Co culture of microalgae and bacteria for wastewater treatment coupling with biomass recovery

1

General context

Biological processes, particularly the activated sludge process (ASP), are widely used in wastewater treatment but face challenges such as the need for pre-treatment, high energy demands for aeration and stirring, and limited nutrient removal efficiency (Longo et al., 2016; Quijano et al., 2017) Traditional methods for phosphorus removal, primarily chemical and physical processes, are costly and inefficient (Cole et al., 2017) In response to these limitations, microalgae-based wastewater treatment systems have emerged as effective solutions, particularly for domestic wastewater, demonstrating significant capabilities in nitrogen and phosphorus removal while facilitating algae biomass recovery (Ruiz-Marin et al., 2010; Li et al., 2019; Chiu et al., 2015) The use of microalgae not only enhances nutrient removal through biomass assimilation but also offers an environmentally friendly, low-cost, and efficient approach to reducing atmospheric carbon dioxide levels (Benemann, 1997; Klinthong et al., 2015; Zhou et al., 2017) Moreover, converting carbon emissions into biomass using microalgae is considered a cost-effective strategy for CO2 reduction, with the resultant algal biomass serving as a valuable resource for biofuels and food products (Chisti, 2007; Chiu et al., 2015).

2015) However, widespread adoption of microalgae-based technology is constrained in terms of design, operation, and biomass harvesting.

Harvesting microalgae biomass presents significant challenges due to their small size (3-30 μm) and low settling rates, which contribute to high operational costs, estimated at 20-30% of cultivation expenses Centrifugation is the most widely used method for microalgae harvesting, offering time efficiency but requiring over 3,000 kWh of energy per ton, making it costly at an industrial scale Coagulation serves as a less energy-intensive alternative but poses issues such as contamination with metal salts and high chemical costs Recent studies suggest that gravitational sedimentation is a low-cost, energy-free harvesting method; however, it suffers from slow settling times and low biomass yield.

The integration of bacteria and microalgae in a single system has gained significant attention from researchers worldwide, aiming to treat COD and nutrients in a compact area while enhancing the settling velocity of microalgae as an alternative co-culture in wastewater management (Su, Mennerich, and Urban, 2012) The concept of "activated algae," first introduced by McGriff and McKinney in 1972, describes the flocculation process between microalgae and bacteria during wastewater treatment (McGriff and McKinney, 1972) Recent studies have tested biological remediation using activated algae in ponds and photobioreactors (PBR), highlighting their potential to address the limitations of traditional methods like activated sludge and microalgae, which include high aeration costs, significant land use, and sludge handling challenges (Boelee et al., 2014; Marcilhac et al.).

The microalgae-bacteria granular system exhibits a favorable settling rate of 21.6 ± 0.9 m/h, high biomass content, and a dense microbial structure, successfully operating in a closed Photobioreactor (PBR) (Liu et al., 2017; Tiron et al., 2015, 2017) Following the transfer of seed microalgae to the aerobic granular sludge system (AGS), microalgae-bacteria granules form, with extracellular polymers (EPS) playing a crucial role in granulation and maintaining particle structure (Liu et al., 2017; Cai et al., 2019) Additionally, the presence of filamentous microalgae and various operational parameters, such as stirring speed and retention time, significantly influence the granulation process (Tiron et al., 2015, 2017) The symbiotic relationship between microalgae and bacteria offers numerous advantages, including carbon dioxide utilization for oxygen production, reduced aeration needs, enhanced nutrient removal rates, and potential for high microalgae recovery efficiency (Tricolici et al., 2014; Tang et al., 2016; Zhu et al., 2019) Nonetheless, research has yet to thoroughly evaluate the maintenance of stable biomass concentrations and wastewater treatment performance under continuous operating conditions, particularly regarding the operational dynamics of co-culture PBRs (Quijano et al., 2017).

Granulation of activated algae is poised to become a key trend in future wastewater treatment technologies The granulation process is significantly influenced by various operational conditions, including reactor configuration, nutrient starvation, and hydraulic shear force (Zhu et al., 2008; Gao et al., 2011) These environmental factors closely relate to the growth rate and cell composition of algae (Schenk et al., 2008), which directly impact the removal efficiency of activated algae in wastewater treatment systems Consequently, further research and information are essential to enhance understanding and optimize the performance of granulated activated algae technologies.

This study investigates the co-culture of microalgae and bacteria in sequencing batch photobioreactors (PBR) using synthetic wastewater, focusing on optimizing agitation speed during granular formation and assessing the effects of organic loading rates Key characteristics of the granular include morphology, settleability, microalgae-bacteria distribution, and extracellular polymeric substances (EPS) content The research will thoroughly evaluate biomass recovery efficiency, as well as organic matter and nutrient removal The findings aim to inform the design of cost-effective, high-efficiency microalgae-based systems for wastewater treatment and algae biomass harvesting.

Objectives of the study

The topic “Co-Culture of Microalgae and Bacteria for Wastewater Coupling with

Biomass Recovery utilizes a photobioreactor (PBR) that co-cultures microalgae and bacteria within a sequencing batch reactor (SBR) to effectively treat domestic wastewater rich in nutrients like nitrogen (N), phosphorus (P), and organic carbon (C) This innovative approach not only enhances microalgae biomass production but also aims to generate valuable bio-products while significantly reducing CO2 emissions.

Research scope

This research was conducted within the laboratory with the following contents:

In a study on flocculation and granulation processes, Chlorella sp was utilized as a biological inoculum, combined with aerobic activated sludge from a supermarket's wastewater treatment system, specifically from the MBR tank The initial biomass concentration was set at 400 mg/L, with a co-culture mixing ratio of microalgae to activated sludge at 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

This article explores the impact of optimal operating parameters and innovative processes in microalgae-activated sludge systems on the development of granulation co-culture It highlights the importance of utilizing wastewater with suitable nutritional content for algae cultivation, aimed at recovering high-yield biomass with diverse potential applications Furthermore, this research lays the groundwork for future in-depth studies on promising microalgae-based technologies.

To address the growing demand for a high-quality living environment, it is essential to implement integrated green technologies that effectively reduce CO2 emissions and nutrient levels in wastewater, particularly in Vietnam, where eutrophication from industrial and agricultural runoff poses significant challenges This approach aims to identify and develop sustainable green technologies that can harness nutrients in wastewater for microalgae biomass cultivation, offering numerous profitable applications while enhancing human life quality at minimal cost.

REVIEW

Co-culture of Microalgae-Bacteria

2.1.1 Wastewater treatment using microalgae-based systems

Microalgae-based technology for wastewater treatment is gaining traction due to its numerous benefits This innovative approach effectively removes nitrogen and phosphorus through the natural photosynthetic processes of microalgae It is also cost-effective and environmentally friendly, as it eliminates the need for additional chemicals, while simultaneously generating oxygen, reducing carbon dioxide, and minimizing metal ions Moreover, the harvested microalgae biomass offers potential applications in producing food, feed, fuel, fertilizers, and pharmaceutical ingredients.

