Luận văn thạc sĩ Kỹ thuật môi trường: Co-culture of microalgae and activated sludge for wastewater treatment

ABSTRACT This work was to make a comprehensive assessment on variety of inoculation ratios and biomass retention times for co-culture systems of microalgae and activated sludge.. The eff

INTRODUCTION

Background

Domestic and industrial activities have discharged wastewater (WW) possessing concerned nitrogen and phosphorus level Those nutrient compounds are the main cause of eutrophication in the receiving water reservoir because they diminish oxygen concentration for aquatic living and trigger algae bloom Consequently, excessive nitrogen and phosphorus imbalance the function of ecosystem It is a critical need to phase out nutrient elements in WW prior to discharging to avoid eutrophication and guarantee healthy water quality for community consumption Generally, activated sludge processes (ASPs) are favorite ones which have been applied widely to target nutrient in the discharged WW stream [1] Such processes are sequencing batch, anaerobic, anoxic, oxic reactors and hybrid systems of those given single technology However, ASPs are restrained by high energy consumption which is mandatory to provide oxygen for bacteria respiration and nutrient metabolism process Energy for aeration accounts for 60% to 80% total used energy as a whole of wastewater treatment process using ASPs [2] Another one-hybrid system (e.g., anaerobic- anoxic-oxic) is a viable option but goes along the high capital cost It is essential to propose a robust single-stage process for dual purposes of nutrient remediation and energy-efficient This process aims to apply for various types of wastewaters as municipal wastewater, winery wastewater, piggery and, fermentation wastewater (Godos et al., 2009; Mujtaba and Lee, 2017; Qi et al., 2018; Higgins et al., 2018a)

Microalgae have demonstrated their feasibility for wastewater remediation thanks to due to their low cost, easy operation and revenue-raising potential (e.g., bioenergy production and pigment extraction) (Rittmann, 2008; Mata et al., 2010) They are prominent candidate to develop the above-mentioned single-stage technology Microalgae can assimilate high-load nutrient level and start producing biomass in a relevant short time less than 24 h [7], [8] Concurrently, photosynthesis of microalgae generates oxygen given a good chance to provide oxygen for ASPs process [6] That is, microalgae can serve as aQ ³DHUDWLRQ GHYLFH´, ideally replace the mechanical aeration system and cut off energy cost for aeration in ASPs [9] Based on this concept, microalgae and bacteria can mutually support each other Studies on the symbiosis of microalgae±bacteria have been accelerating as a mean for domestic and industrial wastewater remediation [10] Given a single-stage technology, microalgae consumes nitrogen and phosphorus in WW for biomass production whilst bacteria metabolize organic matters in a greater extent

As reported for the co-culture system, nutrient remediation and biomass growth are influenced by several factors, encompassing different inoculum ratio, operating condition, wastewater composition and reactor configuration [11] The effects of wastewater matrix and inoculum ratio has been reported in several studies [12] [13] The former reported that 1:3 inoculation ratio (microalgae:bacteria wt/wt from now on) was an appropriate one for both nutrient remediation and biomass production amongst a range of ratio (1:0, 1:1, 1:2, 1:3, 2:1, 3:1 wt/wt) [12] The latter study indicated that COD, total nitrogen (TN), and total phosphorus (TP) in vinegar WW were removed more significantly by adding either 1% (v/v) or 10% (v/v) of Bacillus firmus and Beijerinckia species into the real WW-cultivated microalgae [13]

Compared to a single microalgae culture, COD, TN and TP assimilation efficiency in co-culture system increased by 22.1%, 20%, and 8.1%, respectively [13] The ratio of mixed microalgae-bacteria could affect performance of nutrient remediation However, the co-culture system of microalgae and bacteria still faces inherent obstacles in term of biomass harvest [14] The traditional technologies for biomass harvest are coagulation and centrifugation/separation The efficiency of those traditional technologies remains insignificant and operation cost is still high [15] The drawback in settling and biomass harvest of microalgae could be tackled by adding activated sludge Activated sludge performs better settling ability compared to microalgae and exhibits extensive COD removal than the available bacteria in wastewater [16] Therefore, several studies have integrated microalgae and ASPs to be co-culture system based on the above-mentioned co-benefits for WW remediation

[15]; Mujtaba and Lee, 2017; Zhu et al., 2019) For instance, the 5:1 ratio was found for sufficient nitrogen and phosphorus removal (91.0% and 93.5% respectively) [15] In turn, their findings indicated the inoculum ratio did not impact on COD removal [15] In another study, an optimal ratio primed to 2:1 to attain the highest efficiency of municipal wastewater treatment [1] High removal of COD (82.7%), TN (75.5%) and TP (100%) could be obtained under an inoculum ratio of 1:1 [11] It can be seen the optimal microalgae:sludge ratio of the previous studies varied due to the discrepancies of wastewater composition For instance, Su et al (2012) studied high loading concentration of COD (380 mg L -1 ), TN (50 mg L -1 ) and PO4 3- (8 mg L -1 ) while [1] experimented low loading level of COD (60 mg L -1 ), NH4 +-N (50 mg L -1 ) and TP (1.3 mg L -1 ) Also, COD: N ratio could affect bacteria and microalgae growth

A 4.3:1 ratio could enrich microalgae biomass and nutrient recovery [11] It is important to note that the previous works did not explore thoroughly an optimal ratio for simultaneous nutrient remediation and biomass production In addition, the role and contribution of microalgae and activated sludge in the co-culture system have not been addressed adequately A minor change in microalgae:activated sludge ratio could influence the whole performance Therefore, biomass fraction of both microalgae and bacteria needs to be quantified, and thus to elaborate their role on organic and nutrient remediation

Moreover, existing co-culture systems are relatively low performance [1], [15] Beside of influence of the factors such as inoculum ratio, wastewater compositions (COD:N and N:P ratios), microalgae/ bacteria strain, operating conditions (such as biomass retention time (BRT); hydraulic retention time (HRT); light:dark cycle; light intensity; mixing/aeration) on performance as well as biomass production [17]±[23]

As reported of [24], the foremost factors that can improve the performance of the co- culture system are expressed as supplemental aeration to increases CO2 concentration and the option of suitable BRT However, external mechanical aeration is not a convincing solution due to energy consumption Thus, applying a suitable BRT is an excellent option BRT can be better controlled microbial growth rate in the reactor and amount of biomass wasted from the reactor [25], which therefore affects the biomass concentration, microalgae productivity, and nutrients removal [26] Thus, that very important to improve the algae growth rate and explored as symbiotic mechanism between microalgae and bacteria in co-culture system In practice, the pilot-scale-based algae cultivations are generally open pond and photobioreactor (PBR), but those systems still have some inherent disadvantages Specifically, an pen pond is easily water evaporation, contamination, a requirement of large land, and difficult to control physico-chemical conditions [26] Meanwhile, the limitation in PBR is poor settleability, biomass washout, and harvesting limitation [27] To overcome those obstacles, a membrane photobioreactor (MPBR) was introduced as a promising technology [26] The MPBR is combined of between a conventional PBR and membrane filtration module (external or submerge membrane) and it could offer an alternative approach to prevent biomass washout [23], [28]±[30] Thus, MPBR is used that they can serve as an effective way of combining wastewater treatment with biomass production Compared to a conventional PBR the MPBR system enhances the light accessibility and also provides a sufficient mixing, easily accessible carbon source, an operation at lower hydraulic retention time (HRT), high loading rate, and completed retention of biomass [26]

