2-8.7 and 7-9, and 20- 24 and 16-27 °C respectively. Appropriate salinity of the culture medium depends mostly on the natural environment of the cultivated algae as marine and freshwater species have different requirements for the salt concentration. (Aishvarya et al. 2015).
As microalgal culture media can be expensive, nutrient-rich wastewater has been suggested to offer more affordable medium for large-scale microalgal production. The usage of wastewater can reduce not only the production cost but also the environmental impacts of the process, when the consumption of industrial nutrients and freshwater decreases. (Pires et al. 2013). Microalgae can obtain most of the nutrients, that are needed for algal growth, from wastewater. At the same time the cultivation of microalgae in wastewater can benefit the wastewater treatment industry.
Microalgae can be applied for tertiary wastewater treatment since they can effectively remove nutrients from their culture medium. The efficiency of microalgae in nutrient removal is based on their large surface to volume ratio which allows them to take up large amounts of nutrients.
Microalgal species from Scenedesmus (S.) and Chlorella (C.) genera tolerate wastewater especially well and can reach very high or even almost complete removal of nitrate, ammonia, and phosphate (PO43-) when grown in secondary treated wastewater. (Aishvarya et al. 2015).
Microalgal species, the characteristics of wastewater, cultivation method and conditions, initial algal concentration, and nitrogen to phosphorus ratio affect the performance of microalgae to remove nutrients (Pires et al. 2013). In addition to nutrients, microalgae can remove and break down several chemicals that are present in wastewater. The ability of microalgae to degrade micropollutants such as hormones, antibiotics, and phenolic compounds has been studied recently. (Delrue et al. 2016). Microalgae have also high capacity to accumulate metals. To
utilize this ability, metal-tolerant and highly accumulating microalgal strains can be isolated from polluted waters. (Moreno-Garrido 2008). In the studies microalgae have shown potential for the treatment of municipal wastewater as well as a wide range of industrial wastewaters including wastewaters from pulp and paper industry, agriculture, dairy industry, oil refineries, and alcohol distilleries. Combining microalgal production and wastewater treatment is reasonable also because the scale and production facilities that are needed for these industries are similar. The downside of using wastewater as culture medium is the restrictions it causes for the usage of produced algal biomass. Due to regulations the use of wastewater-grown microalgae in food, pharmaceutical, and cosmetics industries would most likely be impossible, which leaves biofuel and energy industry the most promising field for the application of wastewater-grown microalgae. (Delrue et al. 2016).
Microalgal cultivation can be combined also with carbon sequestration industry. Carbon sequestration is used to mitigate climate change by sequestering greenhouse gas CO2 out of the atmosphere. Microalgae fix carbon to algal biomass and can thus be utilized in carbon sequestration. (Pires et al. 2013). One kilogram of microalgal biomass is reported to be able to fix 1.83 kg of CO2. The ability to sequester carbon has been observed in several microalgal genera including Scenedesmus, Chlorella, Chlorococcum, Nannochloropsis, and Spirulina.
Carbon sequestration with microalgae can be performed by conveying industrial flue gas to microalgal culture, where microalgae use the CO2 of the flue gas in photosynthesis. (Aishvarya et al. 2015).
2.1.3 Methods for Cultivating and Harvesting Microalgae
Before starting the cultivation of microalgae, the cultivated microalgal species needs to be selected. Microalgae can be cultivated as a single species culture or as a natural consortia of either multiple microalgal species or microalgal and bacterial species. It is important to make sure that the selected microalgal species can proliferate in the used culture medium and withstand the variation of the environmental conditions of the cultivation site. (Delrue et al.
2016).
Microalgae is traditionally cultivated either in open ponds or closed systems called photobioreactors. Open cultivation systems i.e. high rate algal ponds or raceway ponds are usually shallow, under half a meter deep raceway-shaped systems, that use paddlewheels to circulate the water. As ponds are located outdoors, sunlight and runoff water from the
surrounding area are utilized as light and nutrient sources for the microalgae. Among artificial cultivation systems raceway ponds are the most commonly used. Compared to open ponds, photobioreactors provide more controllable conditions for microalgal cultivation as temperature, lighting, and hydrodynamics of the culture can be adjusted in a closed system. In photobioreactors microalgae is cultivated in closed containers and the culture is mixed by aeration or circulation within the system. Photobioreactors are available in several designs. The main reactor types include tubular, flat plate, column, and stirred tank. (Aishvarya et al. 2015).