Microalgae offer a diverse range of mechanisms for effectively treating wastewater, including nutrient assimilation, nitrogen volatilization, phosphorus precipitation, and the biodegradation of organic matter They can remove ammonium through nitrification, biosorb heavy metals, and disinfect pathogens by altering pH levels (Wang et al., 2017) Utilizing wastewater as a growth medium, microalgae cultivation has numerous applications, such as producing bio-products, mitigating carbon dioxide, and facilitating bioremediation These systems are capable of treating various waste types, including human sewage, livestock and agro-industrial waste, as well as effluents from food processing and agriculture Additionally, research is underway to enhance microalgae-based systems for the removal of toxic minerals like lead, cadmium, mercury, scandium, tin, arsenic, and bromine (Abdel-Raouf et al., 2012).

Microalgae are highly effective for treating nutrient-rich wastewaters due to their superior nutrient uptake capabilities These systems can serve as post-treatment solutions for removing nitrogen and phosphorus from municipal and anaerobic wastewater effluents, which typically contain significant levels of these nutrients.

Microalgae wastewater treatment systems can be categorized into open and closed systems, with open ponds facing limitations such as evaporation losses, uneven CO2 diffusion, inconsistent light distribution, and significant space requirements Additionally, these systems are prone to contamination from other algae species and bacteria In contrast, closed systems, known as photobioreactors (PBR), offer enhanced control over operating conditions, minimize evaporation losses, and reduce contamination risks while allowing for higher biomass density Key parameters in PBR, including size, design, light intensity, CO2 feeding techniques, and agitation mechanisms, can be optimized for maximum algae growth However, scaling up biomass production presents challenges, such as extended treatment times, large area requirements for efficiency, difficulties in separating algae from treated wastewater, and decreased performance due to bacterial contamination and zooplankton predation.

Microalgae growth and nutrient removal efficiency are significantly influenced by both the specific species of microalgae and various operating conditions Key factors include light intensity and duration, temperature, pH levels, nutrient concentrations, and the presence of oxygen and carbon dioxide Additionally, mixing conditions, the presence of toxic chemicals, and predator species also play critical roles in determining the effectiveness of microalgae cultivation.

Microalgal-bacterial (MABA) processes, which involve aggregates of microalgae, bacteria, and cyanobacteria, present a sustainable and cost-effective alternative to traditional wastewater treatment methods These processes leverage the symbiotic interactions between microalgae and bacteria to enhance growth rates, improve nutrient removal efficiency, and increase the carbohydrates and lipid content in microalgae Additionally, they promote flocculation and facilitate the disruption of microalgal cell walls, allowing for better wastewater management Furthermore, symbiotic microorganisms assist microalgae in adapting to changing environmental conditions, while also enabling them to perform complex tasks for various applications.

The interactions between algae and bacteria extend beyond the simple exchange of carbon dioxide and oxygen, involving mutualism, commensalism, competition, and parasitism, which lead to physiological and metabolic changes in both organisms (Fuentes et al., 2016) Bacteria enhance microalgal metabolism and biomass growth by secreting essential micronutrient metabolites such as vitamin B12, phytohormones, thiamine derivatives, and siderophores (Ramanan et al., 2016) In Microalgae-Based Activated Biofilm Aeration (MABA) processes, microalgae produce oxygen through photosynthesis, utilizing light and CO2, which bacteria then use to biodegrade organic matter in wastewater, subsequently releasing CO2 for microalgal photosynthesis This synergistic interaction can significantly reduce costs and energy demands associated with aeration, improving the treatment efficiency of organic and inorganic substances in wastewater and achieving nitrogen and phosphorus removal efficiencies exceeding 90% under optimal conditions (Luo et al., 2014; Alcántara et al.).

The presence of bacteria in microalgae cultivation can significantly reduce harvesting costs by promoting bioflocculation, which enhances the settleability of the co-culture for easier biomass harvesting A study by Cho et al (2014) demonstrated that cultivating Chlorella vulgaris with various bacteria increased the algal growth rate from 0.22 day -1 to 0.47 day -1 and the final cell mass from 1.3 g L -1 to 3.31 g L -1 Additionally, this increased growth was associated with a modest 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)

Microalgae can negatively impact bacteria by increasing pH levels and dissolved oxygen concentrations due to their photosynthetic activity Rapid microalgal growth can lead to high culture density, creating dark zones where respiration occurs, subsequently reducing oxygen availability for bacteria (Munoz and Guieysse, 2006) Additionally, the interplay between these cultures can be detrimental; photosynthesis raises pH and temperature in aquatic environments, which may hinder bacterial growth (Lian et al., 2018) Furthermore, microalgae grow more slowly than heterotrophic bacteria, limiting pollutant removal due to insufficient oxygen production (Guieysse et al., 2002).

Furthermore, it has been reported that bacteria can excrete metabolites presenting an algicidal effect which inhibit the growth of microalgae.

The relationship between microalgae and bacteria involves intricate interactions beyond mere nutrient exchange Understanding these complexities is essential for optimizing culture conditions that improve wastewater treatment efficiency.

The interactions between bacterial and algal communities in wastewater treatment are complex, despite extensive research Combining microalgae and bacteria offers significant benefits, including the elimination of aeration needs, as these organisms naturally exchange CO2 and O2 through their metabolic processes, leading to reduced treatment plant costs Additionally, microalgae contribute to CO2 fixation, helping to lower greenhouse gas emissions from activated sludge tanks.

Figure 2.2Microalgae-based technology for aerobic wastewater treatment comparing with conventional activated sludge (Tiron et al, 2017)

Implementing activated algae-based technology offers significant economic and ecological advantages over the conventional activated sludge process In the activated algae system, aerobic processes occur naturally through photosynthesis, eliminating the need for mechanical aeration, while the CO2 released during wastewater treatment is utilized by microalgae for growth Additionally, the residual biomass from microalgae can be repurposed for various applications In contrast, traditional aerobic wastewater treatment with activated sludge demands substantial energy for oxygen production, contributes to greenhouse gas emissions like CO2, and has limited options for repurposing residual sludge, which is often restricted to specific uses such as fertilizer.

2.1.3 Wastewater treatment by co-culture

The key distinction between algal systems and microalgal-bacterial consortia in nitrogen removal lies in their mechanisms Algal systems primarily rely on biomass assimilation and ammonium volatilization due to pH changes, whereas microalgal-bacterial consortia utilize additional pathways, including nitrification and denitrification, facilitated by oxygen produced by microalgae This oxygen-carbon dioxide exchange enhances the removal of organic matter and nitrogen through the activity of heterotrophic and nitrifying bacteria Additionally, both open and closed photobioreactors feature dark zones that create anoxic conditions, promoting denitrification by anoxic heterotrophic bacteria.

Phosphorus can be effectively removed from water through both chemical and microbiological processes It serves as a vital nutrient for microalgae, which preferentially absorb phosphorus in the forms of H2PO4 - and HPO4 2- This nutrient is then incorporated into algal cells via phosphorylation, transforming it into high-energy organic compounds Despite its importance, the mechanisms of phosphorus removal in waste stabilization ponds remain poorly understood, with limited literature providing clear insights into the process.

2009) The chemical mechanism of phosphorus removal is through precipitation.

The removal of phosphorus in algal systems is primarily driven by phosphorus assimilation, which is influenced by the pH and dissolved oxygen levels in the bulk liquid High pH and dissolved oxygen concentrations facilitate the precipitation of phosphorus (Cai et al., 2013) Notably, Di Termini et al (2011) demonstrated that microalgae assimilation can achieve phosphorus removal rates of 80-90% in both outdoor and indoor closed photobioreactors.