Recently, there are several previous studies on combining the MPBR with co-culture of microalgae-activated sludge symbiothetic in order to removal the nutrients and organic matter in wastewater [31], [32] However, most of the studies were operated under long BRT (more than 10 days) and external aeration supply In addition, little knowledge is obtained about continuous flow MPBR experiment which is necessary to investigate the performance of practical operation Furthermore, microbial cells are generally considered to be a potential of dramatic fouling when using membrane filtration [33], [34] It is generally recognized that extracellular polymeric substance (EPS) is one of the major factors affecting membrane fouling It is interest to note that the previous works did explore that the EPS concentrations released by algae- activated sludge PBR was decreased compared with only activated sludge PBR [35]

It is tempting to speculate that using co-culture of microalgae-activated sludge in

MPBR may not only performance (nutrients and organic matter removal), prevent biomass washout and decrease the aeration consumption but also alleviate the membrane fouling However, to date, limited report of this approach is available on influent of BRT in co-culture symbiothetic MPBR Furthermore, how different BRT influence the membrane fouling and pollutants removal efficiencies, and which BRT is better for the MPBR performance are still limited

Given the research gaps indicated above, in this study, the co-culture system of microalgae-activated sludge was developed The optimum microalgae-activated sludge inoculum ratio for simultaneous nutrient and organic matter remediation was defind The mechanism of nutrient remediation and the role of microalgae and activated sludge incorporated in a symbiotic system were explored And the effects of biomass retention times in MPBR on the treatment performance and membrane fouling were elucidated.

Objective of study

Given the research gaps indicated above, this study was built to explore the following objectives: i) To determine optimum microalgae and activated sludge inoculum ratio (wt:wt) for simultaneous nutrient/ organic matter removal and biomass production and define the mechanism of nutrient removal and to validate the role of microalgae and activated sludge under a symbiotic system that based on changes of biomass fraction in co- culture systems; ii) To develop a Membrane Photobioreactor, given that extensively investigated for WKH LQÀXHQFH RI Biomass Retention Time (BRT) on the treatment performance; membrane fouling phenomenon under co-culture system of microalgae and activated sludge in municipal wastewater.

Scope of the study

1.3.1 Effect of microalgae:activated sludge inoculation ratio on COD and nutrients removal, as well as in biomass productivity

The first objective of this study was investigated through an application of 5 photobioreactors (PBRs) operated under batch culture conditions for 12 days with 5 different initial microalgae:activated sludge ratios (0, 9:1; 3:1; 1:1; 0:1 (wt:wt)) The initial total biomass concentration was the same in all 5 PBRs This application will be called task 1 in this report The aquatic medium in the PBR was synthetic wastewater containing nutrients (N, P), Chemical Oxygen Demand (COD) in the form of sodium acetate, and trace elements to allow the microalgae growth The objective was to determine which microalgae:activated sludge ratio would reach the highest performances in COD and nutrients removal, as well as in biomass productivity

1.3.2 Effect of biomass retetion time on performance and fouling study of membrane photobioreactor-based microalage-activated sludge culture treating domestic wastewater

The second objective of this study was investigated through an application of long- term performance and fouling study of membrane photobioreactor-based microalage- activated sludge culture treating supermarket wastewater with 4 different biomass retention times (3 days, 5 days, 7 days and 10 days) This application will be called task 2 in this report The objective was to determine which proper biomass retention time would reach the highest treatment performance, biomass productivity and effect of biomass retention times on fouling behavior.

LITERATURE REVIEW

Overview on microalgae

Microalgae is photosynthetic prokaryotes or eukaryotes [36] which can quickly grow and can live in difficult conditions due to diverse cell structure, unicellular or multicellular, prokaryotes such as Cyanophyceae, or eukaryotic organisms such as green algae (Chlorophyta) and diatoms (Bacillariophyta) The main components of microalgae are protein, carbohydrate, and lipids Starches, proteins are used as biofuel, biological material [6]

Chlorella sp is a genus of single-celled green algae, belonging to the Chlorophyta branch The cells are spherical or elliptical in shape, without cilia, 2-10àm in diameter, containing photosynthetic pigments help to absorb light and use CO2 to photosynthesize and create energy plants The microalgae is present in all existing ecosystems on the Earth, not only underwater but also on land (Slade et al., 2013)

Chlorella is considered popular because it lives in many different habitats, both freshwater and seawater Chlorella parasitica is found as cell symbiosis of

Table 2 1 Composition of microalgae based on dry biomass (%) [38]

Microalgae have certain characteristics which make them very valuable as a raw food source Each cell is basically a "plant" of synthetic sugar and other bioactive compounds, which can later be used as a source of nutrition Microalgae are biological resources with high carbon content [29] which is known as a potential and diverse source of materials for biofuel production such as bioethanol and biodiesel due to species diversity and culture conditions Another benefit considered as the potential of microalgae is use cosmetics, pharmaceuticals or be applied in air handling and treatment of contaminated wastewater due to the special chemical composition of algae [1], [39], [40]

Wastewater treatment and reuse of wastewater are important approaches to address the increasingly serious global freshwater scarcity today Moreover, the high concentration of nitrogen and phosphorus in wastewater is considered to be one of the concerned issues which caused the eutrophication in lakes, estuaries, and oceans Most wastewater treatment solutions require assurance of technical and economic issues such as large energy, large area or high operating and maintenance costs Compared to the elimination of chemical nutrients and bacteria, microalgae can be able to remove nutrients by means of biosecurity and cheaper Microalgae can serve as a tertiary treatment after MBRs to further remove nutrients (Nitrogen and Phosphorus) that were not degraded in the MBR process through assimilating nutrients into the microalgae biomass [26], [41] Therefore, the concentration of nutrients in wastewater will be significantly reduced The rapid growth of microalgae allows it to grow even in extreme conditions and has proven to be an advantage in difficult wastewater treatment like industrial wastewater In addition to reducing the impact of nitrogen and phosphorus in wastewater discharged into the environment, microalgae also contribute to aquatic biodiversity [6]

Burning fossil fuel has contributed to greenhouse gas emissions (mainly CO2) and climate change seriously in recent years [42] Therefore, alternative solutions to reduce CO2 emissions have been noticed by research centers around the world Over the next few years, the use of microalgae as a biological method has created a great potential to reduce CO2 by photosynthesis CO2 is fixed into plants and microalgae through photosynthesis so the production of micro-algae biofuels can reduce CO2 emissions by burning fossil fuels (Mata et al., 2010) Moreover, microalgae have many benefits such as higher fixation rates than plants, some microalgae can even grow under conditions in the range of CO2 from 10 to 15% - a common concentration in the furnace burning (Tang et al., 2011) Photosynthesis by microalgae is considered a simple and appropriate process for the O2 cycle on Earth This process has fixed

CO2 into microalgae biomass in the form of carbohydrates and lipids as a promising research direction Researchers and companies focus on microalgae that it is not only an alternative fuel source but also a renewable fuel source