Due to their strengths and weaknesses open pond systems and photobioreactors are suitable for different purposes and are both used in microalgae production industry. Photobioreactors can provide higher volumetric productivities due to the controlled conditions, but the biggest disadvantage of the photobioreactors is the high cost of cultivation. The cost of microalgae production in photobioreactor is over ten times higher compared to open pond system with the same production capacity. For this reason, photobioreactors are best suited for the cultivation of axenic microalgal strains producing high value molecules, that can counterbalance the production cost. Another possible application is the treatment of highly hazardous wastewaters that need to be treated regardless of the cost of the treatment. (Delrue et al. 2016). Open pond systems on the other hand are cost effective, easy to operate, and suit well for the mass cultivation of microalgae. In open pond systems the initial building expenses are lower and the production capacity larger compared to photobioreactors. However, open systems are susceptible to weather conditions and contamination. (Aishvarya et al. 2015). Contaminations can severely threaten the success of microalgal cultivation. Contamination can be caused by other microalgal species, viruses, and bacteria as well as herbivorous protozoa and zooplankton that graze on microalgae. For example, a high concentration of zooplankton can destroy 90%
of the algal biomass in the course of just a few days. (Delrue et al. 2016).
An alternative to the traditional cultivation methods is attached cultivation. In attached cultivation the microalgae are immobilized onto supporting materials which are then immersed into the culture medium. An example of this method is biofilm rotating disc reactor which can be used for combined wastewater treatment and microalgal production. (Delrue et al. 2016).
Regardless of the used cultivation method, ideal characteristics for the cultivation system from the commercial point of view include high area and volumetric productivity, low production costs, easily controllable culture parameters, energy efficiency, and reliability (Aishvarya et al.
2015).
The next step after cultivation is the harvesting of microalgal biomass. Harvesting is a major challenge in microalgae production as the small size and low density of microalgal cells makes them difficult to separate from the culture medium. (Pires et al. 2013). In addition, microalgal cultures are extremely dilute. 99% of the culture medium need to be removed from the culture before microalgal biomass can be further processed. Harvesting consists of two steps, which are concentrating and dewatering the biomass. (Delrue et al. 2016).
Sedimentation, flocculation, and flotation are methods used for concentrating the microalgal biomass. Sedimentation is a simple method that allows microalgae to settle gravitationally to the bottom of the settling basin. The settling rate of algal biomass is species specific and depends on the density and size of the algal cells. Sedimentation can be a slow process and thus flocculation is often used to achieve faster settling of algal biomass. Flocculation is based on the addition of chemicals that induce flocculation i.e. formation of larger aggregates from singular cells. Ferric sulphate, calcium hydroxide, and polymeric substances can be used as flocculants. (Aishvarya et al. 2015). In addition to chemicals, bacteria (bioflocculation), pH change (autoflocculation), or electric field (electroflocculation) can be used to enhance flocculation. Flocculation is an efficient concentrating method with low cost and energy consumption. (Delrue et al. 2016). The last algae concentrating method, flotation, utilizes air bubbles in the separation of algal biomass. Algal cells are attached to the bubbles that are released from the bottom of the basin. Cells and bubbles accumulate to the liquid surface and form foam, which can be skimmed off. Algal cells with high lipid content and low density are best suited for harvesting with flotation. The disadvantage of the method is the high production cost of the bubbles. (Aishvarya et al. 2015).
Dewatering of the algal biomass can be achieved by using centrifugation or filtration.
Centrifugation is an efficient and commonly used method. It is, however, not suitable for processing large quantities of algal biomass due to the high cost and energy requirement of the process. With filtration dewatering can be performed with significantly lower cost and energy consumption. For example belt filter press can be used for this purpose. After concentrating and dewatering the biomass the dry weight concentration of the biomass is 15-25%. If necessary for the following use of microalgal biomass, the biomass can be dried using spray or solar drying. Both of these methods are very effective in drying the biomass without denaturation.
As hot gas is used in spray drying, the energy demand of the method is very high compared to
solar drying. Solar drying on the other hand requires large surface area for drying the biomass.
(Delrue et al. 2016).