Microalgae – Bacteria flocculation

Bio-flocculation is an eco-friendly harvesting method that involves the aggregation of microalgae cells with various microorganisms, including bacteria and filamentous fungi This technique enhances the recovery efficiency of microalgae in wastewater treatment by promoting the formation of microalgae-bacteria flocs A study by de Godos et al (2014) reported that these flocs demonstrated a settling rate of 0.28–0.42 m/h during wastewater treatment.

Bio-flocculation is an eco-friendly and cost-effective technique for harvesting various microalgae In co-cultures, microalgae and microorganisms release extracellular polymer compounds, including polysaccharides and proteins, which lead to the formation of flocs This process, known as bio-flocculation, enhances the efficiency of microalgae harvesting (Ndikubwimana et al., 2015; Nie et al.).

Flocculation enhances the settling rate or flotation by increasing the size of floc cells through aggregation Despite its potential, bio-flocculation is not widely utilized in current harvesting processes due to challenges in controlling it at an industrial scale and the high production costs of bio-flocculants An ideal flocculant should be cost-effective, non-toxic, efficient at low concentrations, and preferably sourced from sustainable, non-fossil fuel materials Recently, innovative methods for microalgae harvesting using bio-flocculation and self-flocculation have been investigated.

- “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 involves the use of algae and bacterial species that produce bioflocculants Notable bacteria such as Bacillus sp (Ndikubwimana et al., 2014), Rhodococcus sp (Peng et al., 2014), Solibacillus sp (Wan et al., 2013), and Pseudomonas sp (Liu et al., 2016) have been identified for their ability to produce bioflocculants, which are effectively utilized in the harvesting of 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);

The flocculation of microalgae can be effectively achieved using bioflocculants such as Chitosan (Lersutthiwong et al., 2009), guar gum (Banerjee et al., 2014), and untreated corn stover (Guo et al., 2017) These organic flocculants offer several advantages, including reduced sensitivity to pH levels, non-toxic characteristics, a broad range of applications, and the necessity for lower dosages during the flocculation process.

Bio-flocculation and auto-flocculation are distinct processes, despite being used interchangeably at times Auto-flocculation typically occurs spontaneously in microalgal cultures when pH levels rise due to the depletion of CO2 during photosynthesis This phenomenon is linked to the precipitation of calcium or magnesium compounds (Vandamme et al., 2013).

The incorporation of bacteria in microalgal cultures significantly enhances the flocculation of suspended algae, leading to improved settling characteristics of the biomass Research indicates that bacterial exopolymers play a crucial role in promoting floc formation within algal-bacterial consortia, which not only increases aggregation but also stabilizes existing aggregates, thereby enhancing settleability The size of algal-bacterial flocs typically ranges from 50 μm to 1 mm, with the majority falling within this spectrum.

The development of activated algae granules, ranging in size from 400 to 800 μm, was reported by Tiron et al (2017) This process utilized pre-formed algal flocs and the existing bacterial population found in raw dairy wastewater as inoculum, highlighting an innovative approach to wastewater treatment.

The study by Tiron et al (2017) highlights the successful formation of activated algae granules in a 1.5 L photobioreactor operated as a sequencing batch, utilizing diluted pretreated dairy wastewater with ammonium concentrations ranging from 15.3 to 21.8 mg NH4+-N L-1 and hydraulic retention times (HRT) between 24 to 96 hours These granules, measuring 600 to 2000 μm, exhibit a settling velocity of 21.6 ± 0.9 m h-1, significantly enhancing biomass harvesting efficiency, which addresses a major limitation of traditional algal systems.

Flocculation of microalgae suspensions involves five primary mechanisms: charge neutralization, electrostatic patching, bridging, and sweeping flocculation, which collectively facilitate the aggregation of particles (Vandamme et al., 2013) This intricate process is influenced by various factors, including cell surface characteristics, culture conditions, growth phase, cell concentration, pH, ionic strength, and flocculant type and dosage (Sayano et al., 2013) Effective mixing is essential for enhancing flocculation efficiency by increasing collision frequency and intensity, which are vital for floc formation (Gregory, 2005) Smaller microalgal species, due to their larger specific surface areas, necessitate higher doses of flocculants relative to biomass weight (Schenk et al., 2008) Additionally, the composition of the culture medium significantly impacts flocculation, with extracellular polymeric substances (EPS) and γ-glutamic acids secreted by microorganisms playing a crucial role in floc formation and stability (Salehizadeh et al., 2000; Rawat et al., 2011; Sheng and Yu).

Bio-flocculants play a crucial role in enhancing wastewater treatment by improving the separation of solids and liquids Various extracellular polymeric substances (EPS) have been developed for this purpose, facilitating cell-to-cell interactions among microalgae and bacteria without causing cell stress or lysis Research indicates that a high polysaccharide-to-protein (PN/PS) ratio in EPS significantly boosts microalgal flocculation Additionally, EPS excretion tends to increase under nutrient stress, promoting lipid accumulation and aiding in nutrient removal within microalgae-bacterial consortia The pH level also affects both the charge of microalgal cell surfaces and EPS production; lower pH values lead to larger floc sizes, while higher pH levels result in weaker flocs.

Microalgae-associated bacteria, including Flavobacterium, Terrimonas, and Sphingobacterium, significantly enhance flocculation by secreting high concentrations of extracellular polymeric substances (EPS) Under specific hydrodynamic conditions, the resulting flocs exhibit strength and uniformity in size Weaker flocs tend to break and re-flocculate until they attain sufficient strength to withstand shear stress Turbulence contributes to bioflocculation by increasing collision frequency and promoting microbial cell aggregation.

The settleability of algal-bacterial flocs is influenced by factors such as calcium concentration, hydrophobicity, and the filament index of filamentous microorganisms, which enhance floc strength Operational parameters like influent composition, hydraulic retention time, sludge retention time, and the presence of predators also play a crucial role in determining floc structure Notably, bacteria promote aggregation through their hydrophobic characteristics, especially under starvation conditions.

1996) Stronger flocs are able to form when sludge retention time increased, resulting in rising cell age in activated sludge (Tchobanoglous, 2003)

Bio-flocculation offers significant benefits by eliminating the reliance on costly and toxic chemical flocculants in water treatment However, this method also presents challenges, including the potential for microbiological contamination, which can disrupt the food supply of algae biomass.

Activated Microalgae granules

2.3.1 Characteristics of Activated Microalgae granules (AMGs)

Activated microalgae granules offer significant advantages over traditional algae species in wastewater treatment They enhance settleability and improve the recovery efficiency of microalgae from effluent Additionally, their use eliminates the need for costly mechanical aeration systems and harvesting steps, thereby reducing overall processing time and costs.

Microalgae-bacteria granules (AMGs) have been developed into spherical formations that incorporate microbial cells, target microalgae, filamentous microorganisms, inert particles such as calcium, magnesium, and iron, as well as extracellular polymeric substances (EPS) This results in a dense, tangled, and globular structure, as highlighted by Cai et al.

