General context
Nowadays, biological processes have been mainly applied in wastewater treatment plants especially activated sludge process (ASP) However, there are still many drawbacks of biological process, for example, the need of pre-treatment, the high energy requirement for aeration, stirring, and the low nutrient removal capacity (Longo et al., 2016; Quijano et al., 2017) Moreover, the removal of P in wastewater, which is usually done by chemical and physical processes (flocculation, flocculation), causes high costs and low treatment efficiency (Cole et al., 2017). From these existing shortcomings, the application of microalgae-based wastewater treatment systems using wastewater as a source of nutrient has been successfully developed in recent years, especially domestic wastewater has brought the positive results in the ability to treat N, P and algae biomass recovery (Ruiz-Marin et al., 2010; Li et al., 2019; Chiu et al., 2015) The advantage of using algae corresponds with the ability to remove nitrogen and phosphorus in wastewater through the assimilative process of biomass which have been studied such as post-treatment process (Honda et al., 2017), urine (Tuan et al., 2014), aquaculture wastewater (Gao et al., 2016), livestock wastewater (Garcia et al., 2017) Besides, microalgae-based technology was considered as an environmental-friendly, low-cost and high- efficiency method to reduce the concentration of carbon dioxide in the atmosphere (Benemann, 1997; Klinthong et al, 2015; Zhou et al, 2017) In addition, utilizing microalgae to convert carbon emission into biomass is regarded as one of the most cost-effective method of CO2 reduction and gained biomass of algae could be used as high-profit product like biofuels, food source, etc (Chisti, 2007; Chiu et al., 2015) However, widespread adoption of microalgae-based technology is constrained in terms of design, operation, and biomass harvesting.
Since the microalgae has very small size (3 - 30 μm) and the settling rate is less than3.6 x 10 -3 m/h, it is difficult and expensive to separate microalgae from cultivation media (Granados et al, 2012; Hu et al, 2017) Thus, harvesting microalgae biomass is a bottlenecks in developing production systems for manufacturing bioproducts or applying microalgae to treat wastewater It is estimated that about 20 - 30% of operating costs during the cultivation of microalgae are from the microalgae biomass harvesting through coagulation/flocculation, centrifugation, flotation, etc. (Renuka et al, 2013) Therein, centrifugation is the most popular method due to its saving time but also the most energy-consuming method when it applies to microalgae harvest at an industrial level, which is supposed to have a high energy consumption (more than 3,000 kWh/ton of microalgae) (Schenk et al., 2008) In addition, the coagulation method is also often used as a substitute for centrifugation because of less energy consumption (Gerde et al 2014) Nevertheless, this method contains several drawbacks, such as the contamination of final product with metal salts, high costs of chemicals and the difficult to operate (Granados et al, 2012) In some recent studies, gravitational sedimentation which is applied to the harvesting of microalgae is the most appropriate method with low cost and no energy consumption However, the main disadvantage of this method is the time it takes to settle naturally as well as the yield and biomass gathering are low.
To present COD and nutrients treatment simultaneously in a compact area, enhance the settling velocity of microalgae, thus providing an alternative co-culture of microalgae in wastewater, the idea of combining bacteria and microalgae inside one system has been developed and attracted attention of many researchers around the world (Su, Mennerich and Urban, 2012) In 1972, the term "activated algae" was used for the first time in experimental research of McGriff and McKinney to refer the product of flocculation process between microalgae and bacteria during wastewater treatment (McGriff and McKinney, 1972) Biological remediation by activated algae has been tested recently in ponds and PBR, demonstrating their potentiality in overcoming the individual application disadvantages of either activated sludge and microalgae, such as high cost of aeration, large area consumption and sludge handling problem (Boelee et al., 2014), (Marcilhac et al.,2014) Currently, the microalgae-bacteria granular, which is considered to have a good settling rate (21.6 ± 0.9 m/h), high biomass content and dense microbial structure has operated successfully in closed culture system Photobioreactor (PBR) (Liu et al., 2017; Tiron et al., 2015, 2017) After transferring the seed microalgae to the aerobic granular sludge system (AGS), the formation of the microalgae-bacteria aerobic granular occurred Therein, extracellular polymers (EPS) plays an important role in the process of granulation and maintaining the internal structure of particles (Liu et al., 2017; Cai et al., 2019) Furthermore, the presence of filamentous microalgae and the operational parameters of the culture system (stirring speed, retention time, etc.) is also confirmed to play a major role in the granulation process (Tiron et al, 2015, 2017) The symbiotic co-culture of microalgae and bacteria has taken a lot of advantages, in consist of the ability to use carbon dioxide to produce oxygen in order to decrease the need for aeration, the increase of nutrient removal rate and the potential for high microalgae recovery efficiency (Tricolici et al., 2014; Tang et al., 2016; Zhu et al., 2019) However, under continuous operating conditions, maintaining a stable biomass concentration, substantial wastewater treatment performance has not been interested in research and evaluation, especially in term of operating condition of co-culture PBR (Quijano et al., 2017).
Therefor granulation of activated algae is expected to be the trend of future wastewater treatment field However, granulation process is influenced by many operational conditions, such as reactor configuration, starvation, hydraulic shear force and so on (Zhu et al., 2008), (Gao et al., 2011) These environmental conditions also have a close relationship with growth rate and cell composition of algae (Schenk et al., 2008), which have a direct correlation with removal performance of activated algae wastewater treatment system Therefore, a new technology as granulation from activated algae also needs more information and research to fulfill knowledge about the performance of this system, adapt utilization.
In this study, the co-culture of microalgae-bacteria is cultivated in sequencing batchPBR containing synthetic wastewater at different operating parameters to determine the optimum agitation speed in the stage of granular formation and the impact of organic loading rate on granular The granular is characterized by morphology, settleability, distribution of microalgae-bacteria and the amount of EPS The efficiency of biomass recovery, organic matter & nutrient removal will be assessed in detail The results from this work will be the premise for the design of microalgae-based systems to treat wastewater and harvest algae biomass, which is high efficiency and low cost.
Objectives of the study
The topic “Co-Culture of Microalgae and Bacteria for Wastewater Coupling with
Biomass Recovery” will be an application of photobioreactor (PBR) with the co- culture of microalgae-bacteria, operating in sequencing batch reactor (SBR) process to treat synthesis domestic wastewater with high content of nutrients (N, P) and organic (C), combining with microalgae biomass production-oriented produce bio- products and reduce CO2.
Research scope
This research was conducted within the laboratory with the following contents:
Biological inoculum used for flocculation/granulation processes was represented by Chlorella sp mixing with aerobic activated sludge from wastewater treatment system of supermarket (in MBR tank) with initial biomass concentration 400 mg/L The co-culture microalgae:activated sludge mixing ratio was 5:1 (%w:%w)
Wastewater used for culture is artificial wastewater with C:N:P mass ratio is about 100:10:1 corresponding to COD, nitrogen and phosphorus concentrations of 384 ± 20 mg L -1 ,40 mgN L -1 and 4 mgP L -1 , respectively.
In the PBR model, co-culture were cultured in four stirred photo-bioreactors with a working volume of 7L under four different agitation speed of 80, 120,
Research content
This study aim to achieve the following contents:
To form fully developed microalgae-bacteria granules.
To find the optimal agitation speed value for flocculation-granulation process in PBR and wastewater treatment performance
Observing the effect of Organic loading rate (OLR) and/or Nitrogen loading rate (NLR) on co-culture granular performance including granular characteristic and wastewater treatment efficiency
The meaning of the topic
The topic contributed, supplemented and understood more about the effects of optimal operating parameters and innovative process of microalgae-activated sludge system on the development of granulation co-culture Thereby, it applies to the utilization of wastewater with appropriate nutritional content in algae culture to recover high yield biomass for many potential applications from algae biomass In addition, the topic also serves as the basis for the next in-depth studies on a promising microalgae-based technology.
With the need to meet the increasingly high quality of the living environment of the society, finding and applying integrated green technology to reduce CO2 emissions and nutrients in wastewater is a very necessary thing today, especially in Vietnam,where the eutrophication is a challenge of industrial, agricultural wastewater This topic will contribute in the selection and development of a viable green technology for utilizing nutrients in wastewater for microalgae biomass culture to serve many potential applications for profit, and improving the quality of human life at low cost.
REVIEW
Co-culture of Microalgae-Bacteria
2.1.1 Wastewater treatment using microalgae-based systems
Microalgae-based technology in wastewater treatment has received growing attention in recent years owing to its outstanding advantages, including (1) simultaneously efficient N and P removal via microalgae photosynthetic assimilation; (2) cost effective with environment friendly due to no additional chemicals are required comparing to conventional treatment method, while oxygen generation, carbon dioxide mitigation, and metal ion reduction can be realized at the same time; and (3) potential utilization of the harvested microalgae biomass for production of food, feed, fuel, fertilizers, food stocks for pharmaceutical industry, etc (Wang et al., 2017).
One of the main advantages of the use of microalgae for wastewater treatment is the diversity of removal mechanisms for different types of pollutants Nutrients assimilation, nitrogen volatilization and phosphorous precipitation, aerobic biodegradation of organic matter, ammonium removal through nitrification, biosorption of heavy metals and pathogen disinfection due to pH fluctuations (Wang et al., 2017) Overall, microalgae cultivation using wastewater as the growth medium has multiple applications in bio-products, carbon dioxide mitigation and bioremediation The microalgae systems can treat human sewage, livestock wastes, agro-industrial wastes and industrial wastes Also, microalgae systems for the treatment of other wastes such as piggery effluent, the effluent from food processing factories and other agricultural wastes have been investigated Also, microalgae based system for the removal of toxic minerals such as lead, cadmium, mercury, scandium, tin, arsenic and bromine are also being developed (Abdel-Raouf et al., 2012)
Microalgae have a high potential to be applied for the treatment of nutrient rich wastewaters due to high capacity for nutrient uptake Consequently, microalgae systems can be used as post-treatment systems for the removal of nutrients from effluents treated in municipal wastewater, anaerobic wastewater, which usually contained substantial amounts of nitrogen and phosphorus in low or medium level (Ruiz-Martinez et al, 2012; Wu et al., 2014).