2.2 IMMOBILIZATION OF MICROALGAE
2.2.1 Advantages and Disadvantages of Immobilization of Microalgae
The immobilization of microalgae into polymer matrices has been suggested to provide a solution to the lack of cost-effective harvesting methods for microalgal cultivation. If microalgal biomass successfully retains within the immobilization matrix, the harvesting process can be avoided completely. (Pires et al. 2013). The immobilization of microalgae has been studied since the 1970s. The purpose of immobilization is to prevent microalgae from moving freely in the culture medium. Instead, the living microalgal cells are entrapped inside the immobilization matrix, often shaped like a spherical bead. The pores of the polymer matrix are smaller than algal cells. Thus, the algal biomass remains within the matrix while the culture medium can still flow through it. (de-Bashan and Bashan 2010). The nutrients of the culture medium are adsorbed onto the matrix and slowly absorbed deeper into the matrix to finally be consumed by microalgae (Tam and Wong 2000). Compared to suspended cells, immobilized microalgae have longer lag period. However, later the specific growth rate of both culture types can be very similar. Immobilization has been observed to affect the algal growth in multiple ways. In some experiments, immobilization enhanced the algal growth whereas in other experiments it didn’t affect the growth or even inhibited it. (Moreno-Garrido 2008).
In addition to concentrating the microalgal biomass and making the harvesting easier, immobilization has many other advantages. Immobilization protects the entrapped microorganisms in several ways. Immobilization can improve the resistance of microalgae to toxic compounds when wastewater is used as a culture medium. Immobilized microalgae are also safe from zooplankton that graze on them. In addition, immobilization reduces the competition for nutrients between microalgae and other microorganisms of the wastewater. In general, immobilization can improve the survival of immobilized microorganisms in the wastewater. This is important as many promising candidates for wastewater treatment lack environmental competitiveness when grown in wastewater. Immobilization has positive effects also on the metabolism and function of microalgae. Compared to suspended cells, immobilized microalgae have shown increased chlorophyll, carotenoid, and lipid content as well as higher dry weight and enhanced photosynthesis. As a consequence of the improved metabolism, immobilized microalgae can remove nutrients (nitrate, ammonia, and phosphate) faster and
more efficiently than suspended microalgal culture. In addition, immobilized culture method is simple to apply. (de-Bashan and Bashan 2010).
Despite the many advantages of immobilization, the method still has some drawbacks and challenges. The high cost of both immobilization matrices and the immobilization process makes especially the large-scale application of the technique costly (Pires et al. 2013, Wang et al. 2016). For successful wastewater treatment, the immobilized system requires longer retention time compared to traditionally used wastewater treatment techniques, which in turn raises the treatment costs. In addition, the treatment efficiency of the immobilized system varies, and unpredictable environmental parameters affect the success of the treatment.
Maintaining the integrity of the immobilization matrix during the wastewater treatment is also important. Microorganisms and contaminants of the wastewater can cause the immobilization matrix to deteriorate during the treatment, which may limit the use of some immobilization matrices in the field of wastewater treatment. (de-Bashan and Bashan 2010). The deterioration of immobilization matrix usually leads to the release of algal cells from the matrix to the culture medium. This cell leakage can be minimized by optimizing the immobilization process to enhance the stability of the immobilization matrix. (Lam and Lee 2012). Most of the results showing high nutrient removal efficiencies with immobilized treatment have been obtained from small-scale laboratory experiments. Thus, more experiments, that are performed in larger scale, are needed to confirm the suitability of treatment with immobilized microalgae in large- scale applications. (Pires et al. 2013).
2.2.2 Immobilization Methods
Immobilization methods can be divided to passive and active immobilization. Passive immobilization can be performed only with microorganisms that naturally attach to surfaces and grow on them. To achieve passive immobilization, suitable surfaces or carriers are offered as substrate for the microorganisms, which then start to grow on them. Passive immobilization is, however, easily reversible process, and loose cells can be detached from the immobilization matrix into the culture medium. (Moreno-Garrido 2008). In active immobilization microorganisms are actively immobilized into or onto the immobilization matrix. Active immobilization can thus be performed with all types of microorganisms regardless of their natural way to grow. More precisely immobilization methods can be classified to covalent coupling, affinity immobilization, adsorption, confinement in liquid-liquid emulsion, capture behind semi-permeable membrane, and entrapment in polymers. The focus of this literature
review is in immobilization into polymers, which is the most commonly used technique among different immobilization methods. (de-Bashan and Bashan 2010).
2.2.2.1 Immobilization Matrices
Several natural and synthetic polymers with different properties can be used as immobilization matrices when microalgae are immobilized into polymers. The most important characteristic of immobilization matrix is hydrophilicity as the culture medium is required to be able to diffuse into the matrix to provide nutrients for the immobilized microorganisms. Natural polymers, that are used in immobilization, are derived from algal polysaccharides and include alginate, carrageenan, and agar. (de-Bashan and Bashan 2010). Among algal polysaccharides alginate is the most commonly used polymer. It is extracted from brown algae and constitutes of different proportions and sequences of guluronic and mannuronic acids. As an immobilization matrix alginate is ideal as it is non-toxic, permeable, and relatively stable. In addition, alginate is transparent, which allows light to penetrate into the immobilization matrix and enables the immobilization of photosynthetic microorganisms. (Moreno-Garrido 2008). Other advantages of alginate include the relatively cheap price of the material and availability also in large quantities. Alginate is also easy to use, and the immobilization is carried out simply by introducing the alginate into a solution containing cations. The immobilization process does not include extreme physicochemical changes, which favors the survival of microalgae through the process. (de-Bashan and Bashan 2010).