Microalgae can produce high oxygen rates (O2) (up to 9 mg/L.m) through photosynthesis for aerobic organic pollutants [15] During photosynthesis, microalgae can take advantage of carbon dioxide (CO2) which produced by aerobic bacteria from the process of organic decomposition Therefore, using microalgae is also capable of mitigating climate change

The use of wastewater as a source of nitrogen and phosphorus has the potential to reduce the cost of cultivation and energy consumption (Honda et al., 2017) Chlorella sp has proven many potential features for future research on renewable energy When compared with plants, Chlorella sp shows rapid growth, low-cost farming, high biomass productivity and energy costs (18,59 MJ/kg) It is a potential candidate for the bioenergy industry, especially biodiesel [37] Chlorella also contains high levels of lipids, proteins, carbohydrates and other micronutrients (45% proteins, 20% lipids,

20% carbohydrates, 5% celluloses, 10% minerals, and vitamins) can apply for many biochemical and production fields

As the quality of life improves, the demand for healthy development becomes more concerned Besides combining diet and exercise to achieve a healthy body, vegetable- based nutritious foods are being used and dominated in the market On the market today, the health benefits of aquatic microorganisms such as microalgae are being studied and recognized and appreciated Compositions of microalgae can be extracted into chemical compounds as colorants, antioxidants, b-carotenes, polysaccharides, triglycerides, fatty acids, vitamins, biomass and applied for various industries (pharmaceuticals, cosmetics, nutraceuticals, functional foods, biofuels) (Mata et al., 2010) Some microalgae are commonly used in functional foods: Nostoc, Botryococcus, Anabaena, Chlorella, Chlamydomonas, Scenedesmus, Synechococcus, Perietochloris and Porphyridium They contain vitamins and essential elements as Potassium, Zinc, Selenium, Iron, Manganese, Copper, Phosphorus, Sodium, Nitrogen, Magnesium, Cobalt, Molybdenum, Sulfur and Calcium (Ventakesan et al., 2015) The growth rate of microalgae is also a noteworthy point that the choice to be functional foods for both humans and cattle

With many discovered and researched characteristics, Chlorella is considered to be an excellent nutrient source and highly effective photosynthesis as well as rapid growth With high commercial value, Chlorella can be used in many areas such as food technology, pharmaceuticals, or wastewater treatment, etc [43] Especially in the food industry, carotenoids, fatty acids, amino acids, and antioxidants in Chlorella increase the nutritional value of human and cattle foods After global fears of an uncontrolled overpopulation, during the late 1940s and early 1950s, Chlorella was considered as a promising food source as a possible solution for the world crisis because of famine

In addition to the main use of CO2 emission reduction and wastewater treatment, algae are also applied to other applications such as fertilizer and pollution control

Some algae can be used as organic fertilizer, or in semi-biodegradable types Algae can be grown in ponds, treat fertilizers influent from farms, microalgae are rich in nutrients so they can be reused as fertilizer - a way to reduce costs in cultivation (Ventakesan et al., 2015) Microalgae have been used to treat wastewater by absorbing nutrients such as nitrogen and phosphorus from cattle wastewater, especially inbred farms [44] Algae can be also used to treat high-polluting heavy metals such as uranium At wastewater treatment plants, microalgae can be used to reduce toxic chemicals and increase the purity of water) (Mata et al., 2010)

Unlike most energy crops, microalgae cultivation does not require large area, and could potentially be scaled up vertically and could be carried out in open or closed systems (Chen et al., 2011) The habitat of green algae is found abundantly in freshwater environments, the two most common systems for cultivating microalgae are open farming systems, such as open ponds, reservoirs and raceways, and systems Closed cultivation is controlled using different types of bioreactors The choice of culture medium depends on the location, space, and available water, cost and desired products

Traditionally, natural water such as lake water and lagoons has been used to grow algae These open production systems are outdoors and depend on providing natural light as the only energy source for the system Microalgae were cultured in open systems include unstirred shallow ponds, stirred circular ponds and paddlewheel stirred raceway ponds Their area can be up to 1200ha, usually are conducted on non- agricultural lands and the depth should be 20-35 cm to ensure adequate exposure to light Paddlewheels provide mechanical energy to the culture, keeping the cells in suspension (Singh, et al., 2015)

The advantages of open systems are less energy consuming for culture mixing, minimal capital and operating costs, convenient to operate and sustainable (Gao et al., 2015) However, an open raceway pond requires large areas to scale up and adverse weather conditions There are lost CO2 and water to the atmosphere (Singh et al., 2015) and the influence of microorganism contamination cannot be neglected (Mata et al., 2009) Besides, the shear stress resulted in the rotation of paddlewheel may affect the photosynthetic capacity (Chiu et al., 2015), which limits the growth of microalgae Thus, high biomass productivities are only achieved with microalgae strains resistant to severe environmental conditions, such as high salinity (Dunaliella), alkalinity (Spirulina), and nutrition (Chlorella sp.) (Singh et al., 2015)

Closed culture systems are used specifically for the growth of unicellular organisms under completely sterile conditions, ensuring the absence of contaminants in plants The development of industrial-scale optical reactors that can be operated in feasible biological and economic parameters is still underway Closed systems are dominated by tubular and flat-plate reactors, usually designed as photobioreactors (PBRs) Moreover, other options are bags, coils or domes

Table 2 2 Comparison between open and closed systems for microalgae production (Singh et al., 2015)

Parameter Open systems Closed systems Environmental impact

Biological issues Algal species Restricted Flexible

Contamination High risk Low risk

Process issues Temperature control No Yes

Use of wastewater Yes Yes

Reactor cleaning Not required Required

Costs Investment costs Low High

Co-culture of microalgae and bacteria system

In a co-culture system mixing microalgae and bacteria, both microorganisms will LQWHUDFW ZLWK HDFK RWKHU DQG LQÀXHQFH WKHLU UHVSHFWLYH JURZWK 7R VLPSOLI\ microalgae will use light as energy source and CO2 as carbon source and produce oxygen and biomass in the process On the other side, bacteria will use oxygen as electron acceptor and organic carbon as energy source to produce CO2 and water through aerobic respiration SimplL¿HGUHDFWLRQVRIWhese two processes can be found below:

Aerobic respiration needs oxygen, that is actually produced by photosynthesis Photosynthesis needs carbon dioxide, that is actually produced by aerobic respiration Microalgae and bacteria have are both bringing necessary chemical compounds to each other and are thus having a symbiosis This is the reason why no aeration is needed in a photobioreactor containing a co-culture Aeration providing O2 and CO2 is here not necessary Figure 2.2 presentsthe symbiosis between microalgae and bacteria and the outlets for the recovered biomass

Figure 2.2 Symbiosis interactions between microalgae and bacteria [16]

However, the interactions between microalgae and bacteria are more complex than a simple nutrients exchange For example, bacteria can use microalgae as habitat, to be protected from GL൶FXOW environmental conditions Furthermore, it has been shown that bacterial and microalgae growths released extracellular polymeric substances that could stimulate the growth of each other species [45] On the other hand, both FXOWXUHVFDQDOVRKDYHQHJDWLYHH൵HFWVRQHDch other For example, the photosynthesis will induce an increase in pH and temperature of the aquatic media, which can have QHJDWLYHH൵HFWVRQEDFWHULDOJURZWK Furthermore, it has been reported that bacteria can excrete metabolites presenting an algicidaOH൵HFW[45]