Microalgae wastewater treatment systems can be open or closed systems Open pond systems show some limitations due to (1) evaporation losses, (2) limited diffusion of CO2 from the atmosphere in the water, (3) unequal light distribution in the pond and (4) large space requirement (Razzak et al., 2013) Contamination of other algae species or bacteria is also likely to happen Closed systems refer to photobioreactors (PBR), in which the operating conditions can be monitored and control easily PBR allow to avoid evaporation losses, have a better control over the operational conditions, optimize the cultivation condition of biomass growth and reduce the contamination risks In photobioreactors, a much greater biomass density can be maintained than in open ponds Parameters like photobioreactor size and design, light intensity and source, depth of light penetration, technique of feeding CO2 and agitation mechanism can be adjusted to ensure the maximum growth of algae (Nicoleta Ungureanu et al 2019) Biomass production is complicated on a higher scale for this technology including (1) Relatively long treatment time or However, one of the main drawbacks is the large area requirements to achieve higher efficiencies and removal rates than conventional systems such as activated sludge (2) Difficult separation of algae with treated wastewater (3) Reduced performance under bacterial contamination and zooplankton predation
The major factors influencing the microalgae growth and nutrient removal efficiency are not only characteristic of microalgae species but also mainly impact by operating condition including light intensity and cycle, temperature, pH, nutrient concentrations, oxygen and carbon dioxide concentrations, mixing condition, presence of toxic chemicals and predator species.
Microalgal-bacterial (MABA) processes have also been extensively studied and they refer to “bio-flocculant of algal-bacterial” or aggregates of bacteria, microalgae and/or cyanobacteria They are a sustainable and cost-effective alternative to conventional wastewater technologies, with cost-free oxygenation potential and effective nutrient removal This is achieved through direct or indirect symbiotic interactions between microalgae and bacteria, which can accelerate the microalgal growth rates coupled to enhanced wastewater removal efficiency, boost the carbohydrates and lipid content in microalgae, promote microalgal flocculation processes, and facilitate microalgal cell wall disruption (Ramanan et al., 2016; Higgins et al., 2018) In addition, symbiotic microorganisms help their algal hosts to cope with changing environmental conditions Apart from playing a role in enhancing microalgae production, associated bacteria can help the algae to perform more complex tasks with diverse applications (Lian et al., 2018).
The interactions between algae and bacteria are not limited to the exchange of carbon dioxide and oxygen (fig 1.) On the opposite, the interactions can be mutualism/commensalism or competition/parasitism (Fuentes et al., 2016) As a result, algae and bacteria are able to change their physiology and metabolism.Besides, bacteria also secrete micronutrient metabolites such as vitamin B12,phytohormones (IAM, abscisic acid, cytokinins, ethylene, and gibberellins),thiamine derivatives, and siderophores to accelerate microalgal metabolism and biomass growth (Ramanan et al., 2016) In MABA processes, microalgae producesO2 by photosynthesis, in the presence of light and CO2, which is used by bacteria to biodegrade the organic matter present in the wastewater In return, CO2 is produced for microalgae photosynthesis This interaction could reduce cost and energy demand from aeration and improves the efficiency of organic and inorganic substances in wastewater treatment, thus shortening the HRT required for wastewater treatment; under optimal conditions, removal efficiencies of nitrogen and phosphorus higher than 90% can be achieved (Luo et al., 2014; Alcántara et al.,2015) Furthermore, the presence of bacteria can reduce the costs related to harvesting operations as aggregation with microalgae might induce bioflocculation which reinforce the settleability of co-culture to easy harvesting biomass In previous study of Cho et al (2014) when Chlorella vulgaris was cultivated with four different bacteria, maximum algal growth rate and final cell mass increased from 0.22 day -1 to 0.47 day -1 and from 1.3 g L -1 to 3.31 g L -1 respectively This increased growth was furthermore accompanied by a slight rise in algal lipid content from 22.4% to 28%.
Figure 2.1Schematic representation of microalgae-bacteria interactions and exchanged molecules during wastewater treatment (Ferro, 2019)
Other negative effects of microalgae on bacteria are the increase in pH due to the photosynthetic activity and high dissolved oxygen concentration The fast growth rate of microalgae in dominant condition can create a high density in the culture that led to the increase of dark zones, in which microalgae can perform respiration and diminish the amount of oxygen for bacteria (Munoz and Guieysse, 2006) On the other hand, both cultures can also have negative effects on each other For example,the photosynthesis will induce an increase in pH and temperature of the aquatic media, which can have negative effects on bacterial growth (Lian et al., 2018).According to Guieysse et al (2002), due to the growth of microalgae is slower than heterotrophic bacteria so that pollutant removal was limited by O2 production.
Furthermore, it has been reported that bacteria can excrete metabolites presenting an algicidal effect which inhibit the growth of microalgae.
The interactions between microalgae and bacteria are more complex than a simple nutrients exchange The complexity of these interactions needs to be understood in order to maximize the positive effects to develop culture conditions that enhance wastewater treatment.
However, numerous interactions occur between the bacterial and the algal communities and understanding all the effects is complex, despite all the studies conducted on the topic The main advantages of combining microalgae and bacteria to treat wastewater are (1) the fact that aeration is not necessary, as microalgae and bacteria exchange CO2 and O2 through their metabolic activities, thus reducing the costs of the treatment plant, and (2) the CO2 fixation by the microalgae, which reduce the greenhouse gas emission occuring in activated sludge tanks.
Figure 2.2Microalgae-based technology for aerobic wastewater treatment comparing with conventional activated sludge (Tiron et al, 2017)
Economic and ecologic advantages that could be obtained by implementing activated algae-based technology in comparison with the conventional activated sludge process Aerobic processes could occur in the activated algae system without mechanical aerations means as the oxygen is generated through photosynthesis processes, released CO2 during wastewater treatment is used in photosynthesis by microalgae, and residual microalgae biomass could be used in a wide range of applications In comparison, aerobic wastewater treatment using activated sludge requires a significant amount of energy for oxygen generation, causes the release of the greenhouse gas such as CO2, and residual activated sludge is limited to recover for other purpose (such as fertilizer) due standard for reusing.
2.1.3 Wastewater treatment by co-culture
The main difference between an algal system and a microalgal-bacterial consortium in terms of nitrogen removal is the removal pathways In algal systems, assimilation into the biomass and ammonium volatilization due to pH fluctuations are the two main removal mechanisms In microalgal-bacterial consortia these are not the only removal mechanisms, but another important pathway of nitrogen removal is nitrification and denitrification, as nitrifiers can make use of the oxygen produced by the microalgae The exchange of oxygen and carbon dioxide allows the efficient removal of organic matter and nitrogen by heterotrophic and nitrifying bacteria. Furthermore, open and closed photobioreactors contain dark zones in which anoxic conditions allow denitrification by anoxic heterotrophic (denitrifying) bacteria.
Phosphorus can be removed from the water either by chemical or microbiological mechanisms Like nitrogen, phosphorus is an essential nutrient for microalgae.Phosphorus is taken up by algae preferably in the forms of H2PO4 - and HPO4 2- , and incorporated into the cell through phosphorylation (transformation into high energy organic compounds) (Martinez et al., 1999) However, there is no a clear description in the literature about how the phosphorous removal is achieved in waste stabilization ponds, as the reasons are not well understood (Powell et al.,2009) The chemical mechanism of phosphorus removal is through precipitation.
This mechanism depends on the pH and the dissolved oxygen concentration in the bulk liquid At high pH and dissolved oxygen concentrations, phosphorus will precipitate (Cai et al., 2013) Phosphorus assimilation is the main biological mechanism of removal in algal systems Di Termini et al (2011) achieved phosphorus removal between 80 - 90% in outdoor and indoor closed photobioreactors through microalgae assimilation.
In conventional wastewater treatment plants, organic matter is used as carbon source by heterotrophic bacteria for their growth Microalgae are considered as photoautotrophic organisms, because they use light as energy source and carbon dioxide as carbon source for their growth Therefore, the COD removal in a co- culture system would be considered as the responsibility of the bacteria However, it has been shown that some species of microalgae can adapt to diverse environmental conditions and use organic matter as carbon source for their growth Numerous studies have investigated the COD removal efficiency of co-cultures to know more about the roles of both microalgae and bacteria Tiron et al (2015) could reach high removal efficiency of chemical oxygen demand (86-98%), by applying sequencing batch mode of 24 hours cycle in PBR and light intensity of 235 umol/m 2 /s Tricolici et al (2014) obtained 95 and 78% of COD removal efficiency, for 360 and 820 umol/m 2 /s light intensity values, respectively Su et al (2012) obtained a COD removal efficiency of similar values (95%) for different microalgae bacteria ratio and concluded that this parameter does not influence the COD removal efficiency. Only algae showed a COD removal efficiency of (66.0% - 6:0), while only activated sludge (73.6% - 5:1), proving the potential of co-culture water treatment compared to only activated sludge or microalgal treatment Furthermore, in study of Zhu et al.(2019) treated low COD/N ratio (4.3) wastewater and had as results COD removal efficiencies of 83.8%, 79.7% and 82.7% in activated sludge,C vulgarisand the co- culture systems, respectively According to study of Huang et al (2015) removed96.1% and 95.2% in his illuminated and not illuminated reactors, respectively.Overall, in the co-culture systems, COD removal efficiency can reach usually higher than 80%, depending on the operating conditions, initial biomass concentration and COD concentrations Although organic matter may be used either by aerobic bacteria or by heterotrophic metabolism of some mixotrophic microalgae, activated sludge seems to play a much more important role in COD removal, as the presence of microalgae in the treatment process seems to not significantly change the COD removal efficiency.