Carrageenan implies collectively to all polysaccharides extracted from red algae. Similarly as alginate, carrageenan is immobilized with cationic solutions. Also agar originates from some species of red algae. The immobilization procedure of agar differs from that of alginate and carrageenan as agar is a thermo-reversible gel. Agar melts at 85 °C and solidifies between 35 and 40 °C. Thus, agar can only be used for the immobilization of species that can survive a short-term thermal shock. Other natural polymers that can be utilized in immobilization include chitosan, which is obtained from the chitin exoskeletons of crustaceans, and proteins including egg white, collagen, and gelatin. Proteins are, however, rarely used for the immobilization of microalgae. (Moreno-Garrido 2008).
For the immobilization of microalgae, natural polymers are more widely used than the synthetic ones, even though the stability of the natural polymers is weaker. Natural polymers are prone to environmental degradation by microorganisms, and also wastewater can negatively affect
their stability. On the other hand, the diffusivity of natural polymers is higher and the production process less hazardous. (de-Bashan and Bashan 2010). Indeed, for the production of some of the synthetic immobilization matrices, toxic monomers are used. This is the case with acrylamide, epoxy resins, polyvinyl foams, and polyurethane foams. Due to the toxic nature of the monomers, these synthetic immobilization matrices are not suitable for the immobilization of living algal cells. However, they can be used for the immobilization of dead algal biomass e.g. for the metal adsorption purposes. (Moreno-Garrido 2008).
2.2.2.2 Immobilization Process for the Production of Alginate Beads
Spherical alginate beads are the most commonly applied form of immobilization for microalgae. To produce beads, algal suspension is first mixed with the monomers of the desired polymer, usually Na-alginate. The monomers are then connected to each other to form a polymeric gel around the microalgal cells. The goal is that the cells inside the matrix remain intact with minimal interaction with the polymer. (de-Bashan and Bashan 2010). Solidifying solutions containing di- or multivalent cations are used to link the monomers together and harden the alginate. Ca2+ is the most widely used cation for this purpose, but Sr2+ and Ba2+ have been suggested to be used when more stable alginate is needed. After the cultivation of algae, the alginate matrix surrounding the cells can be dissolved with sodium citrate or phosphate solution. (Moreno-Garrido 2008).
For a successful treatment with alginate beads, it is important that the alginate is stable enough to not break down and release algal cells into the culture medium during the cultivation. The concentration of alginate monomers in the algae-alginate suspension and the concentration of cations in the solidifying solution affect the stability of the beads. In general, the mechanical strength of the polymer increases when these concentrations increase. (de-Bashan and Bashan 2010). Lam and Lee (2012) examined the effect of alginate concentration on the algal growth by cultivating beads with alginate concentrations ranging from 0.3 to 1% (w/v%). The beads with the lowest alginate concentration were found unsuitable for algal cultivation as the immobilization matrix was thin and unstable, leading to the rupture of beads and cell leakage.
The alginate concentration had an influence on the growth rate of the immobilized microalgae.
An increase in alginate concentration resulted in a decrease in the algal growth rate due to the thicker immobilization matric limiting the nutrient and CO2 diffusion into the bead. Thus, the authors concluded that the alginate concentration of 0.5% (w/v%) was optimal for algal cultivation as it showed the highest growth rate without significant deterioration of the beads.
Wang et al. (2016) used in their experiment higher alginate concentrations between 1 and 3%
and observed that the alginate concentration had a significant effect on both nutrient removal (ammonium, NH4+ and total phosphorus, TP) and cell leakage. Based on the experiment and statistical analysis, the alginate concentration of 1% was optimal for minimizing the cell leakage. For nutrient removal the optimal alginate concentration was 3%, but due to the material costs the authors recommend the use of 2% alginate. This recommended concentration seems to be commonly used in the studies investigating the immobilization of microalgae (Tam et al.
1994, Tam and Wong 2000, Abdel Hameed 2007, Lee et al. 2020).