In 2019, research revealed that non-algae species, such as vorticella and rotifers, were observed adhering to granule surfaces, indicating a diverse biological population within the granules The extracellular polymeric substances (EPS) in the aqueous matrix played a vital role in granulation, facilitating the formation of stable microbial aggregates (Ahmad et al., 2017; Huang et al., 2015a; Liu et al., 2017) Building on the findings of Tiron et al (2015, 2017), it was found that free microalgae cells were captured within a gelatinous network of filamentous microalgae, where the filaments provided biological support for both microalgae and bacteria, enhancing their development within the granules The spherical dense structure and aggregation of microalgae and bacteria biomass significantly improved the wastewater treatment efficiency of aerobic microalgal granules (AMGs), which exhibit distinct aerobic conditions on the outer layer and anaerobic conditions in the central core, allowing for efficient nitrogen removal when oxygen diffusion is limited or when granule diameter is sufficiently large.

The symbiotic relationship between bacteria and microalgae within the granular structure enhances the removal of organic matter without the need for additional air supply, as oxygen and CO2 are provided through the photosynthesis of microalgae and the respiration of bacteria This partnership also facilitates the effective removal of nutrients, aided by cell assimilation and simultaneous nitrification and denitrification processes Furthermore, the concurrent nitrification and denitrification are maintained by variations in oxygen saturation during treatment cycles, which include both light and dark phases, likely supported by the dense granular structure.

Dispersed microalgal biomass settles slowly due to factors such as small cell size (5 to 50 μm), low cell density in wastewater treatment (0.4–0.5 kg m−3 dry biomass), and the negative charge on microalgal cell surfaces that inhibits aggregation In wastewater treatment, bacteria are crucial for organic matter removal and can produce extracellular polymeric substances (EPS) that facilitate the aggregation of microalgae and cyanobacteria The resulting microalgal-bacterial aggregates (MABAs) vary in size from 100 to 5000 μm, significantly enhancing harvesting efficiency without the need for flocculants Additionally, the presence of protozoa and small metazoans around MABAs during wastewater treatment suggests they play a vital role in aggregate size through bacterial grazing on the aggregates' surfaces.

There are three methods for creating algal-activated sludge granules The first involves using activated sludge to develop aerobic granular seeds, which are then cultivated under artificial light or natural sunlight to promote natural algal growth on the granules (Huang et al., 2015; Zhao et al., 2018; Zhang et al., 2018) The second method focuses on the formation of activated microalgae granules through self-coagulation between algae and bacteria The process of establishing algal-bacterial aerobic granular sludge is detailed in studies by Zhang et al (2020), Zhang et al (2018), He et al (2018), and NuramkhAMn et al (2019), which can be summarized in three stages based on granule morphology, particle size distribution, and biomass retention.

During the adaptation stage, algae experience rapid growth due to natural or artificial light, allowing them to outcompete bacteria in activated sludge because of their superior nutrient uptake and growth rates Initially, filamentous microorganisms are absent, and the presence of light promotes the natural growth of algae, which then adhere to the surface of flocculent sludge through the bridging action of extracellular polymeric substances (EPS) This co-existence leads to the breakdown of bacterial flocs into smaller pellets, despite increased EPS production to maintain structural integrity Bacteria also attach to the algae, forming combined flocs that enhance surface aggregation In aerobic granules rich in filamentous bacteria, these microorganisms play a crucial role in forming granular consortia, allowing filamentous microalgae to thrive on the surface and create an environment conducive to their growth.

During the maturation stage, the algal-bacterial activated granular sludge (AGS) exhibits stable growth and increased extracellular polymeric substances (EPS) secretion, resulting in a dense and compact structure This maturation enhances nutrient removal efficiency and improves settleability The process is further supported by the coexistence of filamentous bacteria and algae, along with the self-metabolism of microbes, which stabilizes EPS content.

During the maintenance stage, the balance between the attachment and detachment of algal and bacterial biomass is achieved Although the biomass concentration increases at a slower rate during this phase, it provides an opportunity for viable microorganisms to enhance enzyme activity, resynthesize essential metabolites, and adapt to their environmental conditions (Ni et al., 2009).

The primary challenges faced by activated microalgae granules in application include long-term stability, which is influenced by factors such as the carbon to nitrogen (C/N) ratio, aeration intensity, and the duration of aeration, collectively known as hydrodynamic shear force Additionally, the organic loading rate plays a significant role in this stability (He et al., 2018).

Hydrodynamic shear force significantly influences shear stress, shaping the diversity and structure of microbial communities and the production of extracellular polymeric substances (EPS) Reduced agitation intensity over time enhances microbial richness but decreases diversity and evenness, leading to shifts in dominant microorganisms (He et al., 2018) Optimal shear forces promote the formation of strong, compact granules, while insufficient shear results in weaker, porous granules (Wu et al., 2015) Additionally, the hydrodynamic shear force in reactors is crucial for effective nitrification and denitrification, with adequate shear stress ensuring reliable ammonia oxidation, whereas limited shear may hinder nitrification but improve denitrification (Zhang et al., 2018) Studies indicate that hydrodynamic shear force drives granule formation, affecting species integration and metabolic pathways (He et al., 2018; Zhang et al., 2018) Furthermore, hydraulic selection pressure is influenced not only by agitation and aeration but also by settling time and SBR cycle lengths, with prolonged settling periods minimizing selection pressure and short settling times leading to microbial washout and lack of granulation (Liu et al., 2018).

In 2015, it became essential to assess the mechanisms of hydraulic pressure control to gain a clear understanding of its impact on granulation, which is crucial for achieving granules with desired characteristics Additionally, the role of extracellular polymeric substances in this process warrants further exploration.

EPS are essential for the construction and maintenance of activated granular systems, with their composition influencing granule hydrophobicity through the ratio of PN to PS Increased hydrophobicity, particularly from PN, enhances the structural integrity of granules During granulation, PS contributes to the formation of robust algal-bacterial consortia, while PN facilitates the linkage between microbial cells and the attraction of organic and inorganic materials The EPS content remains relatively stable, indicating a steady state of activated granules under varying operational conditions Moreover, changes in operational parameters, such as hydrodynamic shear force and loading rates, prompt EPS to play a crucial role in maintaining the stability of the granulation process.

Nutrient removal is closely associated with shifts in algal-bacterial populations, impacting competition for nutrients and symbiotic relationships (Zhang et al., 2018) Phosphorus can be assimilated by algae, particularly in the forms of H2PO4– and HPO42–, and is transformed into high-energy organic compounds through phosphorylation According to Zamalloa et al., phosphorus accumulation in biomass is the primary mechanism for its removal, accounting for about 95% of total phosphorus elimination from influent Phosphorus removal efficiencies have been reported between 50% and 90% (Ji et al., 2021; Zhao et al., 2019; Zhang et al., 2019) Additionally, Ahmad et al (2017) observed an increase in green microalgal-bacterial granules when phosphorus concentration was maintained at 20 mg/L Thus, the microalgal-bacterial granule system presents a promising technology for effective phosphorus removal while ensuring stable granule development.

Ammonium is the preferred nitrogen source for algal biomass growth, but nitrate assimilation improves during algal–bacterial granulation due to increased particle size and a larger anaerobic zone The nitrogen removal mechanisms in this system involve assimilation by both algae and bacterial biomass, alongside nitrification and denitrification (Zhang et al., 2018) Additionally, Trebuch et al (2020) noted that high nutrient loading enhances nutrient entry into the granular structure and promotes denitrification A high COD loading, with a COD/N ratio of 8, contributes to larger and more compact granules, as observed by Zhao et al (2018), where dominant granule sizes exceeded 2.5mm Conversely, low carbon influent may restrict the growth of fast-settling bacteria, yet the system demonstrates a rapid adaptation and positive response to nutrient changes, highlighting its potential for effective wastewater treatment.