Overall, numerous interactions occur between the bacterial and the algal communities and understanding all WKHH൵HFWVLVFRPSOH[GHVSLWHDOOWKHVWXGLHVFRQGXFWHGRQWKH 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 ¿[DWLRQE\WKHPLFURDOJDHZKLFKFDQFHOVWKHJUHHQKouse gas emission occuring in activated sludge tanks

Wastewater treatment by co-culture Numerous studies have been conducted on wastewater treatment by co-culture and have shown promising results with high QXWULHQWDQG&2'UHPRYDOH൶FLHQFLHV7KHQXWULHQW UHPRYDOH൶FLHQF\GHSHQGVRQ PDQ\ GLYHUVH IDFWRUV VXFK DV PLFURRUJDQLVPV VSHFLHV V\VWHP FRQ¿JXUDWLRQ operational mode, wastewater source and composition, among others This variability RIFRQ¿JXUDWLRQVPDNHVLWGL൶FXOWWRFRPSDUHWKHGL൵HUHQWVWXGLHVDQd their results LQWHUPVRI&2'DQGQXWULHQWUHPRYDOH൶FLHQF\7KHQH[WSDUDJUDSKVZLOOOLVWDQG compare some previous studies results in terms of COD, nitrogen and phosphorus UHPRYDOH൶FLHQF\

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 only 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 VWXGLHVKDYHLQYHVWLJDWHGWKH&2'UHPRYDOH൶FLHQF\RIFR-cultures to know more about the roles of both microalgae and bacteria

[46] FRXOGUHDFKKLJKUHPRYDOH൶FLHQF\RIFKHPLFDOR[\JHQdemand (86±98%), by applying sequencing batch mode of 24 hours cycle in photobioreactors and light intensity of 235 àmol/m2/s [47] REWDLQHGDQGRI&2'UHPRYDOH൶FLHQF\ for 360 and 820 àmol/m2/s light intensity values, respectively [15] obtained a COD UHPRYDOH൶FLHQF\RIVLPLODUYDOXHVIRUGL൵HUHQWPLFURDOJDHEDFWHULDUDWLRDQG FRQFOXGHGWKDWWKLVSDUDPHWHUGRHVQRWLQÀXHQFHWKH&2'UHPRYDOH൶FLHQF\2QO\ algae showed D&2'UHPRYDOH൶FLHQF\RIZKLOHRQO\DFWLYDWHGVOXdge (73.6 ±5.1%), proving the potential of co-culture water treatment compared to only activated sludge or microalgae treatment Furthermore, [17] treated low COD/N ratio (4.3) wastewater and had as results COD remoYDOH൶FLHQFLHVRIDQG82.7% in activated sludge, C vulgaris and the co-culture systems, respectively

[Huang et al., 2015] removed 96.1% and 95.2% in his illuminated and not illuminated reactors, respectively

Finally, [45] summarized in her literature review that co-culture systems can reach excellent &2' UHPRYDO H൶FLHQF\ XVXDOO\ KLJKHU WKDQ GHSHQGLQJ RQ WKH 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 signL¿FDQWO\FKDQJHWKH&2'UHPRYDOH൶FLHQF\

In a co-culture, nitrogen compounds present in the aquatic medium can be removed E\DVVLPLODWLRQLQWRPLFURDOJDHELRPDVVQLWUL¿FDWLRQDQGGHQLWUL¿FDWLRQE\ EDFWHULD¿[DWLRQRIatmospheric N2 by prokariotic microalgae (cyanobacteria), and (4) NH4 stripping by strong aeration and high pH Nitrogen is essential for microalgae growth, as it composes genetic material, enzymes, proteins, hormones, vitamins, alkaloids, amides, and energy transfer molecules [48] Ammonia-Oxidizing Bacteria (AOB) and Nitrite-Oxidizing Bacteria (NOB) obtain energy through QLWUL¿FDWLRQ E\ using inorganic nitrogen compounds as electron donors Through GHQLWUL¿FDWLRQXQGHUDQR[LFFRQGLWLRQV123 N is reduced and ultimately molecular nitrogen (N2) is produced Diverse studies have been conducted to understand the nitrogen removal process

[35] showed in his study that co-FXOWXUHVZLWKOLJKW H[SRVXUHZHUH OHVVH൶FLHQWLQ ammonium removal than co-cultures without light exposure NH4 +-N was almost completely removed (99%) after 10 days without light exposure and after 40 days with natural light exposure, starting with a concentration of 100 mg/L [49] tried GL൵HUHQW UDWLRDFWLYDWHGVOXGJHPLFURDOJDH$60$WRWUHDWZDVWHZDWHUDQG¿QGRXWWKDWWKHQLWURJHQUHPRYDOH൶FLHQF\LQFUHased with higher microalgae proportion He reached 96.59 ±0.37% and 97.58 ±0.26% with AS:MA of 1:0.75 and 1:1 respectively His conclusion was that co-culture systems clearly show a higher nitrogen removal H൶FLHQF\ WKDQ VROHO\ PLFURDOJDH RU VROHO\ EDFWHULD systems Furthermore, he

VXJJHVWHGWKDWWKHELRGHJUDGDEOH&2'QHHGHGIRUGHQLWUL¿FDWLRQFRXOGEHWhe decayed microalgae biomass present in the co-culture, thus saving the expensive costs of additional COD source supplementation

[50] achieved a 99% NH4 +-N removal, and observed that stronger light exposure (225 àmol/m 2 /s) inhibited the nitrite oxidation, causing a NO2 accumulation in the reactors [15] found out that for 91% of nitrogen removDO H൶FLHQF\ LQ KLV KLJKHVW performance reactor (algae/activated sludge = 5), 60% was assimilation into algal- EDFWHULDOELRPDVVDQGQLWUL¿FDWLRQRQO\WKHUHVWEHLQJSUREDEO\1+4 stripping 7KHORZHVWQLWURJHQUHPRYDOH൶FLHQF\E\FR-culture was 58.6%, which was higher than only sludge culture (18.6%) and only algae (41.7%).