According to Lam and Lee (2012), the concentration of calcium chloride solution (CaCl2), that is used to harden the beads, does not affect directly on the algal growth. However, the effect of CaCl2 concentration is indirect as it significantly influences the stability of the beads and consequently the leakage of algal cells from the beads. Low CaCl2 concentration was observed to cause the deterioration of the beads when beads solidified with 0.5-3% (w/v%) CaCl2
solutions were cultivated in an experiment. The beads solidified with 2 and 3% CaCl2 solution stayed stable throughout the 10-day experiment and 2% CaCl2 solution was selected as an optimal solidifying solution concentration. Similarly as Lam and Lee (2012), Wang et al. (2016) found 2% (w/v%) CaCl2 solution to be optimal for bead solidification in terms of minimizing the cell leakage, when experimenting with beads solidified with 2-4% CaCl2 solutions. The use of solidifying solution with higher CaCl2 concentration was observed to result in an increase in the algal leakage. This is explained by the quick formation of thick polymer layer at the surface of the bead when alginate is introduced into a high-concentration solidifying solution. Due to this dense layer the cations cannot easily penetrate into the inner parts of the bead, leaving the inner alginate monomers unreacted and the bead fragile. The CaCl2 concentration was also observed to have an effect on the nutrient removal. For this purpose, 3% CaCl2 solution was optimal but Wang et al. (2016) still recommend the use of 2% solution to minimize both cell leakage and the costs of the bead preparation.
In practice the bead formation is performed by introducing the algae-alginate suspension dropwise into the solidifying solution. The process can be either manual or automated. In the manual production large syringes are used to drop the alginate solution under gravity flow into the solidifying solution. Manual bead preparation is a slow and laborious process as the dropping of alginate takes time and lab personnel is needed to fill the syringes from time to time. Automated bead production is faster and more effective way to prepare beads. In the
automated process a pump is used to transfer the algae-alginate suspension through tubing and syringe into the solidifying solution. In the large-scale production of the beads, a showerhead- like device with multiple holes is suggested to be used to drop the alginate into the solidifying solution. (de-Bashan and Bashan 2010).
Microalgae can be co-immobilized with other microorganisms or nutrients to promote their growth. Plant growth-promoting bacteria from the genus Azospirillum has been suggested to be used in microalgae cultivation. These bacteria are traditionally used in agriculture to fix nitrogen and enhance the growth and yield of the crop plants. According to recent experiments plant growth-promoting bacteria can also affect the growth of microalgae, improving the growth and nutrient removal ability of the co-immobilized algae. Thus, the co-immobilization of microalgae and plant growth-promoting bacteria may provide new applications for the wastewater treatment industry. (de-Bashan and Bashan 2010). The effect of co-immobilization with nutrients on the growth of C. vulgaris was investigated by Lam and Lee (2012). The nutrient solution used in the co-immobilization experiment was prepared from commercial organic fertilizer that contained nitrogen, phosphorus, potassium, calcium, magnesium, boron, and iron. The co-immobilization had a positive effect on the algal growth as after five days of cultivation the biomass concentration of the beads containing both algae and nutrients was 34%
higher compared to beads containing only algae. Co-immobilization enhanced also the specific growth rate of the immobilized microalgae. As the nutrients are present in the beads already at the beginning of the cultivation, microalgae can take up nutrients from inside of the beads immediately. Thus, the diffusion of nutrients into the beads does not limit the algal growth and higher growth rates can be achieved. In addition, co-immobilization with nutrients is suggested to be used as a means to control the cell leakage from the beads. In the co-immobilization experiment the cell leakage was insignificant after five days of cultivation and only increased later in the experiment, when the oversaturation of beads with algae caused the beads to rupture.
When the nutrients are co-immobilized into the beads, the availability of nutrients is greater for the immobilized cells compared to free cells, which may limit the growth of the leaked cells.
In addition, the amount of culture medium can be reduced as the nutrients are present in the beads, and subsequently the water consumption of the process decreases.
2.2.3 Nutrient Removal Mechanisms of the Treatment with Microalgal Beads
Assimilation by immobilized microalgae is not the only mechanism that removes nutrients from the culture medium during the treatment with microalgal beads. If the conditions are favorable,
ammonia can be removed via ammonia volatilization and phosphorus via precipitation with calcium, magnesium, and iron ions of the culture medium. For both of these phenomena, alkaline pH in the culture medium is needed. The pH of microalgal culture often increases during the cultivation as microalgae consume CO2 from the culture medium in photosynthesis.
Thus, these mechanisms often contribute to nutrient removal in microalgal culture. (Pires et al.
2013). Ammonia volatilization is often enhanced by the aeration of the microalgal culture. As metal ions are needed for the phosphate precipitation, the calcium ions of the alginate matrix can favor the removal of phosphate via this mechanism in immobilized cultures. (Tam and Wong 2000).