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

This research involved three primary experiments: first, the co-cultivation of bacteria and microalgae; second, the cultivation of co-culture granules in a photobioreactor (PBR) at varying agitation speeds using a sequencing batch mode; and third, the evaluation of 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 chosen microalgae strain for this study is Chlorella vulgaris, recognized for its effectiveness in municipal wastewater treatment alongside bacteria, achieving over 90% removal of nutrients and organic matter while demonstrating significant biomass production (He et al., 2013; Cai et al., 2013).

Chlorella, as highlighted by Wu et al (2014), is a promising source of food and energy due to its rich nutritional profile This microalga boasts approximately 45% protein, 20% fat, 20% carbohydrates, 5% fiber, along with essential minerals and vitamins, making it an appealing option for health-conscious consumers.

2007) The highest lipid productivity reported in the literature is about 179 mg/L/d byChlorella sp.(Chiu et al, 2008).

The inoculum microalgae used in this study were sourced from the Aquaculture Research Institute, part of the Ministry of Agriculture and Rural Development, located at 106 Nguyen Dinh Chieu Street, District 1, Ho Chi Minh City These microalgae were cultivated in Bold's Basal Medium (BBM) for 18 days before being combined with seed sludge for the experimental process Chlorella sp was grown in a photobioreactor measuring 20 cm in diameter and 60 cm in height, under controlled laboratory conditions with a constant temperature of 25℃, continuous LED illumination, and aeration to ensure agitation.

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 collected from the MBR tank of a wastewater treatment system in a Ho Chi Minh City supermarket was adapted to synthetic wastewater for Experiment 1 Prior to the start of the experiments, the biomass concentration of the seed sludge was measured, with an initial concentration of 5 gSS/L.

In low C/N ratio wastewaters, such as municipal wastewater, landfill leachate, and anaerobic digestion effluent, organic carbon often becomes a limiting factor for both phosphorus release and denitrification due to competition between denitrifiers and polyphosphate-accumulating organisms (PAOs) (Zhu et al., 2018).

In 2019, it was observed that nutrient removal performance is inadequate in wastewaters lacking sufficient organic carbon This study analyzed synthetic wastewater with quality parameters showing a Chemical Oxygen Demand (COD) of 384 ± 20 mg L-1, total nitrogen (TN) primarily in the form of ammonium at 40 mg L-1, and total phosphorus (TP) at 4 mg L-1, with nitrate and nitrite levels undetectable Based on previous research by Tiron et al., the C:N:P mass ratio was determined to be approximately 100:10:1, indicating the necessary balance of nutrients and organic sources for effective granulation.

To reduce variability in the experiment, synthetic wastewater was created using distilled water The composition of this synthetic wastewater, as detailed in Table 3.2, was adapted from the formulation by Huang et al (2015) to enhance its suitability for the study.

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.

The co-culture of microalgae and activated sludge was achieved through flocculation and granulation in a stirred photo-bioreactor, featuring a working volume of 7 liters This reactor had a height of 41.75 cm, a diameter of 17 cm, and a water level of 30 cm, resulting in an effective height to diameter ratio (H/D) of 2.45 (Tiron et al., 2015).

In a study conducted in 2017, the photobioreactors (PBRs) were illuminated continuously with LED lights (SMD 5050) emitting 3800 to 4000 lux, following a 12-hour light and 12-hour dark cycle Each reactor was agitated using a top-mounted stirring machine equipped with two propellers, each measuring 2.7 cm in diameter and a stirring shaft length of 34 cm To minimize light loss and maintain a stable temperature between 27°C and 32°C, the PBR system was housed in a wooden box with a thickness of 10 mm.

Biological inoculum used for flocculation/granulation processes was represented by

In a study involving Chlorella sp and aerobic activated sludge from a supermarket's wastewater treatment system, a co-culture was established with an initial biomass concentration of 600 mg/L The optimal mixing ratio of microalgae to activated sludge was determined to be 5:1 (%w:%w) for experiments (1) and (2), which demonstrated improved nutrient removal efficiencies ranging from 5% to 40% and enhanced biomass growth rates compared to other inoculation ratios (Su et al., 2012).

The feeding substrate consisted of 3.5 liters of synthetic wastewater with a volume exchange ratio of 50% To promote optimal organism growth, the initial pH of the liquor was maintained between 7 and 8 The flocculation and granulation processes were conducted in a sequencing batch mode, utilizing varying agitation speeds of 200, 160, and 120 RPM.

80 rpm in experiment (1) to determine the optimum agitation speed corresponding to optimum shear stress.

Hydraulic retention time was systematically reduced from 72 hours to 48 and then to 24 hours to optimize COD and nutrient removal while ensuring a continuous carbon supply and preventing biomass starvation Concurrently, settling time was decreased from 3 hours to 1 hour and ultimately to 15 minutes, based on the observed settling rate and settleability of the biomass This adjustment aimed to minimize suspended biomass by discharging excess material, which can hinder flocculation and granulation Effluents were collected in Erlenmeyer flasks for water quality analysis, and the settling time was determined to retain only flocs with a settling velocity greater than 0.36 m/h (Granados et al., 2012) As mature granules developed, the settling time was progressively shortened to focus on retaining 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 is adjusted based on the duration of the batch cycle and a reduction in settling time This adjustment is informed by settleability measurements and morphology analysis As settling time decreases, there is a corresponding increase in reaction time.

Analysis methods

COD, Ammonia, NO3 -N, NO2 -N, TP, MLSS, and TSS concentrations will be analyzed in accordance with standard methods (APHA, 2012).

To assess the removal rates of COD and nutrients, samples will be collected from each reactor at 24-hour intervals over three days, with measurements taken at 72 hours, 48 hours, and 24 hours Biomass analysis will be conducted at the conclusion of each batch period.

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

Chlorophyll-a was extracted from a mixture of bacteria and microalgae using an acetone solution, as demonstrated by Lee et al (2015) To analyze the characteristics of sludge and algae, a 20 mL sample of well-mixed sludge was collected from the reactor The methodology followed specific procedures to ensure accurate results.

- 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.

The suspension was centrifuged at 4000 rpm for 10 minutes, and the resulting supernatant was analyzed for chlorophyll-a (Chl-a) content Chl-a concentration was measured using ultraviolet spectrophotometry at specific wavelengths of 630, 645, 663, 750, 772, and 850 nm, with a 90% acetone solution serving 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:

The study identified two types of extracellular polymeric substances (EPS): soluble EPS and bound EPS To analyze EPS in terms of polysaccharides (PS) and protein (PN) per milligram of volatile suspended solids (VSS), it is essential to determine the mixed liquor volatile suspended solids (MLVSS) Typically, only bound EPS is examined in the context of 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 assess the settleability of co-culture systems, it is crucial to quantify the settling behavior of activated flocs Total Suspended Solids (TSS) concentration serves as a valuable parameter for evaluating settleability, alongside the settling rate of biomass.

Before the settling phase, a 250 mL homogeneous sample is transferred into a graduated cylinder The activated algae aggregates are allowed to settle, and their biomass position is recorded throughout the settling time The settling velocity of the activated algae aggregates is then calculated in meters per hour (m/h).

The height (P) of biomass is measured in meters and represents the growth observed between the initial time (T0) and the final time (Tf), both in hours This measurement is influenced by the speed of the granulation process, which is scheduled to occur weekly.