In his literature review, [48] FRQ¿UPHGWKHH൶Fiency of a co-culture system to remove nitrogen from wastewater, by listing some studies, that showed high performance QLWURJHQUHPRYDOH൶FLHQF\-100%)

Phosphorus is an essential macronutrient in the growing process of microalgae Phosphorus compounds can be removed from aquatic medium by (1) assimilation into microalgae biomass, (2) activity of Polyphosphate-Accumulating Organisms (PAOs) and (3) chemical precipitation at high pH values (above 8) and high oxygen concentration [45] POAs have the ability to consume simple carbon compounds (energy source) without the presence of an external electron acceptor (such as nitrate or oxygen) Numerous studies took interest in the pKRVSKRUXVUHPRYDOH൶FLHQF\DQG PHFKDQLVPVDVZHOODVLQWKHLQÀXHQFHRIRSHUDWLQJSDUDPHWHUV

[50] showed WKDW7RWDO3KRVSKRUXV73UHPRYDOH൶Fiency increased with stronger light intensity (42% with 225 àmol/m 2 /s compared to 31% without light exposure) and concluded it was due to a faster algae growth [35] observed that algae growth PLJKWD൵HFWWKHDFWLYLW\RINOB (nitratation process), resulting in Free Nitrous Acid (FNA) accumulation and subsequent inhibition on PAOs

[51] VWXGLHG WKH LQÀXHQFH RI WKe activated sludge/microalgae ratio (AS/MA) and reached 92% and 89% phosphate removal with AS/MA ratio of 0.5 and 1, respectively [15] UHDFKHG D SKRVSKRUXV UHPRYDO H൶FLHQF\ RI ZLWK D microalgae/sludge ratio of 5, showing better treatment performances than only algae system (54.4%) and only activated sludge (10.6%) The phosphorus removal capacity of the sludge depends on the presence of POAs The photosynthesis of the algae was probably limited in the system with only algae, because of the lack of CO2 supply Indeed, no sludge was present to release CO2 and the only supply was from the air Furthermore, [52] REWDLQHGDSKRVSKRUXVUHPRYDOH൶FLHQF\DQGVXJJHVWHGWKDW phosphorus assimilation by the biomass was the main mechanism for its removal [17] FRQ¿UPHGZLWKKLVUHVXOWVWKDWDFR-culture system reaches higher phosphorus removal performances than only bacteria or only microalgae systems He also mentioned another important mechanism to understand decrease or increase in phosphorus concentration in the aquatic medium: some bacterial species are able to assimilate and store phosphate in the form of polyphosphates under aerobic environment, and release them from the biomass into the medium under anaerobic environment, thus increasing the P concentration in the medium Finally, [45] listed results from previous studiHV LQ KHU OLWHUDWXUH UHYLHZ DQG FRQ¿UPHG WKH H[FHOOHQW potential in phosphorus removal by a co-culture system

2.2.1 Microalgae-activated sludge under photobioreactor

A microalgae-activated sludge treatment system is GH¿QHG E\ LWV RSHUDWLQJ parameters and its operational mode In previous studies about co-culture systems using photobioreactors, the most common operating parameters include (1) light intensity and cycle (dark and light phases), (2) Height/Diameter (H/D) of the reactors, (3) agitation speed (shear stress), (4) wastewater composition, particularly in terms of COD/N and N/P ratios, (5) initial biomass concentration and microalgae/activated sludge ratio and (6WHPSHUDWXUHDQGS+LIPRQLWRUHG7KHVHFWLRQ´,QÀXHQFHIDFWRUV on co-FXOWXUH´ZLOOJLYHVRPe common values of these parameters, and explain their LQÀXHQFHRQWKHFR-culture systems

Domestic wastewater

Domestic wastewater is mainly comprised of water (99.9%) together with relatively small concentrations of suspended and dissolved organic and inorganic solids Among the organic substances present in sewage are carbohydrates, lignin, fats, soaps, synthetic detergents, proteins and their decomposition products, as well as various natural and synthetic organic chemicals from the process industries Table 2.4 shows the levels of the major constituents of strong, typical and weak domestic wastewaters Domestic wastewater also contains a variety of inorganic substances from domestic and industrial sources, such as nitrogen and phosphorus, also including a number of potentially toxic elements such as arsenic, cadmium, chromium, copper, lead, mercury, zinc, etc However, from the point of view of health, a very important consideration in agricultural use of wastewater, the contaminants of greatest concern are the pathogenic micro- and macro-organisms Pathogenic viruses, bacteria, protozoa and helminths may be present in raw municipal wastewater and will survive in the environment for long periods

Table 2 3 Composition of Untreated Domestic Wastewater [55]

Note: All units in mg/L

Membrane Photobioreactor

A typical membrane photobioreactor (MPBR) is commonly known as a continuously operating system, combined conventional PBR with an external filter or submerged membrane using microfiltration or ultrafiltration membranes (hollow fibers and flat sheets) for solid-liquid separation (Honda et al., 2012) The MPBR is designed and operated to increase access to light (high surface/volume ratio) and facilitate the microalgae development, such as mixing feed, CO2 absorption capacity, and reduce the construction and operation costs (Chiu et al., 2015) MPBR allows for optimal control of operating conditions or increased CO2 absorption capacity into the culture medium and reduced the microbial contamination These are the conditions that cannot find in closed PBR systems (Chiu et al., 2015) Cultivation microalgae

Chlorella for biomass and lipid production using wastewater as nutrients resource

Using of membranes aims to provide the maintenance of algae cells, avoid leaching and is capable of separating the biomass retention time (MRT) and hydraulic retention time (HRT) (Honda et al., 2012) The separation of HRT and SRT allows MPBR to produce 3.5 times the biomass concentration of PBR (Marbelia et al., 2014)

MPBR is classified by two functions (Bilad et al., 2014a) (2): (1) Membrane Photobioreactor is responsible for maintaining biomass (BR-MPBR) and (2) Membrane Photobioreactor used to transport CO2 into the farming environment (Bilad et al., 2014)

Advantages of MPBR technology compared to PBR: x High biomass concentration: Use membranes to maintain microalgae cells thus solving biomass leaching problems (Chiu et al., 2015) Increasing the value of biomass if it continues processing into raw materials, producing biofuel, human and animal nutrition, using biological fertilizers, pharmaceuticals, cosmetics due to the chemical composition of algae x Flexibility in operation: With the filtration effect of the submerged membrane module, suspended microalgae cells in the reaction can be completely isolated from the wastewater Therefore, the concentration of algae and large wastewater can be achieved simultaneously, and the maintenance time of HRT and biomass (BRT) of biochemical photosynthesis can be completely separated Therefore, we can operate and maintain optimal growth conditions for microalgae (Judd, 2010) x Effluent quality: High concentrations of microalgae and high nutrient content in membrane photobioreactor to maintain the rapid growth of microalgae In this way, microalgae yields and high nutrient removal rates when the reaction operates at a rate that provides a large culture environment With higher biomass yield, MPBR increases the ability to absorb nutrients in the culture medium, which is the potential for treating wastewater combined with microalgae cultivation (Honda et al., 2012) x Space saving: Due to the higher biomass concentration, the system size is smaller than MPBR (Chiu et al., 2015) result in a reduced construction area

Disadvantages of MPBR technology: x High costs: During operation, energy consumption for disturbance and lighting is higher due to high biomass concentration in the tank (Judd, 2010) n addition, the flux is reduced due to congestion, MPBR is regularly cleaned to restore permeability through backwash, chemical cleaning, and replacement, resulting in high maintenance and operation costs (Chiu et al., 2015) x Membrane durability: During operation, the membrane is affected by many IDFWRUVVXFKDVGLUWZDVWHZDWHUTXDOLW\HWFôGXUDELOLW\UHGXFHGRYer time of operation The membrane after a while will be clogged with dirt (Judd, 2010), Reducing permeability and speed of microalgae production leads to a reduction in biomass production efficiency