In addition, nutrients can be adsorbed to the alginate matrix or assimilated by the indigenous bacteria of the culture medium. Tam and Wong (2000) observed that the removal of ammonia from synthetic wastewater with blank alginate beads (beads without microalgae) was faster compared to control aerated wastewater. They suggest that the adsorption of ammonia to the alginate matrix was the reason for the faster ammonia removal. The results obtained by Cruz et al. (2013) indicate that assimilation by bacteria is more important phosphorus removal mechanism than adsorption to alginate, when immobilized microalgae are cultivated in secondary treated municipal wastewater. In the experiment blank beads and beads containing microalgae were cultivated in sterilized and non-sterilized wastewater. Both treatments with non-sterilized wastewater as culture medium resulted in significant phosphate removal whereas the removal of phosphate from sterilized wastewater was minor in both treatments. As the phosphate removal with blank beads was significantly more efficient from non-sterilized wastewater, the bacteria of the wastewater were found responsible for the phosphate removal.
The phosphate concentration in the blank bead treatment was also lower compared to control wastewater which indicates that the beads enhance the nutrient removal by bacteria by offering them surfaces for biofilm formation.
2.2.4 Applications of Immobilized Microalgae
Similarly as suspended microalgae, immobilized microalgae can be cultivated for a wide variety of applications including metabolite production, wastewater treatment, and pollutant removal.
Immobilized microalgae can be utilized in metal recovery. In the process the metals are either absorbed into the algal cells or adsorbed onto the cellular surfaces. Immobilization matrix can contribute to the metal removal by offering additional binding sites for the metals. At the end of the treatment the metals can be desorbed from the cellular surfaces and immobilization
matrix with acid treatment. (Moreno-Garrido 2008). In the experiments immobilized microalgae have been used to remove cadmium, chromium, cobalt, copper, lead, mercury, and nickel from their culture medium. It is also possible to separately immobilize several microalgal species and use them together to treat wastewater. This approach is beneficial especially when the treated wastewater contains multiple pollutants that require specialized microalgae for the degradation of the compounds. Immobilized microalgae have also potential to be utilized in wastewater treatment. However, the technique still needs development and can currently be used as an auxiliary technology combined with other wastewater treatment techniques. (de- Bashan and Bashan 2010).
For some applications immobilized microalgae may be better suited than suspended microalgal culture. In the experiments immobilized microalgae have been used in field toxicity measurements to provide information of the toxicity of effluents or sediments on the organisms.
It is important to conduct the toxicity tests in environmentally relevant conditions. With suspended algal cultures in situ tests may be difficult to perform, while immobilized culture is easy to maintain also in the field conditions. In addition, microalgae can be utilized in biosensors to detect pollutants in the environment. Microalgae have been observed to stay viable in immobilized form for extended time periods. With this feature it is possible to ease the maintenance of microalgal culture collections, which is a laborious task with traditional culture methods. (Moreno-Garrido 2008). In the experiments by Chen, it was observed that Isochrysis galbana immobilized in alginate beads survived the storage of one year without culture medium in darkness at 4 °C (Chen 2003) and S. quadricauda even three years when immobilized and stored similarly at 48 °C (Chen 2001). In both experiments the long-term storage did not affect the number of algae in the beads and the algae started to proliferate when transferred to culture medium after the storage. Some changes in the algal cells were observed after the storage, but the cells soon recovered to their original form when transferred to culture medium.
2.3 OPTIMIZATION OF TREATMENT WITH MICROALGAL BEADS 2.3.1 Optimization of the Initial Algal Concentration of the Beads
During the last few decades the effect of initial algal concentration of the beads on the nutrient removal from wastewater has been investigated using microalgal species from Chlorella and Scenedesmus genera. In an experiment by Abdel Hameed (2007) initial algal concentration of 1.5 106 cells/bead was found optimal for nutrient removal from primary treated wastewater.
The treatment with optimal concentration of immobilized C. vulgaris removed nutrients significantly more effectively than the other treatments (3 105 and 3 106 cells/bead), reaching the removal of 94, 96, and 100% of phosphate, nitrate, and ammonia respectively after 48-hour treatment (Table 1). Increasing the algal concentration of the beads resulted in decreased nutrient removal efficiency and leakage of the algal cells after 32 hours of treatment.