2.1.4 Co-culture systems and configurations in wastewater treatment
Microalgae – Bacteria flocculation
Bio-flocculation represents an environmentally friendly harvesting technique comprising microalgae cells aggregation with different microorganisms, such as bacteria, autoflocculating microalgae and filamentous fungi Therefore, use of microalgae species in wastewater treatment processes leads to an increase in microalgae recovery efficiency by developing the microalgae-bacteria flocs For instance, a study observed the microalgae-bacteria flocs used for wastewater treatment exhibited a settling rate of 0.28–0.42 m/h (de Godos et al., 2014).
Bio-flocculation has been developed as a cost-effective and environment-friendly method to harvest multiple microalgae When being appeared together in one co- culture, microalgae and other microorganism can secret extracellular polymer compounds such as polysaccharides and proteins to form flocculation This phenomenon is called “bio-flocculation” (Ndikubwimana et al., 2015; Nie et al., 2011) During flocculation, sizes of the floc cells are increased by aggregation of cells through flocculation process that can enhance the settling rate or flotation. However, bio-flocculation is currently not widely applied in existing harvesting processes because it remains challenging to control bio-flocculation at industrial scale and high production cost of bio-flocculants An ideal flocculant should be inexpensive, nontoxic and effective at low concentrations and it should preferably be derived from non-fossil fuel sources, thus being sustainable and renewable. Recently, several novel and original approaches for microalgae harvesting using bio-flocculation and self-flocculation have been explored, such as:
- “bacterial floc” from one agal species and a consortium of bacteria originating from activated sludge (Su et al., 2012; Medina, 2006).
- “activated algae” from several algal species and several bacterial species(Doran and Boyle, 1979; Tiron et al, 2015);
- Self-aggregation of several cells of one microalgal species, also termed colonial microalgae, such as Scenedesmus sp (Jung et al., 2014), Pediastrum sp (Park et al., 2011b) and Phormidium sp (Olguín, 2003);
- Flocculation of microalgae sp with fungi (Zhou et al., 2012; Zhang and
- Flocculation was made of produced algae and bioflocculant-producing bacterial species Some bacterial species likeBacillussp (Ndikubwimana et al., 2014), Rhodococcus sp (Peng et al., 2014), Solibacillus sp (Wan et al., 2013), andPseudomonas sp (Liu et al., 2016) have been reported to produce bioflocculants, and applied to harvest algae.
- Flocculation of a non-flocculating microalga species with a second self- aggregating microalgal species, it is also known as auto-floculation (Salim et al., 2012);
- Flocculation of microalgae by bioflocculants, e.g Chitosan (Lersutthiwong et al., 2009), guar gum (Banerjee et al., 2014), untreated corn stover (Guo et al., 2017), etc The organic flocculants are reported to give an advantage in terms of lesser sensitivity to pH, non-toxic nature and wide range of applications and requirement of lower dosages for flocculation process.
Although sometimes bio-flocculation and auto-flocculation can be used interchangeably, actually they are two different phenomena Auto- flocculation often occurs spontaneously in microalgal cultures as a result of a pH increases due to photosynthetic CO2 depletion Autoflocculation is associated with the formation of calcium or magnesium precipitates (Vandamme et al., 2013).
The presence of bacteria in microalgal cultures improves the flocculation of suspended algae Some studies have reported the improvement in the settling characteristics of the biomass in microalgal-bacterial cultures through the formation of granules or aggregates (Gutzeit et al., 2005; Lee et al., 2013; Van Den Hende et al., 2014) The formation of flocs in an algal-bacterial consortium is promoted by the bacterial exopolymers, increasing the aggregation and stabilizing the already existing aggregates, while increasing settleability (Subashchandrabose et al., 2011). Algal-bacterial flocs vary from 50 μm to 1 mm, but the predominant size is between
400 - 800 μm (Gutzeit et al., 2005) Tiron et al (2017) reported the development of granules or as the author calls them ”activated algae flocs”, for this already formed algal flocs and the bacterial population already present in the raw dairy wastewater were used as inoculum The developed activated algae granules had a size between
600 – 2000 μm, and a settling velocity of 21.6 (} 0.9) m h-1 (Tiron et al., 2017). Figure 2.2 presents an example of an activated algae granule This positive effect tackles one of the drawbacks of solely algal systems: efficient biomass harvesting. Tiron et al (2017) the formation of the granules was achieved in a 1.5 L photobioreactor operated as sequencing batch using diluted pretreated dairy wastewater (15.3 – 21.8 mg NH4 +-N L-1) with an HRT between 96 - 24 hours.
In general, flocculation of suspensions of particles, such as microalgae, can be attributed to five common mechanisms: (1) charge neutralization (negative surface charge is cancelled by positively charged ions or polymers), (2) electrostatic patch mechanism (charged polymer binds to a particle of opposite charge), (3) bridging (polymers or charged colloids binds to the surface of two particles), (4) sweeping flocculation (particles are entrapped in a massive precipitation of minerals) (Vandamme et al., 2013) Flocculation is a complex process influenced by cell surface properties (species, culture conditions and growth phase), cell concentration, pH of the environment, ionic strength, and type and dosage of flocculant (Sayano et al., 2013) Crucial for flocculation efficiency is also mixing, which defines the number and intensity of collisions, enabling floc formation and influencing its properties (Gregory, 2005) Smaller species have higher specific surface areas thus requiring a higher flocculants dose per biomass weight (Schenk et al., 2008) In addition, flocculation is affected by the composition of the culture medium In accordance to Salehizadeh et al., (2000) and Rawat et al., (2011), the bio- flocculation strongly effected by secretion of EPS and γ - glutamic acids from living cells of microorganisms, microbial flocculation process is being formed EPS plays a key role in the formation of flocs and maintaining floc structures (Sheng and Yu, 2006), which significantly affects the separation of solids and liquids and further influences the efficiency of wastewater treatment In the bio-flocculant applications for water treatment processes, plenty of bio-flocculants have been produced from various EPS, which are responsible for contacting cells to cells without cell stress or lysis over a period of time between microalgae and bacteria (Lee et al., 2009). According to Cai et al (2018), high EPS with high PN/PS ratio enhance the flocculation of microalgae Excretion of EPS is generally higher under nutrient stress, which is also enhancing lipid accumulation, nutrient removal and settlement of microalgae–bacterial consortium (Xu et al., 2015) The pH influences the charge of not only the microalgal cell surface but also of EPS production (AngelAMlincy et al., 2017), the mean flocs size increases with a decrease in pH, and the flocs become much weaker at a higher pH value Moreover, from the study of Lee et al., (2013), the present of microalgae-associated bacteria, such as Flavobacterium, Terrimonas, and Sphingobacterium which secrete high EPS concentration, play an important role to enhance flocculation Under a certain hydrodynamic condition, the formed flocs are strong and similar in size, as the weaker flocs would break and re- flocculate until they are strong enough to resist the shear stress, i.e., turbulence can promote bioflocculation by increasing the collision frequency and shear stress (by stirring) promoted microbialcells aggregation (Henriques and Love, 2007; Xu et al., 2015; Tiron et al., 2017)
The settleability of the algal-bacterial flocs is affected by the calcium concentration of the biomass, hydrophobicity and filament index representing for the filamentous microorganism,which is considered as a backbone in order to improve the floc strength (Medina, 2006; Jenkins et al., 2004) In addition, there are many operational parameters, including the influent composition, hydraulic retention time,sludge retention time and predators to determine the floc structure (Jenkins et al.,2004; Medina, 2006) It is noticeable that bacteria assists to favor aggregation due to its hydrophobic characteristic under starvation condition (Bossier and Verstraete, 1996) Stronger flocs are able to form when sludge retention time increased, resulting in rising cell age in activated sludge (Tchobanoglous, 2003)
Even though bio-flocculation exhibits obvious advantages in eliminating the need for chemical flocculants, which represent an expensive, toxic alternative solution for water treatment, this mechanism still has some disadvantages (Barros et al., 2015).Flocculation by using microbes can help prevent chemical impurities, but simultaneously contain the risk of microbiological contamination, interfering with the food supply of algae biomass (Vandamme et al., 2013)
Activated Microalgae granules
2.3.1 Characteristics of Activated Microalgae granules (AMGs)
Activated Microalgae granules had much more advantage than most of the representative algae species used for wastewater treatment processes Moreover, the use of activated algae granules proved to have several advantages besides the conventional microalgae flocs: it significantly improves the settleability; ensures recovery efficiency of the microalgae from effluent; for wastewater treatment without additional costs for in the mechanical aeration systems, harvesting step and reducing the time of the process.
Figure 2.3 Microalgae-bacteria granular formation Regarding to previous studies, the AMGs have been modified as a spherical granules including microbial cells (bacteria), target microalgae, filamentous microorganism, inert particles (metals like Ca, Mg, Fe) and extracellular polymeric substances (EPS) in a tangled, dense, globular structure According to Cai et al. (2019), some non-algae species, especially vorticella and rotifers were also noticed to attach/adhere to the surface of the granules, indicating a rich biological population in the granules Among other particles, an aqueous matrix of EPS play a crucial role in the granulation which allowed various microbial species to form stable aggregates (Ahmad et al., 2017; Huang et al., 2015a; Liu et al., 2017).Following Tiron et al studies (2015, 2017), the free target microalgae cells were captured in a dense network of gelatinous matrix of filamentous microalgae, in which the presence of filaments acted as a biological support for both the microalgae and bacteria cells, promoting the development of these species inside granules Depending on spherical dense structure and aggregation of microalgae and bacteria biomass, which promoted high wastewater treatment performance of AMGs have specific characteristic of aerobic sludge in outer part and anaerobic sludge in inner part so nitrogen could be easily removed if oxygen diffusion is limited or diameter of granule is large enough Hereafter there were two conditions of aerobic granules Anaerobic condition was in central core and aerobic condition in outer part (figure 2.3).