Most membrane technologies used in the cultivation and harvesting of microalgae are limited by membrane clogging Membrane fouling is the result of interactions between membrane materials and biomass (including substrate components, cells, debris, and microbial metabolites) that shrink the membrane size or clogged, leading to a decrease in permeability flux flow as well as an increase in current resistance through the membrane (Chang et al., 2002) Membrane fouling control can improve membrane filtration, so that specific studies are needed to optimize operating conditions In microalgae cultivation, membranes are commonly used MF, UF, and

Membrane blockage is characterized by increased membrane transfer pressure (TMP) or reduced throughput during continuous operation Membrane dirt can be caused by physical or biological mechanisms and is increased by the polarization concentration thereby increasing the concentration of contaminants in the area approaching the membrane The degree of dirt is determined by membrane materials, environmental characteristics and operating parameters (Bilad et al., 2014a) Membrane fouling depends on the specific characteristics of the material, the filtration method and the configuration of the membrane, but the biological membrane fouling is mainly due to the growth of microorganisms, in particular, due to the two main components : protein and colloidal particles (colloids) Membrane dirty mechanism of the environment containing cells living in MPBR is summarized briefly as follows (Drews, 2010): x In the initial filtration process, dirt (including colloids, solutes, bacteria (if any) and microalgae cells are retained on the membrane surface due to the convective flow of seepage flow The dirt is smaller than the size of the diaphragm, it will penetrate the membrane and be stuck there, while the contaminants larger than the diaphragm will block the membrane x After that, a layer of cake is deposited on the membrane surface This cake can also act as a secondary filter, often called a membrane Living cells then accumulate on the surface of the cake to form a complex biofilm

After a period of use, the membrane will reduce permeability due to many reasons, so it is necessary to have the membrane cleaning method to increase treatment efficiency and membrane durability Common membrane clearning method (Lim and Bai, 2003):

(1) Sound waves: remove plaque by breaking larger smaller walls by ultrasonic waves

(2) Backwash: remove biological particles and dirt from the membrane

(3) Chemical cleaning: use HCl and NaOCl to wash membranes

(4) The cleaner combination: Summary of above membrane cleaning methods

Table 2 4 Flux recovery performance by membrane cleaning methods (Lim and

METHODOLOGY

Microorganism, wastewater and membrane module

The microalgae strain used in this study was taken from Aquaculture Research Institute 2- Ministry of Agriculture and Rural Development, Vietnam This strain is

Chlorella sp which is capable of nitrogen and phosphorus uptake as nutrient sources

The strain was cultivated and maintained in a sterilized Bold´s Basal Medium (BBM)

Chlorella sp was incubated in a bubble column photobioreactor (PBR) (20 cm diameter, 60 cm height) under typical conditions: the light LQWHQVLW\RIȝPROP -2 s -1 and at room temperature and air aeration, which has been fully described previously [56] [57] The pre-cultured algae cells were taken during the log phase Such action was ¿rstly settled down for 12 h to remove the supernatants, followed by centrifuged at 3600 rpm for 10 min and washed twice with deionized water before it was used for inoculation in the following experiments

The activated sludge was collected from an aerobic reactor in a local wastewater treatment system Mixed liquid suspended solids (MLSS) has a concentration of about 4000 mg L -1 Prior to experiments, the activated sludge was also settled down for 3 h to remove the supernatants, followed by centrifuged at 3600 rpm for 10 min The settled solids washed twice with the distilled water before it was used as source of bacterial consortium

The synthetic wastewater (using for task 1) was prepared with the detail components denoted in Table 3.1 The main components are acetate (medium A), NH4CL (medium B), KH2P04 (medium C) used as the carbon and nutrient sources for cultivation As prepared the synthetic wastewater contains COD 500 mg L -1 , NH4 +-N

200 mg L -1 , TP 45 mg L -1 and is remained at pH 7.6, N/P ratio of 4.4 A low COD/N ratio remained about 2.5, which facilitate for microalgae biomass enrichment and nutrient recovery

Table 3.1 Components of synthetic wastewater

20 Medium I -The trace elements stock solution 1 mL

The composition of the trace element stock solution was:

H3BO3 2.86 g L -1 , MnCl2ã4H2O 1.81 g L -1 , ZnSO4ã7H2O 0.22 g L -1 , CuSO4ã5H2O 0.079 g L -1 , CoCl2ã6H2O 0.05 g

Supermarket wastewater (using for task 2) after preliminary screening, grit removal and primary sedimentation process from the same plant was feed into the MPBR to investigate the organic PDWWHU UHPRYDO DQG QXWULHQW UHPRYDO HI¿FLHQFLHs The characterization of wastewater was COD 185±42 mg L -1 , TKN 35.2±7.3 mg L -1 ,

NH4 +-N 19.4±4.2 mg L -1 , TP 4.9±1.3 mg L -1 , NO3 - -N 0.2±0.2 mg L -1 , NO2 - -N 0.1±0.1 mg L -1 and pH 7.6±0.3 Low relatively COD/N ratio about 5.2±1.5 and N/P 10±2, which facilitates for microalgae biomass enrichment and nutrient recovery

The hollow fiber column membrane used to set up the MPBR was fabricated by inserting a cluster of 18 polypropylene hollow fiber membranes (UF 06-12 S2 F, Polymem, France) into U-PVC shelland sealing them at both ends of the shell The SRUHVL]HRIWKHPHPEUDQHPRGXOHZDVȝPDQGWKHH[WHUQDOVXUIDFHDUHD was 0.0102 m 2 (with the inner diameter of 0.39 mm, and external diameter of 0.74 mm).

Overall reseach content

The experiment work of this research was divided into 2 main parts corresponding to

1) Co-culture of microalgae:activated sludge at different microalgae:activated sludge ratios 1:0, 9:1; 3:1; 1:1; 0:1 (wt:wt) in Photobioreactors to determine the optimum microalgae:activated sludge inoculation ratio for nutrients removal, COD removal Based on the results of the decreased in pollution concentration (nutrients such as nitrogen, phosphorus and organic matter) over time to evaluate the efficiency treatment Biomass evaluation base on chlorophyll-a was measured and converted to dry weight biomass to determine the specific growth and yield of microalgae and activated sludge

2) Operational Membrane Photobioreactor with real wastewater (supermarket wastewater) at optimum microalgae:activated sludge inoculation ratio obtained in (1), fixed HRT of 1 day and various BRT of 3, 5, 7, 10 days to evaluate treatment performance and membrane fouling

During the experiment, water quality parameters (COD, TP, TKN, NH4 +-N, NO3 N,

NO2 N); culture environmental conditions (pH, temperature, DO, light flux density) and biomass evaluation (TSS, biomass base on chlorophyll-a, cell counting, microscope) were measured and analyzed In addition, for task 2, membrane fouling behaviour was investigated base on concentration of soluble EPS (protein and polysaccharides) and transmembrane pressure (TMP), particle size distribution (PSD) were considered to be at steady-state conditions in MPBR system The process could be described as the following figure 3.2

(Batch culture with synthetic wastewater)

Microalgae:Activated sludge ratios of : 1:0, 9:1, 3:1, 1:1, 0:1

To define optimal inoculum ratio

(for simultaneous nutrient/ organic matter removal and biomass production)

(Continuous operation with supermarket wastewater)

Effect of BRT (Biomass retention time) various of 3, 5, 7, 10d

To evaluate treatment performance (N, P, COD removal), biomass production and fouling behavior under various BRTs