Granule shape, size and color

The development of flocs and granules was analyzed using an Olympus DF Plan microscope with magnifications of 4X, 10X, 40X, and a maximum of 100X This microscope enabled detailed observation of the shape, size, and color of the formed granules.

The size distribution of target microalgae cells, activated algae flocs, and activated algae granules will be analyzed using the Mastersizer, employing the laser diffraction method for precise measurement.

The flow profile of a fluid in a packed bed reactor (PBR) demonstrates a turbulent flow, characterized by layers of fluid sliding over one another Additionally, the co-culture of microalgae and bacteria behaves as a non-Newtonian fluid, exhibiting variations in viscosity that change over time during cultivation (Yatirajula et 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 systems, turbulent flow is observed when the Reynolds number exceeds 10,000 (Metcalf and Eddy, 2014) Furthermore, flow becomes turbulent when the Reynolds number surpasses the critical threshold of approximately 2,400 (Avila et al., 2011) The Reynolds number is a key parameter in determining the flow regime.

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.

Statistical analyses were conducted using IBM SPSS Statistics software version 20 One-way and two-way analysis of variance (ANOVA) were employed to identify significant differences among sample groups and to perform regression analysis A p-value of less than 0.05 demonstrated the significance of the ANOVA model, indicating a confidence level of 95%.

Dissolved oxygen (DO) concentrations and pH levels were assessed using a DO meter (Hanna HI 9146, Italy) and a pH meter (Hanna HI 9813-6, Italy) Additionally, light intensity was measured with a 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

The study demonstrated that activated microalgae (AMGs) consistently achieved a COD removal efficiency exceeding 90% across all four agitation speeds, indicating their potential for organic pollutant treatment Statistical analysis revealed that agitation speed had an insignificant effect on COD treatment capacity (p > 0.05), yet significant performance variations were observed throughout the five experimental stages (p < 0.05) Among the tested speeds, R160 exhibited the highest removal efficiency In the initial stages, COD removal improved gradually, but a reduction in settling time from 1 hour to 15 minutes in stage 3 led to decreased COD treatment across all speeds, with mean efficiencies dropping from 94.75% to 87.63% for R80, 93.33% to 90.08% for R120, 94.67% to 89.5% for R160, and 92.67% to 91.06% for R200 The co-culture of microalgae and bacteria facilitated simultaneous COD and nutrient removal, with the latter contributing to biomass formation However, the observed decline in COD removal efficiency was attributed to bacterial washout due to the shortened settling time, which also resulted in decreased biomass concentration across all PBRs.

In stages 4 and 5, the co-culture adapted to a reduced settling time from 15 minutes to 5 minutes, showing no negative impact on COD treatment During stage 4, AMGS emerged in all three PBRs with medium to high agitation speeds, appearing in the order of R160, R200, and R120 This indicates that organic substances and agitation speed do not significantly affect organic removal in co-culture PBRs; however, the formation of AMGS contributes to maintaining a stable organic removal efficiency exceeding 90% Additionally, the SBR cycle plays a crucial role in achieving consistent organic removal across the four reactors The reactors were effectively operated to minimize effluent organic matter, ensuring complete COD utilization by bacteria for nitrification and biomass growth, as stress from limited organic and nutrient availability promotes EPS secretion, filamentous microbial growth, and enhances the 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

Inoculum from the local wastewater treatment plant's membrane bioreactor (MBR) demonstrated effective nitrification During the initial stage, the ammonium (NH4) removal efficiency in the two photo-bioreactors (PBRs) with medium agitation speeds (R160, R200) was significantly higher compared to those with low agitation speeds (R80, R120) The effluent concentrations of nitrite and nitrate confirmed that nitrification was more efficient in the PBRs with medium agitation speeds in the early experiment stages This suggests that the increased oxygen content, resulting from higher agitation speeds, favored the dominance of activated sludge (AS) in the bacterial community during the first and second stages.

The mean nitrate concentrations observed at agitation speeds of R80, R120, R160, and R200 were 2.93, 2.81, 8.01, and 9.86 mg/L, respectively These findings indicate that higher agitation speeds can enhance the nitrification process in activated sludge (AS) The increased shear stress from high agitation creates greater turbulence within photobioreactors (PBRs), which improves mixing and ensures sufficient oxygen levels—primarily produced by microalgae—for optimal bacterial activity.

During stage 2, the mean NH4 removal rates for R80, R160, and R200 increased to approximately 13-14%, while nitrate concentrations across all four photobioreactors (PBRs) remained relatively stable This uneven enhancement in NH4 removal, coupled with a rise in Chl-a concentration, suggests that microalgae played a crucial role in improving NH4 removal efficiency, particularly as bacterial activity diminished after the initial days Consequently, microalgae became the dominant species in the co-culture system of all PBRs This effective nitrification in the co-culture system enhances the metabolic processes of both microalgae and bacteria, as CO2 produced from organic matter decomposition by bacteria supports microalgal photosynthesis, ultimately ensuring adequate oxygen levels are maintained.

During stage 3, the mean NH4 removal efficiency of R120 significantly increased to over 80%, up from 57.65% in stage 2 All four PBRs demonstrated a stable NH4 removal capacity ranging from 70% to 94%, with a clear distinction between the two high agitation speeds (R160 and R200) achieving over 90% efficiency and the two low stirring speeds (R120 and R80) maintaining efficiencies between 70% and 85% Statistical analysis indicated no significant difference in NH4 treatment efficiency between high and low agitation speed groups (p-value > 0.05) By the end of stage 4, the total biomass across all four PBRs exceeded expectations.

2500 mg/L with values ranging from high to low of R80, R120, 200, and R160.

In the later stages of the study, increased biomass concentration and chlorophyll-a (Chl-a) levels led to reduced NH4 removal due to self-shading of microalgae, which inhibited light penetration Conversely, higher agitation speeds and improved flocculation in stages 4 and 5 enhanced light exposure for microalgae within photobioreactors (PBRs), thereby promoting NH4 uptake Additionally, low ammonium concentrations during stage 4 contributed to decreased nutrient removal efficiency in stage 5 Overall, the findings demonstrate that hydraulic shear force from agitation significantly impacts ammonium removal performance, facilitating granule formation.

Figure 4.3 a) TP removal performance b) Remaining TP concentration in effluent of 4 different agitation speeds during the whole experiment

The phosphorus removal performance mirrored the trends observed in COD treatment, showing a significant decline in total phosphorus (TP) removal efficiency when the settling phase was reduced from 1 hour to 15 minutes However, TP removal efficiency gradually improved in stages 4 and 5 of the experiment Throughout the entire study, TP removal efficiency consistently remained above the established threshold.

The study highlights the effectiveness of co-culture systems in phosphorus (P) removal, achieving a potential efficiency of 85% Throughout the experiment, pH levels were maintained between 7 and 7.5, which limited phosphorus removal via precipitation, typically effective at higher pH levels (9-11) The processing of P by arbuscular mycorrhizal (AM) fungi or AMGS occurs primarily through two pathways: extracellular uptake and intracellular adsorption However, the negligible amount of P removed through extracellular uptake is attributed to the role of extracellular polymeric substances (EPS), which vary significantly during the experiment, indicating differences between granular and non-granular phases Consequently, the primary mechanism for P removal in this study is identified as intracellular uptake by both microalgae and bacteria, with microalgae capable of absorbing excess phosphate and storing it as polyphosphates within their cells.