Wang et al. (2016) conducted a study with orthogonal array experiment design to determine the optimized conditions for maximal nutrient removal and minimal algal leakage. Among other factors, initial algal concentration of the beads was included in the experiment in which immobilized S. obliquus was cultivated in synthetic wastewater with two-stage cultivation (semi-continuous and batch stage) for 10 days. Similarly as Abdel Hameed (2007), the authors observed that the initial algal concentration significantly impacts the nutrient removal efficiency. The removal of TP, total nitrogen (TN), and ammonium followed the same pattern in all the treatments (0.2 106, 106, and 5 106 cells/ml): during the semi-continuous stage the nutrient concentrations declined slowly, followed by a faster decline when the batch stage was started. The highest of the algal concentrations, 5 106 cells/ml, performed best both in terms of nutrient removal and algal leakage. Even though the algal growth rate was observed to be low in this treatment, the amount of algal biomass was high already at the beginning of the cultivation, and thus high nutrient removal could be achieved. As the high initial algal concentration may hinder the algal growth, the authors recommend the use of lower initial algal concentration of 0.2 106 cells/ml regardless of the more effective nutrient removal achieved with higher initial algal concentration.
Significant differences in the nutrient removal between treatments with different initial algal concentrations were not observed in some of the experiments. Chevalier and de la Noüe (1985) cultivated three concentrations of immobilized S. quadricauda in secondary effluent from wastewater treatment plant for 9 hours, but did not observe any significant differences in the nutrient removal between the treatments. In the experiment semi-continuous culture mode was used and the culture medium was changed in 3-hour cycles. After the first cycle almost all the phosphate and ammonium were removed from the culture medium, but the removal efficiency decreased in the following cycles. Also Tam et al. (1994) only obtained minor differences in the phosphate and ammonium removal when cultivating C. vulgaris in the concentrations of 5.2 105 and 4.7 106 cells/bead in primary treated wastewater for 7 days (Table 1). The treatment with higher algal concentration (4.7 106 cells/bead) removed especially phosphate
faster than the treatment with lower algal concentration (5.2 105 cells/bead) as after two days of cultivation the phosphate removal percentages of the treatments were 33 and 0.9, respectively. The removal percentage of nitrate at this time was around 45 in both of the treatments. However, the nutrient removal was not particularly fast even in the treatment with higher algal concentration. After two days of cultivation of the same algal species in a closely similar experimental set-up, Abdel Hameed (2007) could reach clearly higher nutrient removal even in the least effective treatment of his experiment. The algal leakage reported in the beads with high initial algal concentration in Abdel Hameed’s experiment, was not observed in the experiment by Tam et al. even though higher initial algal concentration was used. The immobilized algae were not responsible for all of the nutrient removal in the experiment by Tam et al. (1994), as also blank beads performed well, reaching 92 and 49% removal of ammonia and phosphate, respectively at the end of the treatment.
Two of the abovementioned studies (Tam et al. 1994, Wang et al. 2016) also examined the effect of the initial algal concentration of the beads on the algal growth. Both of the experiments concluded that algal growth was faster in treatments with low initial algal concentration compared to treatments with high initial algal concentration. In the experiment by Tam et al.
(1994) a photosynthetic rate measurement also indicated that the cells in the treatment with low initial algal concentration had higher physiological activity. However, this treatment had longer lag phase than the treatment with high initial algal concentration. On the other hand, Wang et al. (2016) reported that in their experiment the growth trends of all the treatments were similar and the lag phase was short. The lower growth rates of treatments with high initial algal concentration were explained by self-shading effects i.e. the prevention of light penetrating deep into the culture medium or deep into the bead due to the high concentration of algal cells in the beads. Also Dos Santos et al. (2002) observed that the treatment with high initial algal concentration exhibited slower growth rate compared to treatments with lower initial algal concentrations. In this experiment three concentrations of Phaeodactylum tricornutum were cultivated for three days in artificial seawater (Table 1). According to the authors, in addition to the higher light penetration, the better availability of nutrients and CO2 inside the beads may have affected the enhanced growth rate of the treatments with lower initial algal concentrations.
Table 1. Description of the experiments studying the effect of the initial algal concentration of the beads on the algal growth and nutrient removal.