The symbiotic relationship between bacteria and microalgae inside the granular structure contributed to: (1) proficient removal of organic matter, no need for air supply with oxygen & CO2 provided through photosynthesis of microalgae and respiration of bacteria; (2) the mostly completed removal of nutrients, with the performance promoted through cell assimilation and nitrification with simultaneous denitrification processes; and (3) the simultaneous occurrence of nitrification and denitrification sustained by the variation of oxygen saturation during the conducted treatment batches (light phase and dark phase circulation) and possibly by the dense granular structure.
As above mentioned, dispersed microalgal biomass settles slowly due to well- identified factors: (a) their small cell size (ranging between 5 and 50 μm), (b) the relatively low cell density achieved in wastewater treatment processes (in the order of 0.4–0.5 kg m−3 of dry biomass), and (c) the negative charge of microalgal cell surface that prevents aggregation of microalgal cells in suspension (Molina-Grima et al., 2003; Uduman et al., 2010) In the case of wastewater treatment, bacteria play a key role in the removal of organic matter and it has been observed that they might produce extracellular polymeric substances (EPS) that mediate their aggregation with microalgae and cyanobacteria (De Schryver et al., 2008) The resultingMABAs present sizes ranging from 100 to 5000 μm, depending on the operating conditions (Arcila and Buitrón, 2016; Gutzeit et al., 2005; Lee et al., 2013; Tiron et al., 2015; Van Den Hende et al., 2016) Such increase of up to three magnitude orders in the size of biomass particles improves dramatically the harvesting without requiring the addition of flocculants as in the case of dispersed biomass systems(Gutzeit et al., 2005) The presence of other organisms such as protozoa and small metazoans in the surroundings of MABAs has been consistently observed during wastewater treatment (De Schryver et al., 2008; Gutiérrez et al., 2016) (Fig 2) De Schryver et al (2008) proposed that protozoa and metazoa play an important role in the size of MABAs as they are associated with bacterial grazing from the surface of the aggregates.
There are 3 methods to create algal - activated sludge granules The first method is using the activated sludge and then developing the aerobic granular seeds After that, aerobic granular seeds were cultivated under artificial light or natural sunlight to develop the untargeted natural algal growth on aerobic granules (Huang et al., 2015; Zhao et al., 2018; Zhang et al., 2018) In he second method, activated microalgae granular formed by self–coagulation between algae and bacteria The detailed mechanism of establishing the algal-bacterial aerobic granular sludge was described in the research of Zhang et al (2020), Zhang et al (2018), He et al (2018), and NuramkhAMn et al (2019) and can be consolidated as following three stages based on the morphology, particle size distribution, and biomass retention of granules.
(1) Adaptation stage: during the initial day algae can grow quickly due to light illumination from natural sunlight or artificial source and compete against the bacteria in activated sludge because of its higher nutrients uptake potential and faster growth rate During this period, there are no obvious filamentous microorganism Exposure to light sources induces algae to grow naturally and then adhere onto to the surface of flocculent sludge due to Extracellular polymeric substances (EPS) bridging In response to the co-existence of algae cells, the bacterial flocs is easily broken with small pellets or flocs generated although moreEPS are excreted to keep its structure Bacteria adhere onto the algae to create bacteria and algae flocs adsorbed onto the aggregation surface If the aerobic granules contain abundant filamentous bacteria whose binding functions are responsible for enhancing the formation of granular consortia, in AMGs filamentous microalgae will grow, occur on the surface, and create a space for the algae to initiate and grow naturally.
(2) Maturation stage: with the stable growth of algae and further increase of EPS secretion, the algal-bacterial AGS becomes mature with a dense and compact structure, demonstrating stably efficient nutrients removal and excellent settleability. This maturation process may be promoted by the co-existence of filamentous bacteria and algae combining with the self-metabolism of microbes, stabilization of EPS content.
(3) Maintenance stage: the attachment and detachment of the algal and bacterial biomass are in balance The biomass concentration continued to increase at a slower rate during the maintenance phase, a period that allowed viable microorganisms to complement the enzymes to resynthesize essential metabolites and adjust to the conditions (Ni et al., 2009).
Main challenges for activated microalgae granular in application is its long-term stability affected by several factors such as carbon to nitrogen (C/N), aeration intensity and the time duration for aeration (totally referred to as hydrodynamic shear force), and organic loading rate (He et al., 2018) a) Hydrodynamic shear force
The hydrodynamic shear force leading to the shear stress which determine shaped and the diversity and structure of dominant microbial community regulation, patent of EPS in microbial, briefly, reduced agitation intensity with increased time led to higher microbial richness, lower diversity and evenness, and shifts of predominant microorganisms (He et al., 2018) Under appropriate hydrodynamic shear force results in stronger and more compact granules to form a heterogeneous A porous and weaker granules is formed when the shear force is too weak, not enough hydrodynamic shear stress (Wu et al., 2015) On the other hand, induced hydrodynamic shear force in reactor is closely related to the removal performance,especially for the nitrification and denitrification processes Sufficient shear stress leads to reliable ammonia oxidizing efficiencies, while limited hydrodynamic shear stress might lead to insufficient nitrification efficiency but better denitrification process (Zhang et al., 2018) A significant results of hydrodynamic shear force could drive the formation of granules in term of structures, integrating styles of species, as well as the metabolism pathways of substrate was found via several studies (He et al., 2018; Zhang et al., 2018) Besides,not only agitation or aeration caused hydraulic pressure, the settling time together with SBR cycle lengths induced hydraulic selection pressure It has been reported that long settling and cycle periods induced minimal hydraulic selection pressure and hence did not promote the propagation of microbial granules, while too short settling times resulted in washout of microbes and small granules hence no granulation (Liu et al., 2015) Therefore, it is necessary to evaluate the mechanism of hydraulic pressure control to understand clearly its influence to granulation which led to ensure the formation of granules with targeted characteristic. b) Extacellular polymeric substances
EPS play a critical role in the construction and maintenance of activated granular. The profiles of contents and compositions of EPS in terms of the PN, PS reflect the hydrophobicity of granules (in ratio of PN/PS) which the increasing of hydrophobicity led to the stronger structure of granules, especially PN In the granulation process, PS might act as a contributor to the strong and sticky formation of algal-bacterial granular consortia (Adav et al., 2008; Show et al., 2012 and Sarma et al., 2017) Whereas, PN has long been considered as a major contributor for enhancing the linkage of neighboring microbial cells and attraction of organic and inorganic materials in the granulation process (Adav et al., 2008). However, the content of EPS will be kept relatively stable, implying the steady state of the activated granules under different operational condition (He et al., 2017). Besides, if there are any change on operational condition such as hydrodynamic shear force, loading rate, etc the EPS content will contribute to keep the stable of granulation process as a responsive mechanism. d) Organic and nutrient loading rate
Nutrient removal is also thought to be linked to changes in the algal–bacterial population in terms of competition for nutrients or symbiosis between algae and bacteria (Zhang et al., 2018) In addition to the activity of the PAOs, phosphorus can be assimilated by algae, preferably in the form of H2PO4– and HPO42–, and incorporated into the algal cell through phosphorylation (transformation into high energy organic compounds) Zamalloa et al also suggested that phosphorus accumulation into the biomass was the main removal mechanism for its removal, which accounted for approximately 95% of the total phosphorus removal from the influent High nutrient removal efficiency of phosphorus was obtained and ranged from 50% to 90% (Ji et al., 2021; Zhao et al., 2019; Zhang et al., 2019) Ahmad et al., 2017 also recorded greater green microalgal-bacterial granules than initial operation with phosphorus concentration 20 mg/L Therefore, the microalgal- bacterial granules system could be an excellent technology for phosphorus removal with stable development of granules.
Although ammonium is the preferred form of nitrogen for algal biomass growth,nitrate assimilation was enhanced during algal–bacterial granulation This is probably due to the increase in particle size as well as the enlarged anaerobic zone.Therefore, it seems that the mechanisms for nitrogen removal in the algal–bacterial system were assimilation by the algae and bacterial biomass as well as nitrification/denitrification (Zhang et al., 2018), Moreover, Trebuch et al., 2020 also pointed out that high nutrient loading could lead to better entrance of nutrients in the granular structure and increased denitrification A high COD loading (COD/N ratio of 8) could also help the granules become bigger and more compact as Zhao et al., 2018 received dominant granules size > 2.5mm In contrast, low carbon influent might limit the growth of fast-settling bacteria in the algal-bacterial granules system but the system still quickly adapted and positively responded with the change of nutrients, which make this technology become promising in treating wastewater e) Photoperiod condition
Photoperiod is known to be one of the main factors influencing biomass production, cell composition, growth rate, and lipid content (Krzeminska et al., 2013) For the optimum biomass productivity, various studies have focused on increasing the light period during a daily photoperiod regime, for example, from the natural photoperiod (12h:12h dark light cycle) to the continuous illumination (0h:24h dark-light cycle) (Krzeminska et al., 2013, Bouterfas et al., 2006) However, there may be some practical constraints to provide sufficient light for financial reason in the lab-scale photobioreactor or capricious weather condition in the on-site algal wastewater treatment system For the reasons, we investigated effects of photoperiod including prolonged dark conditions to represent intermittent or limited illumination.