In the transition from stage 2 to stage 3, microalgae became the dominant species across all four photobioreactors (PBRs), leading to a decrease in phosphorus (P) removal efficiency as the amount of activated sludge (AS) diminished The consistent trend in P removal efficiency alongside chemical oxygen demand (COD) removal suggests that phosphorus is primarily removed through the biomass accumulation of bacteria derived from the activated sludge.

Microalgae activity profile

Figure 4.4Concentration of Chlorophyll a (àg/L) at various stages in the PBRs

In a microalgae-bacteria co-culture system, chlorophyll-a (Chl-a) serves as a key indicator of microalgae biomass, as it is a unique compound found exclusively in microalgae The concentration of Chl-a is utilized as an indirect measure of microalgae growth, showing significant increases from stage 2 to stage 3 Specifically, in the experimental conditions R80, R120, R160, and R200, Chl-a concentrations rose dramatically from 450, 505, 863, and 1018 to 3808, 7037, and 5574, respectively.

After reducing the settling phase from 1 hour to 15 minutes, the Chl-a concentration at various agitation speeds increased significantly, with the highest increase at R120 and the least at R200, showing a 6-14 times enhancement This indicates that, unlike bacteria in activated sludge, microalgal cells tend to form larger flocs that settle quickly, preventing washout Additionally, total biomass in photobioreactors (PBRs) increased significantly from stage 2 to stage 3, rising from approximately 500-700 mg/L to 900-1000 mg/L, highlighting the growth of microalgae biomass as a key factor in this increase.

A one-way ANOVA analysis revealed that varying agitation speeds significantly influenced Chl-a concentration (p-value < 0.05), while total biomass remained unaffected This suggests that shear stress from mechanical agitation impacts microalgae activity in co-culture, altering the granulation process of AM Throughout stages 1-4, Chl-a concentration consistently increased across four photobioreactors (PBRs), surpassing 10,000 µg/L by stage 4 Notably, three PBRs exhibited AMGS, with R160 (day 165 - batch 67) appearing first, followed by R200 (day 172 - batch 70) and R120 (day 188 - batch 77) While Chl-a levels at R160 and R120 were similar, R120 reached 15,000 µg/L, indicating that high agitation speeds facilitate granulation under moderate microalgal biomass, whereas lower speeds promote growth but require more time for granule formation In stage 5, a significant decline in Chl-a concentration across all PBRs was attributed to intracellular Chl-a degradation under stress conditions, particularly in R160, which experienced a dramatic reduction of 64 times, resulting in a Chl-a concentration of only 173.2 µg/L This highlights the direct impact of AMGS formation on microalgal activity Additionally, in R80, where AMGS did not occur, a sevenfold decrease in Chl-a concentration was primarily due to self-shading caused by excessive biomass, limiting light penetration necessary for optimal photosynthesis.

In stage 5, as depicted in Fig 4.7, Chl-a levels gradually decreased while biomass remained stable across all four reactors, indicating that self-shading contributed to the reduction of Chl-a in microalgae, allowing them to adapt to a constant biomass concentration Without photosynthesis, Chl-a becomes less crucial for microalgae survival Notably, dense and compact granules formed in three photobioreactors (R120, R160, R200) during stage 4 (Fig 4.5) Microalgae can autonomously decrease intracellular Chl-a levels due to increased co-culture biomass and insufficient light for growth Instead of cell lysis or death, the reduction of Chl-a occurs under low light conditions, and the emergence of AMGS indicates that algae cells are increasingly limited in light exposure, particularly in the core of AMGs Consequently, Chl-a becomes less essential for microalgae growth Total biomass remained consistent with stage 4 (p-value > 0.05), suggesting no loss in the microbial community Although COD removal performance remained high, nitrification performance declined, evidenced by lower nitrate and nitrite levels in the water, resulting from reduced oxygen production and decreased competition between nitrifying bacteria and microalgae, which led to a lower maximum microalgae biomass/Chl-a ratio (Rio-Chanona et al., 2018).

The emergence of AMGS and low light conditions, when Chl-a concentrations exceed a specific threshold, lead to a shift in microalgae's nitrogen consumption patterns and a decrease in chlorophyll-a levels (Huang et al., 2019) In this study, the AM/AMGS exhibited a dark green color across four photobioreactors (PBRs) (fig 4.6).

Controlling the agitation rate is essential for balancing the growth of microalgae and bacteria, while also enhancing the growth advantage of microalgae Future research should focus on the impact of settling time and sequencing batch reactor (SBR) cycles to optimize biomass concentration, ultimately promoting improved microalgae growth and facilitating rapid 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, influenced by a fluid's shear rate and viscosity, plays a crucial role in the behavior of microalgal cells in photobioreactors (PBRs) It is essential to identify the critical shear stress level, which indicates the threshold beyond which microalgal cells experience detrimental effects Understanding this threshold helps assess the sensitivity of these cells to shear stress, as excessive shear can lead to decreased cell viability, extended recovery periods, lower growth rates, diminished photosynthetic activity, and even cell lysis.

The critical shear stress experienced by microalgal strains varies based on cultivation conditions and the source of shear stress Turbulent flow, generated by micro-eddies of different sizes, impacts the energy content of the flow, with smaller eddies producing more significant shear stress on microalgal cells Micro-eddies that are equal to or smaller than the cells can effectively transmit turbulent forces, increasing the risk of cell damage when their sizes are comparable Conversely, when micro-eddies are larger than the cells, the likelihood of damage decreases, highlighting the importance of managing shear stress in microalgal cultivation (Camacho et al., 2001; Hadiyanto et al., 2013).

Michels et al (2010) found that shear stress negatively impacts microorganism cell viability, with a threshold value varying by strain Their study revealed that shear stress exceeding 1.26 Pa results in a significant decline in microalgae viability, indicated by reduced chlorophyll concentration Additionally, microalgae morphology changes with shear stress, transitioning to a granule structure as stress increases The critical shear stress range for granulation was identified between 1.26 Pa and 2.49 Pa Figure 4.8 illustrates the kinetic coefficients of shear stress and their correlation with granule formation In this study, agitation of AMGs was achieved through centrifugal and inverted mixing forces to ensure uniform biomass distribution.

The particle size distribution (PSD) of four photobioreactors (PBRs) was analyzed at different stages, revealing significant changes in particle size and distribution Measurements were taken when active microbial granules (AMGS) first appeared in R160 and just before the conclusion of stage 4 While PSD remained consistent at lower agitation speeds, higher speeds in R160 and R200 exhibited a narrower distribution and the emergence of larger particles The mean diameters of particles in PBRs R80, R120, R160, and R200 varied significantly, with R200 showing the largest increase in size Following AMGS formation, a decrease in mean diameter indicated a tighter structure, while the growth of AMGS at higher agitation speeds suggested enhanced potential for granulation Additionally, microalgal cell size in relation to turbulent flow micro-eddies highlighted the risk of shear stress-induced cell damage At elevated shear stress, microorganisms tended to cluster to mitigate environmental stress, leading to a gradual increase in particle size However, excessive shear stress risks destabilizing granule structure, as lower shear conditions may hinder microalgal and bacterial growth, corroborating findings from Wang et al (2021) regarding microalgal-bacterial interactions.