Reference Cultivation conditions Culture medium Bead preparation Microalgal species
Treatment time
Initial algal
concentration Nutrient removal Algal growth Chevalier
and de la Noüe,
1985
350 ml culture, 20 ± 1 °C, 14/10 h light/dark cycle, aeration with atmospheric
air, semi-continuous operation: the culture medium was changes in
3-hour cycles
secondary effluent from
wastewater treatment plant
2.5% carrageenan solution, 3mm beads
S. quadricauda 9 h
0.885 g/l dry weight no significant differences between the treatments, the removal efficiency of NH4+-N and PO43--P decreased from first to third cycle
in each treatment
not studied 1.89 g/l dry weight
3.29 g/l dry weight
Tam et al., 1994
1 l culture, 20 ± 2 °C, 16/8 h light/dark cycle,
aeration
primary treated wastewater, PO43--P 3.6 mg/l,
NH4+-N 36.2 mg/l
2% alginate solution, 2% CaCl2 solution,
4 mm beads
C. vulgaris 7 d
5.2 105 cells/bead PO43--P 68.3 %, NH4+-N 99.7%
4-fold increase (2 106 cells/bead) 4.7 106 cells/bead PO43--P 70.6%,
NH4+-N 100%
2-fold increase (8.9 106 cells/bead)
Dos Santos et al., 2002
100 ml culture, 20 ± 1 °C, continuous illumination,
shaking 100 rpm
artificial seawater
1.25% alginate solution, 1% CaCl2 solution, 2.5-3.5 mm beads,
initial algal concentration 104 cells/ml of culture
medium in each treatment
Phaeodactylum
tricornutum 3 d
0.61 106 cells/ml of alginate
not studied
54-fold increase (54 104 cells/ml
of culture medium) 1.54 106 cells/ml of
alginate
60-fold increase (60 104 cells/ml
of culture medium) 2.59 106 cells/ml of
alginate
47-fold increase (47 104 cells/ml
of culture medium)
Abdel Hameed,
2007
1.6 l culture, 25 ± 2 °C, 16/8 h light/dark cycle, aeration with filtered air
40 ml/min
primary treated wastewater, PO43- 11.9 mg/l,
NO3- 4.4 mg/l, NH3 40 mg/l
2% alginate solution, 0.1 M CaCl2 solution,
4 mm beads C. vulgaris 48 h
3 105 cells/bead
PO43- 67%, NO3- 36%, NH3 75%
not studied 1.5 106 cells/bead
PO43- 94%, NO3- 96%, NH3 100%
3 106 cells/bead
PO43- 76%, NO3- 55%, NH3 82%
27
2.3.2 Optimization of the Bead Concentration of the Culture Medium
In the literature the bead concentration is usually reported either as a number of beads per ml of culture medium or as a ratio of the volumes of the beads and the culture medium. In this review the ratio of the volume of the beads to the volume of the culture medium is mostly used.
As can be seen from Table 2, a wide range of bead concentrations have been used in the experiments investigating the effect of bead concentration on the nutrient removal. Both Abdel Hameed (2007) and Tam and Wong (2000) determined 1:3 beads to culture medium ratio to be optimal for nutrient removal. In his experiment, Abdel Hameed (2007) cultivated C. vulgaris immobilized in beads with initial algal concentration of 1.5 106 cells/bead in ratios of 1:3, 1:2, 1:1, and 2:1 in primary treated wastewater for 48 hours. Treatments with ratios of 1:3 and 1:2 removed ammonia significantly more effectively than treatments with higher ratios, and also reached the highest nitrate removals. In the treatments with high bead concentrations, the removal of N-compounds was most likely affected by the self-shading effects caused by the large number of beads. The phosphate removal on the other hand was not significantly affected by the self-shading effects or bead concentration, and all the treatments were equally effective reaching about 95% phosphate removal. Between the most effective treatments, 1:3 beads to culture medium ratio was selected to be the most ideal and economical solution for wastewater treatment.
The experiment by Tam and Wong (2000) was closely similar to that of Abdel Hameed (2007) as C. vulgaris immobilized in beads at concentration of 106 cells/bead was cultivated in synthetic wastewater that simulate settled domestic wastewater. Unlike Abdel Hameed (2007), Tam and Wong (2000) tested also lower bead concentrations than the optimal 1:3 beads to culture medium ratio, as the tested ratios ranged from 1:9 to 1:1.8. However, the treatments with low bead concentrations did not contain enough algal biomass for efficient nutrient removal as after 24-hour treatment both ammonium and phosphate concentrations in the treatment with the lowest beads to culture medium ratio (1:9) were significantly higher compared to other treatments. Similarly as Abdel Hameed (2007), Tam and Wong (2000) observed that in phosphate removal the differences between the other treatments were minor, and in ammonium removal the treatment with 1:3 beads to culture medium ratio performed best, reaching 100% ammonia removal (Table 2). Some of the beads in the treatments with high bead concentrations did not stay suspended in the culture medium during the cultivation and settled to the bottom of the bioreactor, reducing the surface to volume ratio of the beads and the efficiency of the light utilization. This in addition to the self-shading effects had a negative