When forming AMGs, along the radius of the granules, there will be division of microorganisms including algae and different groups of bacteria, thereby leading to differences in P removal by intracellular adsorption (by microorganisms) or extracellular uptake (by EPS) of AMGS or AM aggregates Numerous studies have indicated the outperform treatment ability of AMGS with other biological technology such as ASP or AGS systems In study of (Guoet al., 2021), the mean P removal of AMGS (97.0%) was higher than that of AGS process (94.1%) during the stable operation period due to the phosphorus accumulating algae in AMGS AMGS in study of (Zhang et al., 2020) also exhibited the better performance in nutrients removal, averagely 66.21 ± 5.18% of total nitrogen (TN) and 63.96 ± 6.00% of total phosphorus (TP) in comparison to 53.36 ± 2.37% and 48.87 ± 7.79% by the conventional AGS reactor (RB), respectively However, long reaction time (e.g 3–
7 days), and intensive aeration (e.g 3 L/min) appeared to be the major barriers encountered in reported AMGS processes (Ansari, Abouhend and Park, 2019; Meng et al., 2019; Trebuchet al., 2020). f) Oxygen and carbon dioxide
AND METHOD
Overall reseach content
Parameters of analysis & measure in microalgae – activated sludge co-culture:
TSS, microscope, microalgae biomass evaluation (base on Chl-a concentration), EPS, PSD
Parameters of analysis & measure in water:
COD, TP, NH4+-N, NO3 N, NO2 N, pH, temperature, DO
Parameters of analysis & measure for investigate membrane fouling rate:EPS and TMP
The experiment work of this research was divided into 3 main experiments: (1) cultivation co-culture of Bacteria - Microalgae, (2) Cultivation of co-culture granules in PBR at different agitation speed with sequencing batch mode, (3) Evaluate the characterization of Activated microalgae Granules (AMGs)
+ Morphology development + Biomass productivity + Microalgae recovery efficiency
+ Settleability + Extracellular Polymeric Substance (EPS)
Stirred PBR with co-culture
Characterization of activated microalgae granules
Characteristics Evaluation of treatment performance
Co-culture granulation under Variation of Agitation Speed (rpm): 80, 120, 160, 200
Co-culture of Microalgae:Bacteria
Microbial and synthetic wastewater
3.2.1 Microalgae and symbiotic bacteria strain
The selected microalgae strain was Chlorella vulgaris, based on successful studies on applying microalgae in municipal wastewater with co-existing bacteria on the removal of nutrients and organic matter, which proved its efficient biomass production and nutrients removal (more than 90%) (He et al, 2013; Cai et al., 2013;
Wu et al., 2014) Moreover, Chlorella has the potential to be used as a source of food and energy This is an attractive food source because it contains about 45% protein, 20% fat, 20% carbohydrate, 5% fiber, 10% minerals, and vitamins (Chisti, 2007) The highest lipid productivity reported in the literature is about 179 mg/L/d byChlorella sp.(Chiu et al, 2008).
The inoculum microalgae in the study were provided by Aquaculture Research Institute 2- Ministry of Agriculture and Rural Development (106 Nguyen Dinh Chieu Street, District 1, Ho Chi Minh City) and cultivated in BBM (Bold´s Basal Medium) for 18 days before mixing with seed sludge to operate following the experiment 1 Chlorellasp was cultivated in a photobioreactor (20 cm diameter, 60 cm height) under lab condition with constant temperature (25℃), continuous illumination (led light) and agitated by aeration.
Table 3.1.Component of BBM medium (Bold 1949, Bischoft & Bold 1963)
No Component Stock solution (g L -1 dH 2 O)
Addition per 1 Litre culture medium
Preparation of Culture Solution: Add chemicals and stock solutions as indicated above to 1000 ml of bidistilled water The final pH should be 6.6.
The seed sludge obtained from the wastewater treatment system (MBR tank) of the Supermarket(Ho Chi Minh City, Vietnam).The seed sludge has been adapted to the synthetic wastewater of experiment 1 (table 2.2) The biomass concentration of the seed sludge was determined prior to start-up of experiments The initial seed sludge of the experiment was 5 gSS/L.
Due to the competition for organic carbon between denitrifiers and polyphosphate- accumulating organisms (PAOs), organic carbon usually acts as a limiting factor for phosphorus release and denitrification, especially in low C/N ratio wastewaters(Zhu et al, 2018) Many wastewaters are characterized as low C:N ratios such as municipal wastewater, landfill leachate, and anaerobic digestion effluent(Gao et al, 2019), which means the nutrient removal performance is inefficient in insufficient organic carbon wastewaters This study uses synthetic wastewater quality parameters were 384 ± 20 mg L-1 of COD, Total nitrogen (TN) was present in the form of ammonium at 40 mg L-1, TP of 4 mg L-1, nitrate and nitrite were not detected The C:N:P mass ratio is about 100:10:1 for the sufficient nutrient and organic source during granulation process based on previous studies of Tiron et al. (2015, 2017) In order to minimize variability in the experiment, synthetic wastewater was prepared using distilled water The composition of the synthetic wastewater in experiment was described in table 3.2 based on the synthetic wastewater composition of Huang et al (2015) but modified to be more suitable.
Table 3.2Components of feed synthetic wastewater in experiment (1) + (2)
No The trace elements stock solution 1 mL/L
Experimental set-up and operating conditions of photobioreactors (PBR) system32
Figure 3.2Schematic diagram of photobioreactor (PBR) system
(Remarks: 1 Agitator, 2 Feeding port, 3 Lights, 4 Effluent valve, 5.1st 3-blade impeller, 6 2nd 3-blade impeller, 7 Discharge valve, 8 Wooden box, 9.
Co-culture of microalgae and activated sludge was represented by flocculation/granulation developed in a stirred photo-bioreactor with 7 L working volume columns with a height of 41.75 cm, a diameter of 17 cm, and the water level of 30 cm The effective height to diameter ratio (H/D) was 2.45 (Tiron et al., 2015; 2017) The PBRs were continuously illuminated by 3800 – 4000 lux LED lights (SMD 5050) with a cycle light:dark cycle of 12:12 (h) The agitation of each reactor was implemented by a stirring machine placed on the top consisting of two propellers (2.7 cm in diameter), 34 cm in length stirring shaft The PBR system was installed in a wooden box with a thickness of 10 mm to prevent loss of light and temperature fluctuation (maintained around 27-32 o C).
Biological inoculum used for flocculation/granulation processes was represented by
Chlorella sp mixing with aerobic activated sludge from wastewater treatment system of supermarket (in MBR tank) with initial biomass concentration 600 mg/L. The co-culture microalgae:activated sludge mixing ratio was 5:1 (%w:%w) for experiments (1) + (2) due to the higher nutrient removal efficiencies (5-40%) and biomass growth rate comparing to other inoculation ratios (Su et al, 2012).
Feeding substrate was represented by 3.5 L of synthetic wastewater (volume exchange ratio was 50%) The initial liquor’s pH value ranged between 7 and 8 to enhance the growth of organism The flocculation/granulation process was performed in sequencing batch mode, at different agitation speed of 200, 160, 120,
80 rpm in experiment (1) to determine the optimum agitation speed corresponding to optimum shear stress.
Hydraulic retention time was decreased consecutively from 72 to 48, and 24 h,depending on the time needed for almost complete COD and nutrient removal This operational adjustment was conducted in order to ensure a continuous carbon supply and to avoid biomass starvation In addition, the settling time was also decreased consecutively from 3h to 1h, and to 15 minutes, depending on settling rate and settleability of biomass which were observed by measurement The settling time was reduced in order to decrease the amount of suspended biomass by discharging with outlet which inhibits the flocculation and granulation The effluents were stored in erlenmeyer flask for water quality analysis The settling time was chosen based on the settling velocity of settled biomass, such that only flocs with a settling velocity higher than 0.36 m/h (Granados et al, 2012) was effectively retain in the reactor When matured granules formed, settling time was reduced progressively to retain only high settling granules.
Table 3.3Operating condition for experiment (1) + (2)
Operating parameters Agitation speed (rpm)
Batch cycle: gradually decrease from 72h-48h-24h (volumetric exchange ratio: 50%).The batch cycle is operated following four main phases described in table 3.4.
Table 3.4Batch cycle of SBR mode
Settling time 3h 1h 15 mins 5 mins 5 mins
Stage Stage 1 Stage 2 Stage 3 Stage 4 Stage 5
The reaction time was adjusting follow the duration of batch cycle and decrease of settling time The adjustment of settling time was implemented by the results from settleability measurement and morphology analysis When the settling time decrease, the reaction time increase contemporarily.
Analysis methods
COD, Ammonia, NO3 -N, NO2 -N, TP, MLSS, and TSS concentrations will be analyzed in accordance with standard methods (APHA, 2012).
To get information about the removal rate of COD and nutrients, these measurements will be taken during batch duration, meaning as 72h, 48h and 24h we will take one sample/day of each reactor for 3 days, 2 days and 1 day, respectively. Biomass will be collected and analyzed at the end of each batch.
In this research, biomass evaluation were conducted base on chlorophyll-a in mixture of bacteria and microalgae(Tang et al., 2018).
Chlorophyll-a concentration was used to represent the growth of algae biomass
(Hecky and Kling, 1981) Chlorophyll-a in mixture of bacteria and microalgae was extracted by acetone solution (Lee et al., 2015) 20 mL well-mixed sludge sample was taken from reactor to analyze sludge and algae characteristics The procedure have done the following:
- Firstly, the sample was centrifuged at 4000 rpm for 10 minutes.
- Secondly, the supernatant was discarded, and the residual was suspended in 20 - 40 mL again with 90% acetone solution and 0.05 g CaCO3 and then the suspension sheared by a vortex mixer for 1 min.
- Thirdly, the above suspension was stored at 4 °C for 24 h in darkness.
- Finally, the suspension was centrifuged at 4000 rpm for 10 minutes and the supernatant was used to determine Chl-a content Chl-a concentration was measured at wavelengths: 630, 645, 663, 750, 772 and 850 nm by ultraviolet spectrophotometry; 90% acetone solution was used as the blank.
Total chlorophyll-a concentration (ct, àg/L) was calculated:
Where V is the volume of each sample (L), and V1 is the volume of acetone-based extract (mL) OD is the absorbency at corresponding wavelength and σ is optical path of cuvette (cm).