Experimental design

3.3.1 Task 1: Defind optimal inoculum ratio of microalgae:activated-sludge under photobioreactor

The stirred photobioreators (PBRs) were used for all batch experiments The transparent glass reactors have the working volume of 14 L and the dimension of length x diameter = 60 cm x 20 cm A wooden box with a thickness of 100mm was installed to cover the PBRs and this helps preventing the loss of light Led lamps were

Task 2 rolled around the PBRs so as to provide certain light intensity of 100 ȝPROP -2 s -1 All batch reactors were operated under a cycle of 12 h light±12 h dark Constant mixing was maintained using a stirrer (100 rpm) to avoid algae sedimentation Detail schematic diagram of the PBRs system is illustrated in Figure 3.3

Figure 3.3 Schematic diagram of the PBRs system

Five reactors were prepared with different inoculum ratios: pure culture of microalgae (1:0 wt/wt); co-culture of microalgae and activated sludge under the ratios of (9:1, 3:1, 1:1 wt/wt); and only activated sludge (0:1 wt/wt) Total initial biomass concentration was remained about 400 mg L -1 for all reactors Detailed initial concentrations of microalgae and activated sludge were presented in Table 3.2

Table 3.2 Initial concentration of microalgae and activated sludge under different inoculation ratios

Microalgae: activated sludge inoculation ratios

Initial conc of microalgae (mg L -1 ) 400 360 300 200 0 Initial conc of activated sludge (mg L -1 ) 0 40 100 200 400 Initial conc of inoculum biomass (mg L -1 ) 400 400 400 400 400

3.3.2 Task 2: To evaluate treatment performance (N, P, COD removal), and biomass production and fouling behavior of MPBR under various BRTs

The experiments were performed in MPBR as a stirred tank PBR which were made of transparent acrylic plastic (60 cm in height x 10 cm in internal diameter) combined a hollow fiber column membrane MPBR operating under continuous conditions, the working volume was 4 L The PBR reactor was installed led lamps rolled around were used to evenly irradiate the PBR with about 100 àmol m -2 s -1 (measured at the top of liquid surface) for a 12 h light±12 h darkcycle Constant mixing was maintained using a stirrer (100rpm) to avoid biomass sedimentation The hollow fiber column membrane used to set up the MPBR was fabricated by inserting a cluster of 18 polypropylene hollow fiber membranes (UF 06-12 S2 F, Polymem, France) into U- PVC shelland sealing them at both ends of the shell The pore size of the membrane PRGXOHZDVȝPDnd the external surface area was 0.0102 m 2 (with the inner diameter RIPPDQGH[WHUQDOGLDPHWHURIPP7KHIHHGDQGRXWÀRZRI the PBR were pumped at the same rate using a multi-channel peristaltic pump In the PHPEUDQH PRGXO WKH RXWÀRZ FRQVLVWV RI D QXPEHU RI FKDQQHOV FRQVLVWLQJ RI permeate lines from the membrane and of retentate line TMP sensor was installed on a pipe connected with the pump and retentate line The schematic diagram of the MPBR is presented in Figure 3.4

Figure 3.4 The schematic diagram of the MPBR

Particularly, for the operating conditions, the MPBR maintained 24 hours for hydraulic retention time (HRT) and changed of 10, 7, 5, 3 days for biomass retention time (BRT) To control the BRT, correspoding BRT of 10, 7, 5, 3 days, the volume of waste biomass were 0.40, 0.57, 0.80, 1.33 L d -1 , respectively The MPBR was operated with a flux of 16.5 L m -2 h -1 under cross flow velocity (CFV) passed into the membrane was 1 m/s Once the MPBR reached 40kPa, physical cleaning was applied to clean the membrane module with tap water Chemical cleaning for membrane module was conducted when BRT changed to recover membrane permeability The membrane was firstly washed with tap water to remove the cake layer on the membrane surface before immersed in sodium hypochlorite the solutions (NaOCl 0.5% v/v) and sodium hydroxide (NaOH 4% v/v) for 8 hours

Analytical parameters

The dissolved oxygen (DO) concentration was measured using a DO meter and the pH was measured by a pH meter Light intensity was directly measured using a submersible spherical light sensor (US-SQS/L, ULM-500, FA Walz, Germany) The trans-membrane pressure (TMP) was measured by a digital pressure gauge, used as an indicator of the membrane fouling propensity

Prior to analyses, 250 milliliters of samples were collected at the midway of the PBR with a valve every day and at the moment near the end of the dark phase Such parameters as COD, TP, TKN, NH4 +-N, NO3 N, NO2 N, MLVSS were analyzed according to standard methods [58] In addition, to determine the size flocculation of biomass, a particle size distribution (PSD) analysis was performed using a Laser Scattering Particle Analyzer (Horiba LA-950, Japan) with ranged from 0.01 ± 3000 ȝP

Microbial biomass analyses

The total biomass in reactors was defined through measuring the dry weight In detail,

10 mL of mixed liquid samples for all reactors were taken every day for analyses All samples were filtered using a membrane with a pore size of 0.45 àm (Fisher Whatman puradisc-25 mm), followed by being dried at 105 °C for 2 hours and then weighed Dry biomass was defined based on a change in weight between before and after filtered samples For the co-culture system, the dry biomass includes microalgae and activated sludge features (C = C m + C b ) Where C is the total biomass concentration

(g L -1 ), C m is the microalgae biomass concentration (g L -1 ), C b is the activated sludge concentration

For the microalgae biomass (C m ), it was measured through Chlorophyll-a content extracted from the microalgae cell Chlorophyll-a concentration was defined based on the previous method [54] Such concentration was then converted to dry weight through a standard curve with the equation: y = 4216.4 x ± 302.43 This equation performs the correlation between Chlorophyll-a concentration and the dry weight of microalgae The standard curve was presented in Fig S-2 Where y is the concentration of Chlorophyll-a, and x is the dry weight of microalgae

Chlorophyll-a content was extracted using an acetone solution Firstly, a 40 mL sample taken from either pure culture or co-culture systems was centrifuged at 4000 rpm for 10 minutes After supernatants were discarded, the residual features were mixed with 90% acetone solution and 0.05 g CaCO3 and then was sheared using a vortex mixer for 1 minute Secondly, such suspension was stored at 4 °C for 24 h in darkness before it was centrifuged at 4000 rpm for 10 minutes for supernatant recovery These supernatants were used to determine the Chlorophyll-a content In detail, Chlorophyll-a concentration was measured using ultraviolet spectrophotometry under different wavelengths: 630, 645, 663, 750, 772, and 850 nm 90% Acetone solution was used as the blank As reported the Chlorophyll-a concentration of microalgae in a co-culture system was defined as Eq (1) [51]: ୫ ൌ ሾଵଵǤ଺ସሺ୓ୈ లలయ ି୓ୈ ళఱబ ሻିଶǤଵ଺ሺ୓ୈ లరఱ ି୓ୈ ళఱబ ሻା଴Ǥଵ଴ሺ୓ୈ లయబ ି୓ୈ ళఱబ ሻିଶହǤଶሺ୓ୈ ళళమ ି୓ୈ ఴఱబ ሻሿ୚ భ ୚Ǥ஢ (1)

Where V is the sample volume (L), V1 is the volume of acetone-based extract (mL), 2' 2SWLFDO 'HQVLW\ LV DEVRUEHQF\ DW D FRUUHVSRQGLQJ ZDYHOHQJWK DQG ı LV the optical path of the cuvette (cm) Also, such parameters as total biomass productivity, specific growth rate, and specific uptake rate were expressed as Eq (2), (3), (4)

Total biomass productivity (ȕmg L -1 day -1 ): ߚ ൌ ௑ି௑ ೚ ௱௧ (2)

The specific uptake rate (݅, g gbiomass -1 d -1 ): ᢡ ൌ ஼ ೚ ି஼ ሺ௑ି௑ ೚ ሻ௱௧ (4) Where, X 0 , X are total biomass at the initial and final time of the log phase; C 0 and C represent for the substrate concentration at the initial and final time of phase; ǻW is the interval days e.g., from the initial time to a time which total biomass reach steady state.