Figure 4.8c illustrates that while the AM floc size at R80 and R120 began large under lower shear stress conditions (0.04 – 0.15 Pa), the linear trend in AM size for both photobioreactors (PBRs) suggests a consistent decrease over time at similar rates.

In R160 and R200, with high shear stress ranging from 0.35 to 0.69 Pa, the aggregation mechanism (AM) began with small flocs that progressively increased in size Following the formation of the aggregated microgel (AMG), a reduction in mean diameter indicated a more compact structure Additionally, at higher agitation speeds, the AMG exhibited greater potential for growth compared to its current size The stable AMG sizes for R160 and R200 were observed to range from 400 micrometers.

At day 218, the analysis revealed that granules with a diameter of 600 μm exhibited a shaped structure, while those in the R120 range of 200 μm to 400 μm displayed an unshaped structure (Fig 4.12) A comparison of different agitation speed PBRs indicated that shear stress significantly influenced the shape, structure, and size of anaerobic microalgal granules (AMGs) In the R160 setup, stable granules were characterized by a round shape and dense structure, featuring a distinct biomass layer where bacteria formed the core, surrounded by dense Chlorella cells The extracellular polymeric substances (EPS) were thought to facilitate the connection between microbes and microalgae, aiding in the formation of granules through a filamentous microalgae network Granule sizing in R160 was noted to be uniform and consistent (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

In reactors 4 and 5, a notably high settling rate was observed, particularly in R160, where granule formation occurred first, leading to the highest settling rate among the reactors While shear stress did not significantly impact the settling rate, it facilitated the aggregation of filamentous microalgae, microalgae cells, and bacteria into larger structures, enhancing the settleability of AM/AMGs Conversely, in R80, despite the absence of granule formation, the presence of dense flocs of AM resulted in a comparatively high settling rate relative to other reactors that did produce granules.

The PN/PS ratio for all four PBRs exhibited significant fluctuations across different stages In stage 1, the PN/PS ratios for R80, R120, R160, and R200 peaked at 11.08, 6.64, 5.5, and subsequently dropped to 2.83, 2.19, 1.66, and 1.61, respectively After day 47, this ratio continued to decline, coinciding with a decrease in total EPS concentration until the end of stage 2 The increase in PS concentration indicated heightened bacterial activity in later stages, driven by the high metabolic rates of bacterial cells favoring PS production over PN (Huang et al., 2015) Previous research by Tay et al (2001) demonstrated that enhanced aeration and increased shear stress promoted PS formation and granulation, a trend observed in this study during stage 2 when larger microalgal flocs formed, particularly in R160 and R200, which experienced the highest shear stress From day 62 onward, the EPS per unit biomass gradually decreased as total biomass increased, suggesting microorganisms were entering a starvation phase due to insufficient substrate for normal growth Research by Li et al (2006) indicated that prolonged starvation during granulation compels microorganisms to utilize EPS as a substitute, thereby maintaining a controlled EPS concentration to facilitate granulation.

During stage 2, the reduction of HRT from 6d to 4d led to an increased OLR and a slight enhancement in COD treatment capacity, indicating improved organic utilization by the anaerobic microbes (AM) Despite a reduction in settling time that resulted in higher washout biomass, the high COD consumption by AM allowed for effective microbial growth, keeping the mean biomass concentration in the PBRs relatively stable compared to stage 1 Notably, the production of particulate nitrogen (PN) and particulate solids (PS) varied significantly during this period, with the PN/PS ratio increasing across all four PBRs, peaking at 11.08, 6.64, 5.5, and 4.32 for R80, R120, R160, and R200, respectively This aligns with findings from Yang et al (2018), which suggested that nutrient availability significantly influences the PN/PS ratio, highlighting that bacteria preferentially convert excess carbon into storage compounds like PS at high OLRs, while at lower OLRs, more carbon is allocated for biomass synthesis rather than PS production.

Recent studies have shown that reducing hydraulic retention time (HRT) from 6 days to 4 days leads to a 1.5-fold increase in organic loading rate (OLR), with extracellular polymeric substance (EPS) secretion dominated by proteins (PN) over polysaccharides (PS) This finding aligns with previous research on microalgae (Sawayama et al., 1992; Zeng et al., 2013) Chlorella sp cultivated in wastewater with high nitrogen levels under phosphorus-limited conditions produced a significant amount of protein-rich EPS due to excessive nitrogen uptake (Wang et al., 2014) PS acts as a bio glue, enhancing cell-to-cell interactions and reinforcing microbial structures within granules (Liu, Liu, and Tay, 2004) Meanwhile, the PN surrounding granules contributes to the formation of cell walls with high surface hydrophobicity, which is essential for promoting granulation (Liu et al., 2004).

Research indicates that the secretion of PN and a high PN/PS ratio in pools promote the granulation of AM (Cai et al., 2019b; Huang et al., 2015; Ahmad et al., 2017) Notably, the PN/PS ratio tends to fluctuate between 2 and 3.5 over a period of 100 days (Huang et al., 2015) and reaches approximately 7 by day 60 (Cai et al., 2019a).

The PN/PS values remain relatively stable throughout the operation, suggesting that the elevated PN/PS ratio of 11.08 at R80 signifies an imbalance between bacterial and microalgal activity, which ultimately hinders granule formation in subsequent stages.

During stage 3, the PN/PS ratios of the four PBRs were recorded at 2.83, 2.19, 1.66, and 1.61 Following day 47, there was a gradual decline in the PN/PS ratio alongside a decrease in total EPS concentration until the end of stage III The observed increase in PS concentration indicates heightened bacterial activity in the later stages, as the high metabolic activity of bacterial cells promotes PS production over PN (Huang et al., 2015).

Enhanced aeration increases shear stress on granules, promoting polysaccharide (PS) formation and granulation In this study, large microalgal flocs were observed at agitation speeds of 160 rpm and 200 rpm, which corresponded to the highest shear stress and PS concentration However, from day 62, the number of extracellular polymeric substances (EPS) per unit of biomass gradually decreased due to biomass growth, indicating a starvation phase where substrate availability was insufficient for normal microbial growth This starvation prompted microorganisms to utilize EPS as an alternative, leading to a significant reduction in EPS concentration Research by Li, Kuba, and Kusuda (2006) supports this, showing that prolonged starvation during floc formation and binding resulted in a notable decrease in EPS concentrations, including proteins and polysaccharides, thereby promoting controlled EPS levels essential for granulation.

AND RECOMMENDATIONS

Conclusions

The study titled “Co-culture of Microalgae and Bacteria for Wastewater Treatment Coupled with Biomass Recovery” utilized lab-scale photobioreactors (PBRs) to explore the mechanisms behind activated microalgae granule formation while assessing their wastewater treatment capabilities over a 250-day period Findings revealed that the formation of activated algae granules is influenced by extracellular polymeric substances (EPS) and filamentous microalgae, which can be modulated by varying agitation speeds and shear stress levels Specifically, lower shear stress required more polysaccharides (PS), while higher shear stress increased the need for proteins (PN), leading to larger particle sizes due to PN secretion The optimal granule formation occurred at agitation speeds of 120, 160, and 200 rpm, with the largest particle size observed at 200 rpm (~600 µm) Notably, an agitation speed of 160 rpm (shear stress of 1.26 Pa) yielded the best results for particle formation time, granule stability, and 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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