Chlorophyll-a of photosynthetic bacteria (c b , àg/L) was calculated:
Chlorophyll-a of algae (c, àg/L) was calculated:
There were two types of EPS as soluble EPS and bound EPS The sample was determined EPS in term of polysaccharides (PS) and protein (PN) per milligram of VSS so MLVSS must be determined in this analysis Normally, only bound EPS was considered in microalgae and activated sludge.
The procedure to take bound EPS have done the following:
Step 1: 50 ml of suspended biomass centrifuge 4000 rpm, 20 min
Step 2: Take bound EPS + 50-100 mL of NaCl 0.9 %
Step 3: Heat at 80°C, 1h Leave it cool
Step 4: Centrifuge 4000 rpm, 20 min to separate bound EPS solution
Figure 3.3Scheme of sample preparation for bound EPS determination
The procedure for PS determination:
The procedure for PN determination:
Solution B: 1000 mL of (20 g Na2CO3 + 4 g NaOH)
Solution C: 1 mL of solution A + 50 mL of solution B
Solution D: 10 mL of Folin-Ciocalteu phenol reagent + 10 mL of deionized water
To evaluate the performance of an co-culture settleability, it is essential to quantify the settling behaviour of the activated flocs in the system, TSS concentration is an useful parameter to determine the settleability beside settling rate of the biomass.According to Valigore et al (2012),
Before the settling phase, 250 mL of homogenous sample will be transferred into a graduated cylinder (250 mL, 0.23 m height) The activated algae aggregates will be allowed to settle and the biomass position in the cylinder will be recorded during the settling time The settling velocity of the activated algae aggregates is calculated as follows: where Srepresents the settling velocity of the activated algae aggregates (m/h) and
P is the height (m) crossed by biomass between initial time (T 0 ) (h) and final time (T f ) (h) The occurrence of this measurement will depend on the speed of the granulation process but will start on a basis of once a week.
Granule shape, size and color
Flocs/granules development was observed and determined by microscope Olympus DF Plan 4X, 10X, 40X and 100X This microscope had maximum magnification of 100x.We will be able to observe the shape, size and color of the formed granules using this type of microscope.
For more detail on size of flocs and granule, the size distribution of the target microalgae cells, activated algae flocs and activated algae granules size will be measured using analyzer Mastersizer, based on the laser diffraction method.
Figure 3.4Flow profile of a fluid in PBR shows that the fluid acts in layers that slide over one another (turbulent flow) Co-culture of microalgae-bacteria is non-Newtonian fluid due to its viscosity changes following cultivation time (Yatirajulaet al., 2019)
Shear stress exposed to the microalgae can be determined with (Wang and Lan, 2018): τ = γ˙.η (9) where τ is the shear stress (Pa), γ˙ is the shear rate (s −1 ), and η is the apparent viscosity (Pa s).
Agitation is commonly expressed by revolutions per minute (rpm) (Hodaifa, Martínez and Sánchez, 2010) or tip speed (m s -1 ) (Leupold et al., 2013) Tip speed is determined by
Utip= (2π r n)/60 (10) where n is the rotation rate in rpm and 60 is used to convert rpm to rps, r is the radius of the stirring apparatus (m)
In PBR, system, turbulent flow occurs when the Reynolds number greater than 10,000 (Metcalf and Eddy, 2014) In addition, Reynolds number is higher than a critical value of approximately 2,400, flow is turbulent (Avila et al., 2011) where the Reynolds number is defined as:
According to (Hall and Stephen, 2012) and (Metcalf and Eddy, 2014), in turbulent flow, power required for rapid mixing was defined as:
P: power input of impeller (W, kg.m 2 /s 3 ) ρ: density of the liquid (kg/m 3 )
D: diameter of the impeller (m) (in this study is 0.054) n: rotation speed (rps)
K: power number for impeller, depend on blade characteristics and independent of liquid viscosity In addition, the scale or size of an impeller and the elevation of the impeller above the tank bottom have a very small effect on its power number In this study, two impellers are used on the same shaft with the ratio of distance between two impeller (S) over diameter of impeller (D): S/D = 4, a combined power number of two impeller was calculated to be approximately 166% compared to single-impeller power Therefore, the power number of two impeller in this study was chosen K combined = 0.5644 (Paul et al., 2003; Jirout & Rieger, 2011; Stephen, 2012)
Table 3.5Shear stress of 4 different agitation speeds based on above formulations
Rotation Tip speed Shear η Re P γ˙
The volume distribution of the target microalgae cells, activated algae flocs and activated algae granules size will be measured using analyzer Mastersizer, based on the laser diffraction method.
All statistical analyses were performed using the package IBM SPSS Statistics software 20 Parametric one-way and two-way analysis of variance (ANOVA) was used to examine significant differences among groups of samples and to obtain the regression analysis The significance of the ANOVA model is evidenced by the lesser p-value < 0.05, which indicated the significance at 95%.
The dissolved oxygen (DO) concentrations and pH were measured by DO meter (Hanna HI 9146, Italia) and a pH meter (Hanna HI 9813-6, Italia) Light intensity will be measure by submersible spherical light sensor (US-SQS/L, ULM-500, FA Walz, Germany).
AND DISCUSSION
Treatment performance
Figure 4.1a) COD removal performance b) Remaining COD concentration in effluent of 4 different agitation speeds during the whole experiment
Fig 4.1a showed that the AMGs COD removal ability in all 4 agitation speeds although there were fluctuations during the whole experiment, mean removal efficiency was always above 90%, which suggests that the combination of microalgae and AS (a.k.a activated microalgae) creates a potential system for the treatment of organic pollutants The one-way ANOVA statistical values showed that the effect of different agitation speeds on the COD treatment capacity of activated microalgae (AM) in both aggregates and granules formation was insignificant (p value > 0.05) However, when evaluated over the entire experiment (5 stages), the removal performance of AM in each stages showed the a b opposite results (p value < 0.05) Among four agitation speed, the R160 showed highest performance through 5 stages In stage 1 and stage 2 with floc form, the ability of AM to remove COD in 4 agitation speeds tends to increase gradually. Through stage 3, reducing the settling rate from 1h to 15 mins caused a slight decrease in COD treatment at all 4 PBRs Specifically, the mean COD removal efficiency of R80, R120, R160, R200 in stage 2 is 94.75%, 93.33%, 94.67%, 92.67% all reduced to 87.63%, 90.08%, 89.5%, 91.06% in stage 3, respectively In the co-culture system of microalgae and bacteria, simultaneous removal of COD and nutrients in a single reactor is enabled if the pair of microorganisms is symbiotic In this case, nutrients are converted to biomass components of microalgae and bacteria played a key role in treating organic substances Therefore, the reduction in COD removal is caused by the washout of bacteria when settling time was decreased Biomass concentration after 15 min of settling phase also decreased in all PBRs of 4 agitation speeds (fig 4.6).
Through stages 4 and 5, after the co-culture has gradually adapted to changes in settling time, the reduction from 15 mins to 5 mins of settling no longer has a negative effect on COD treatment Also at stage 4, AMGS appeared in all 3 PBRs with medium to high agitation speeds, in order of appearance from before to later, respectively R160, R200, and R120 In conclusion, organic substances are not a key factor in the formation of AMGS as well as the agitation speed do not influence the organic removal in co-culture PBR However, as the formation of AMGs will lead to the stable organic removal efficiency over 90% Besides, one factor should be consider in stable organic removal performance of four reactor was SBR cycle In this study, four reactor was operated properly to maintain the effluent organic matter was lowest, in order to ensure COD was fully utilized by bacteria for nitrification and biomass growth The explanation was based on the purpose of making stress for microorganism in AM, lack of organic and nutrient in cultivation culture will enhance the EPS secretion, filamentous microbes growth, and enhance granulation process.
Figure 4.2Nitrogen removal performance in terms of a) NH4 removal efficiency (%) and effluent concentration (mg/L) of b) NO2and c) NO3
During 245 days of the experiment, except for some days of initial 36 days (stage 1- 2), nitrite concentration was always in the negligible value (< 1mg/L) The high
NO2 values in the first days of the experiment showed that the bacteria in the
AS inoculum were efficient in nitrification (AS was taken from the local WWTP's MBR) At stage 1, NH4 removal efficiency of the two PBRs with medium agitation speeds (R160, R200) was significantly superior to that of the two PBRs with low agitation speeds (R80, R120) Effluent concentrations of nitrite and nitrate of the 4 PBRs showed that nitrification was more effective in PBRs with medium agitation speeds in the early stages of the experiment It can be understood that higher oxygen content confused in cultivation culture due to higher speed which is leading to AS become dominant in bacterial community in 1st & 2nd stage. b c
Specifically, mean nitrate concentrations of R80, R120, R160, R200 were 2.93, 2.81, 8.01, 9.86 mg/L, respectively This suggests that high agitation speeds has the potential to promote the nitrification of AS, because shear stress generated by high agitation speed will increase turbulence for the liquid inside PBRs, thereby increasing mixing capacity and maintain enough oxygen (produced by microalgae mostly) for bacteria activity.
Through stage 2, mean NH4 removal of R80, R160, R200 all tended to increase from about 13-14% while nitrate concentration at all 4 PBRs did not change too much An uneven increase in NH4 removal capacity and nitrate concentration, accompanied by an increase in Chl-a concentration during stage 2 to end of stage 3 also confirmed that the increase in NH4 removal efficiency during this period is carried out by the uptake of microalgae, which showed that bacteria are only active in the first days, later on, the growth of microalgae gradually being dominant in the microbial community of the co-culture system of all 4 PBRs It is ensured that, higher performance of nitrification in co-culture system bring to better metabolism of microalgae and bacteria CO2 production from the decomposition of organic matter by bacteria is continuously added for photosynthesis by the microalgae leading to the formation of the required oxygen level.
Through stage 3, the mean NH4 removal efficiency of R120 increased sharply, reaching over 80% compared to 57.65% in stage 2 The NH4 removal capacity of all 4 PBRs still increased and remained stable in the range of 70-94% with a distinct partition between 2 high agitation speeds R160, R200 (>90%) and 2 low stirring speeds R120, R80 (70-85%) (statistical value of comparison also found that the high or low agitation speed groups had no difference in NH4 treatment efficiency, p- value > 0.05) At the end of stage 4, the total biomass of all 4 PBRs reached over
2500 mg/L with values ranging from high to low of R80, R120, 200, and R160.
In this stage, the higher biomass concentration & Chl-a leading to the reduce ofNH4 removal due to the self-shading of microalgae caused light penetration inhibition In other words, high agitation speeds combining with better flocculation of R160 in stage 4 & 5 help the microalgae inside PBRs increase their exposure to light, promoting microalgae in NH4 uptake The other reason could be found in this study, under low ammonium concentrations (during stage 4), (Nils, 2003; Choi et al., 2010) leading to the decrease of nutrient removal performance in stage 5 In the conclusion, a clear evidence indicated that hydraulic shear force caused by agitation has significant effect on the removal performance of AM, set the stage for the formation of granules.
Figure 4.3 a) TP removal performance b) Remaining TP concentration in effluent of 4 different agitation speeds during the whole experiment
Phosphorus removal performance also had the same trend as the COD treatment when TP removal efficiency also decreased sharply after reducing the settling phase from 1h to 15 mins and gradually increased when entering stages 4, 5 (fig 4.1b).However, during the whole experiment, TP removal efficiency was always above
85%, indicating the potential of co-culture systems in treating treatment In the study, pH was always controlled between 7-7.5, which means that phosphorus removal by precipitation (normally occurred at pH 9–11) was not sufficient in this study As discussed in the literature review, the processing of P by AM or AMGS is accomplished by two main pathways: Extracellular uptake and intracellular adsorption The amount of P removed through the extracellular uptake pathway is considered to be negligible because EPS is one of the key factors and clearly reflects the granulation process of AM The trends in EPS secretion (both bound and soluble EPS) varied continuously throughout the experiment and there was a clear difference between the granular and non-granular phases (section 4.3.2), which cannot be guaranteed P removal efficiency is always above 85% Therefore, the main pathway for P removal of AMGS in this experiment is considered to be through intracellular uptake of both microalgae and bacteria Microalgae can excessively take in phosphate-P over its actual demand and store P in the form of polyphosphates (poly-P) in cells (Solovchenko et al., 2016) However, during stage
2 to stage 3, where microalgae became dominant at all 4 PBRs, P removal efficiency decreased with the decrease of AS Since the P removal efficiency during the whole treatment has mostly the same trend with COD removal efficiency, it is highly likely that P is removed mainly by biomass accumulation of bacteria fromAS.
Microalgae activity profile
Figure 4.4Concentration of Chlorophyll a (àg/L) at various stages in the PBRs
In co-culture system of microalgae-bacteria, Chl-a is considered to be a characteristic compound that occurs only in microalgae biomass Therefore, Chl-a concentration is considered as an indirect parameter to assess the biomass growth of microalgae Chl-a concentrations in the co-culture also increased significantly during this time, from stage 2 to stage 3 (fig 4.4) Specifically, in R80, R120, R160, R200, Chl-a concentration increased from 450, 505, 863, 1018 to 3808, 7037, 5574,
6193 àg/L In other words, after reducing the settling phase from 1 h to 15 min, theChl-a concentration of different agitation speeds increased from 6-14 times, the highest increase was R120, the least increase was R200 This change shows that,compared with bacteria in AS, microalgal cells tend to agglomerate into larger flocs,which can settle rapidly within 15 minutes, avoiding being washout Total biomass inside PBRs from stage 2 to stage 3 showed a significant increase in all of 4 reactors,from about 500-700 mg/L increased to 900-1000 mg/L (fig 4.7) Therefore, the increase in total biomass is attributed to the growth of microalgae biomass.
One-way ANOVA statistics showed that different agitation speeds had an effect on Chl-a concentration (p-value < 0.05), while total biomass did not show this difference In other words, shear stress generated by mechanical agitation affects microalgae activity in co-culture, thereby causing changes in AM's granulation process During the period from stage 1-4, Chl-a concentration at 4 PBRs all increased continuously and reached over 10,000 àg/L at stage 4 Also in stage 4, there were 3 PBRs appeared AMGS in order from soonest to latest: R160 (day 165 - batch 67), R200 (day 172 - batch 70), and R120 (day 188 - batch 77) During the granulation stage, if Chl-a at R160 and R120 are approximately the same between 10,500 and 11,000 àg/L, Chl-a at R120 is 1.5 times this value, up to about 15,000 àg/L This result shows that, if the high agitation speed provides sufficient shear stress for granulation with moderate microalgal biomass, the low one is more favorable for microalgal growth and requires a longer period of time for AM to form granules Through stage 5, the sharp decrease in Chl-a concentration in all 4 PBRs, as explained in section 4.1, was due to intracellular Chl-a degradation under stress condition (high biomass concentration but maintain under low loading rate, and lack of light penetration) Specifically, this decrease was most evident in R160, the PBR with the earliest AMGS appearance with a record reduction of 64 times, Chl-a in AMGS was now only 173.2 àg/L, which indicated that AMGS formation directly affects and changes the activity of microalgae In addition, at the agitation speed without AMGS of R80, the decrease of about 7 times the concentration of Chl-a was mainly due to the effects of self-shading phenomenon, too much biomass leading to the lack of light penetration to enough for microalgae to grow according to the normal photosynthesis mechanism.
Also in this stage 5, as Fig 4.7 showed that Chl-a was decreased gradually, however,the biomass were maintained as stable concentration in four reactor Therefore, a clean evidence for self-shading created the degradation of Chl-a content in microalgae to minimum value to adapt with constant biomass concentration.Without photosynthesis, Chl-a will no longer be an important compound for microalgae to survive Indeed, granules appeared at 3 PBRs of R120, R160, R200 at stage 4 with dense and compact structure in shape (fig 4.5) Microalgae cells have the ability to reduce the amount of intracellular Chl-a on their own, which can be explained by the excessive increase in co-culture biomass, the light supply from the beginning for AMGS and microalgae growth is no longer sufficient However, instead of lysis and death, Chl-a content in the cell of microalgae was reduced upon on the lack of lighting condition In addition, the appearance of AMGS, which means that the algae cells will be distributed along with the biomass layers of the seeds, especially in the core of AMGs, are gradually more limited with light exposure Therefore, Chl-a gradually becomes a substance that does not need too much to ensure the growth of microalgae Total biomass at this time was no different from that in stage 4 (p-value > 0.05), indicating no loss in the microbial community Moreover, in this stage, even the COD removal performance was kept at high level but the nitrification performance slipped down (less nitrates and nitrites in the water), a clear results from not as much produced oxygen, in this case NH4-N removal mostly by microalgae assimilation and the competition of nitrifying bacteria with microalgae is reduced leading to the lower maximum microalgae biomass/Chl-a value (Rio-Chanona et al., 2018).
The appearance of AMGS and insufficient light when the Chl-a concentration reaches a certain value will cause microalgae change the tendency to consume nitrogen, and reduce the photosynthesis pigment, which is Chl-a in this study (Huang et al., 2019) The color of AM/AMGS at 4 PBRs was dark green (fig 4.6).
Therefore, the agitation rate control could not only balance the growth rate between microalgae and bacteria but also improve the growth advantage of microalgae But,future study should consider about the settling time effect and SBR cycle to control better biomass concentration which is leading to better growth of microalgae and quick granulation.
Figure 4.5Activated algae in form of (a) flocs R80, and AMGS (b) R120, (c) R160,
(d) R200 in day 218 under microscope x10 magnification
Figure 4.6Comparison between AM/AMGs (left) with seed microalgae (right) a b c d
Figure 4.7Total biomass concentration (mg/L) in a) mixed culture, b) Correlation between Chl-a concentration and biomass
Impact of shear stress on granulation process of AMGS
Shear stress is a function of a fluid's shear rate and viscosity, as shown in Eq (8). Shear stress was continuously imposed on cells in different stages of the life cycle in PBRs The critical shear stress level, represented as critical shear stress (Pa) or critical impeller tip speed (m/s), is the level over which microalgal cells are adversely impacted It can be used to figure out how sensitive microalgal cells are to shear stress Lower cell viability, longer recovery time, reduced cell growth rate, reduced photosynthetic activity (PA), or cell lysis are all negative consequences of shear stress on cells.
The critical shear stress of each microalgal strain is dependent on cultivation conditions, and the source of shear stress The turbulence of flow generated by eddies of varying sizes and with the decrease in their sizes, their energy content proportionally affected Agitated "shear stress" values are generated by micro- a b eddies of similar or smaller sizes than microalgal cells Micro-eddies that are equal to or smaller than cells can transmit turbulent forces - including shear force - to these cells (Camacho et al., 2001) When cells are of similar dimensions as surrounding micro-eddies, there is greater risk for cell damage Less damage occurs when the micro-eddies are larger than the cells (Hadiyanto et al., 2013).
According to Michels et al (2010), Shear stress had an adverse effect on the viability of microorganism cells with a threshold value dependent on different strains In this study, it is showed that the shear stress value more than 1.26 Pa leading to cause a sudden decrease in viability of microalgae cell based on chlorophyll concentration decrease The morphology of microalgae also change by the rule of shear value With the increase of shear stress, the moderate to form as granules structure However, it could clear find out that a the critical range of shear stress stimulation on granulation were in the range of 1.26 Pa to under 2.49 Pa. Kinetic coefficients of shear stress on size distribution in Figure 4.8 to show the relation of shear stress to formation of granules In this study, AMGs agitation was irritated by centrifugal and upside down mixing force in order to have evenly distributed biomass.
Figure 4.8Particle size distribution (PSD) of 4 PBRs in a) day 165 th (batch 67 th ), b) day 216 th (batch 90 th ) and c) linear correlation between shear stress and size distribution during the whole treatmentPSD was measured when AMGS firstly appeared in R160 (fig 4.8a) and right before ending stage 4 (fig 4.8b) While PSD at the lowest agitation speed did not show any difference, PSD at the higher one of R160, R200 showed a narrow distribution and the appearance of large particles (AM/AMGS) This figure shows that AMGS at high agitation speeds of R160, R200 is gradually uniform and growing On the other hand, the mean diameter of particles inside 4 PBRs R80, R120, R160, R200 was 178.71, 179.3, 326.97, 303.53 àm at batch 67th Except R200 increased to 339.1 àm, mean particle size of R80, R120, R160 then decreased to 145.98, 160.08, 259.56 àm at batch 90th, respectively After the AMGS was formed, the decrease in mean diameter showed a tighter structure, while the size increase of the AMGS at higher agitation speed showed more potential for graining growth than the present size The size of microalgal cells relative to micro-eddies caused by turbulent flow can play a role in cell damage by shear stress Therefore, at high shear stress, microorganisms show a greater tendency to cluster and join together to reduce their resistance alone to environmental physical stress, reducing cell damage Since then, with the gradual increase of shear stress, the particle size tends to increase gradually Although shear stress is considered as an essential physical stress for granulation, excessive shear stress poses a high risk of imbalance and disruption of grain structure This indicated that the stability of AMGs was likely destroyed due to blocked growth of microalgae and bacteria under lower shear stress and lack of light penetration, same results with Wang et al (2021) study on the interaction microalgal-bacterial granular.
As can be seen from figure 4.8c, although initially AM floc at R80, R120 with lower shear stress (0.04 – 0.15 Pa) were large, the linear representation of AM size nearly parallel in these two PBRs indicated their decrease over time at equal rates.
In R160, R200 with high shear stress (0.35-0.69 Pa), AM started with small sized flocs, then gradually increased in size After the AMG was formed, the decrease in mean diameter showed a tighter structure, while the size increase of the AMG at higher agitation speed showed more potential for graining growth than the present size In this study, the stable AMGs size of R160 & R200 ranged from 400 um to
600 um with shaped structure and of R120 ranged from 200 um to 400 um with un- shaped structure (day 218, Fig 4.12) Comparing between the structures and sizing of different agitation speed PBR, a clear influence of shear stress value on shape,structure, and size of AMGs In R160 with stable granules, it has round shape,dense structure with clear biomass layer including the core was bacteria and surrounded by dense target Chlorella cells, the EPS bridge is believed to be the connection between microbes and microalgae, supporting with the filamentous microalgae network to form granules Sizing of granules in R160 were uniform and even (Fig 4.11j).
Figure 4.9 Settling rate (m/h) of AM/AMGs during the experiment
The settling rate of AM during the stage 1-3 were low which only ranged from 2.5 m/h – 5 m/h However, when the flocculation and granules formation occurred during stage
4 & 5, a significant high settling rate were observed, especially in R160, the granules was form firstly and promote the highest settling rate compared to the others The shear stress did not have any significant effect on settling rate but it stimulate the attachment of filamentous microalgae, microalgae cells, bacteria cells into one big structure and promote the settleability of AM/AMGs In the case of R80, even there was not any granules formation but dense flocs of AM could make it have high settling rate comparing to the other reactor which formed granules.
The PN/PS ratio of all 4 PBRs had many changes over different periods Specifically, if in stage 1, PN/PS in R80, R120, R160, R200 rose with the highest values of 11.08,6.64, 5.5, they reduced to 2.83, 2.19, 1.66, and 1.61 respectively After day 47 th , thePN/PS ratio gradually decreased and was followed by a decrease in the total EPS concentration until the end of stage 2 The increase in PS concentration suggested that bacteria became more active in the later stages due to the high metabolic activity of bacterial cells favors the production of PS rather than PN (Huang et al., 2015). Previous work by Tay et al (2001) indicated that enhanced aeration, which induced higher shear stress on granule, led to increase promotion of PS formation and enhanced granulation This was similar to the present study wherein stage 2, when big microalgal flocs were formed, PS was highest in R160 and R200, corresponding to the highest shear stress From day 62 nd onward, the number of EPS calculated per unit of biomass tended to decrease gradually due to the growth of total biomass This result indicated that microorganisms were in the process of starvation - a phenomenon in which the environment's substrate was not enough to provide for the normal growth of microorganisms Research by Li et al (2006) confirmed that during granulation process, the longer starvation forced microorganisms to use EPS as a substitute, resulting in a properly controlled EPS concentration to promote granulation.
During stage 2, when reducing HRT from 6d to 4d, although OLR was increased, the slight increase in COD treatment capacity in stage 2 compared to stage 1 indicates an increase in the organic utilization ability of AM The reduction of settling time increases the amount of washout biomass, however, the COD consumption of the AM is still high, the microbe still grows effectively, leading to the mean biomass concentration in the PBRs not having much change compared to stage I However, the production of PN, PS in the tanks in this period has many differences Specifically, in the first stage after reducing HRT (stage 2 and the initial days of stage 3), the PN/PS ratio of all 4 PBRs rise with the highest values in R80, R120, R160, R200 are 11.08, 6.64, 5.5, 4.32 respectively In a study on sludge only (Yang et al., 2018), they demonstrated that the ratio of PN/PS was significantly affected by the nutrient substances, and supported the viewpoint that bacteria tended to convert excess carbon substrates into intracellular storage compounds, like PS, print EPS at high OLRs;whereas at low OLRs, the majority of the carbon source was used in biomass synthesis instead of PS production This contrasts with the results in this present study when
HRT was reduced from 6d to 4d, (OLR increased by 1.5 times), EPS secretion shows the dominance of proteins (PN) over polysaccharides (PS), which is similar to the results from the study of microalgae only (Sawayamaet al., 1992), (Zeng et al., 2013). Chlorella sp grown in the wastewater with high nitrogen concentration and P-limited condition produced a large number of protein-rich EPS due to nitrogen over-uptake (Wang et al., 2014) Since PS is a bio glue, facilitating cell-to-cell interaction and further strengthening microbial structure in granules (Liu, Liu and Tay, 2004) and PN surrounding the granule generate cell walls with high surface hydrophobicity, which is considered as a triggering force for granulation (Liuet al., 2004), the trend of increased
PN secretion and high ratio of PN/PS in pools were indicated favored the granulation of AM (Caiet al., 2019b), (Huanget al., 2015), (Ahmadet al., 2017) However, also in the above studies, the PN/PS ratio often fluctuated between 2-3.5 within 100 days (Huang et al., 2015) or approx 7 at day 60 th (Cai et al., 2019a), and actually those
PN/PS values do not change too much during the whole operation Therefore, the rather high PN/PS ratio at R80 (11.08) indicates an imbalance in the activity of bacteria and microalgae, leading to the inability to form granules at a later stage.
During stage 3, the PN/PS ratios of the 4 PBRs are 2.83, 2.19, 1.66, and 1.61 respectively After day 47, the PN/PS ratio gradually decreased and was followed by a decrease in the total EPS concentration until the end of stage III The increase in PS concentration suggests that bacteria are gradually becoming more active in the later stages due to the high metabolic activity of bacterial cells favors the production of PS rather than PN (Huang et al., 2015) Previous work by (Tay, Liu and Liu,
2001)indicated that enhanced aeration, which induces higher shear stress on granule, leads to increased promotion of polysaccharide (PS) formation, which will enhance granulation This is similar to the present study wherein this stage 3, large microalgal flocs were formed, the highest PS concentration was in the PBRs with agitation speeds of 160 rpm and 200 rpm, corresponding to the highest shear stress From day 62, the number of EPS calculated per unit of biomass secreted tended to decrease gradually due to the growth of total biomass This result indicated that microorganisms are in the process of starvation - a phenomenon in which the environment's substrate is not enough to provide for the normal growth of microorganisms As they progressed to the AMGS formation, the starvation forced microorganisms to use EPS as a substitute, leading to a sharp decrease in the EPS concentration Research by (Li, Kuba and Kusuda, 2006) confirmed that during the period from the formation of the flocs to the binding of the flocs for granulation, the longer starvation promoted a significant decrease in the EPS concentrations consisting of PN and PS used by microorganisms, resulting in a properly controlled EPS concentration to promote granulation.
AND RECOMMENDATIONS
Conclusions
Thesis “Co-culture Of Microalgae And Bacteria For Wastewater Treatment Coupling With Biomass Recovery” implemented with lab-scale PBRs, shear stress provided mechanically by agitator without aeration to investigate the mechanism of activated microalgae granules formation, and at the same time evaluate the ability of activated microalgae to treat wastewater and biomass profile during granulation process Over 250 days of operation The results indicated that the mechanism of activated algae granule formation is mainly based on two factors: EPS and filamentous microalgae, which can be adjusted by agitation speed and shear stress value The lower the shear stress the more PS was needed, the higher the shear stress the more PN was required Particle size increase based on PN secretion. Activated algae granule was formed at 3 higher agitation speeds of 120, 160, 200 rpm with the largest particle size at R200 (~ 600 àm) R160 (shear stress 1.26 Pa) gives optimal results in terms of particle formation time, stable granules structure, as well as wastewater treatment efficiency.
Recommendations
In the experimental process, there are still some limitations in operation, especially related to time Based on the results of the study, the thesis makes the following recommendations:
- The control of loading rate of organic components, nutrient ratio in each batch of operations should be studied for further granulation control.
- Using real wastewater to ultilize the available source of nutrient in wastewater for granulation
- Combined research on the ability to recover algal biomass of granules for bio-product manufacturing.
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