Nitrogen mass balance

Mechanism removal of nitrogen was defined according to a mass balance Total nitrogen was calculated as follows: TN = TKN + NO2 - + NO3 - The contributions to nitrogen mass include TN uptake by biomass, TN stripping, TN-denitrification, and residual TN Since all batch reactors was operated under the stirred condition and remained a pH of 7.5-9, TN stripping has negligible contribution Thus total nitrogen mass balance can be defined as follows:

Initial TN = Residual TN + TN-denitrification + TN uptake by biomass (5) Whilst TN uptake by biomass might be due to contributions of either microalgae or bacteria assimilation, TN-denitrification is obtained from bacteria metabolism The nitrogen content in biomass was referred from a past work that used a synthetic wastewater for cultivation [17] As reported the nitrogen content in biomass cultivated with activated sludge and microalgae system were 4.04% and 7.73%, respectively whereas this content was 8.25% for co-culture system Such values were used to calculate the TN uptake by biomass.

Determination of membrane resistances

The membrane resistances were determined based on our previous work [59] The calculation was employed with the Darcy equation (Eq 6 and Eq 7) In brief, recording flux -DQG703ǻ3ZHUHXVHGWRGHWHUPLQHWKHUHVLVWDQFHEDVHGRQ(T6 and Eq 7 After completed every BRT investigation, and TMP reached 40 kPa, the membrane module was taken out to filter with the pure water, which was used to determine the total resistance (Rt) The cake resistance (Rc) regarded as deposition of the cake layer onto membrane surface that can be completely flushing using tap water Thus, the total of (Rf+Rm) can be defined by the filtration of pure water with removing the cake layer Subsequently, Rc can be calculated by subtraction of the total resistance (Rt) and the total of (Rf+Rm) After that, the membrane was soaked in the cleaning agents of 0.5% NaOCl and 4% NaOH for 8 hours to determine the membrane resistance Finally, Rf is determined using Eq 7

Where, J is WKHSHUPHDWHIOX[ǻ3LVWUDQV-PHPEUDQHSUHVVXUH703ȝLVWKHYLVFRVLW\RI permeate; Rt is the total resistance; Rm is the intrinsic membrane resistance; Rc is the cake layer resistance and Rf is the fouling resistance caused by the adsorption of soluble matters and the pore blocking.

Statistical analysis

Results was showed as the average value ± standard deviation Parametric one-way analysis of variance (ANOVA) was used to examine significant differences among groups of samples using the IBM SPSS Statistics software 20 p < 0.05 indicated significance at 95% confidence.

RESULTS AND DISCUSSION

Task 1: Defind optimal inoculum ratio of microalgae:activated-sludge under

4.1.1 Effect of inoculum ratios on biomass growth and variation of dissolved oxygen and pH

As denoted in Fig 4.1, it was generally accepted that after 5 d total biomass concentration increased from 0.4 g L -1 to 1 g L -1 for either single microalgae culture or co-culture systems i.e., inoculum ratios of 1:0, 9:1 and 3:1 wt/wt (Fig 4.1a, b, c) For 1:1 ratio, the total biomass concentration reached to 1 g L -1 after 10 d On the other hand, total biomass concentration of 0:1 ratio obtained a slight increase to 0.6 g L -1 The maximum total biomass concentrations were obtained at day 6 th yielding 1.12 g L -1 and 1.07 g L -1 for 1:0 and 3:1 ratios, respectively However, this fact occurred a later stage i.e., on day 9 th for 9:1 ratio (1.1 g L -1 ), on day 11 th for 1:1 ratio (1.08 g L -1 ) Such findings indicate the initial inoculum ratio impacted on biomass growth in some certain extent Higher initial fraction of microalgae using for co- culture system could be proper condition so as to shorten acclimatization period The biomass fractions of microalgae and activated sludge also changed following the inoculum ratios (Fig 4.1) Only the fractions of 9:1 ratio were unchanged, being around 90%, during experimental period (Fig 4.1b) It is important to note that a major change of biomass fraction was observed between the initial and final stage of experiment for 3:1 and 1:1 ratios (Fig 4.1c, d) Especially for latter ratio, the fraction of activated sludge remained stable (50%) for the first 4 d, then it decreased gradually to 18% on the last day The reason laid on the composition of feeding wastewater for microalgae and sludge which comprised a low COD/N ratio (2.5:1) This low COD/N ratio could augment biomass yield of microalgae rather than bacteria [11] Microalgae could also assimilate organic carbon competitively with bacteria in photoheterotrophic condition [60] Thus, upon organic matter in feeding wastewater exhausted since day 4 th , bacteria growth depleted and their fraction in total biomass decreased (Fig 4.1)

Figure 4.1 Total biomass, biomass fraction of microalgae and activated sludge under different inoculation ratios (microalgae: bacteria wt/wt): (a) 1:0, (b) 9:1, (c) 3:1, (d) 1:1, (e) 0:1 and (f) Total biomass productivity under ratio conditions

Another point, it was found that the PBRs operated with higher initial activate sludge concentration could render to decrease the maximum microalgae biomass This fact implies suspended activated sludge would interfere photosynthesis process of microalgae by reducing light intensity for microalgae cells As a result, photosynthesis yield was diminished and biomass yield of microalgae dropped Our calculation showed that the specific growth rates of microalgae decreased proportionally with the decrease of microalgae:sludge ratios (Table 4.1)

Table 4.1 6SHFL¿FJURZWKUDWHRIPLFURDOJDHDQGDFWLYDWHGVOXGJHXQGHUGLIIHUHQW inoculum ratios

Specific growth rate (d -1 ) Inoculum ratios

In practice, high specific growth rates of microalgae in 1:0, 9:1 and 3:1 ratios offered benefit as this fact reduced retention time and reactor volume To further define a proper inoculum ratio for biomass production, total biomass productivities of all ratios were calculated and compared (Fig 4.1f) The result was all pure microalgae culture (ratio 1:0) or co-culture (ratios 9:1, 3:1, 1:1) possessed superior total biomass productivity than the single activated sludge culture (ratio 0:1) (One-way ANOVA, p < 0.05) Such findings reinforce the vital role of microalgae on the biomass production under the following typical conditions: wastewater having low COD/N (2.5:1), photoheterotrophic, stirred condition and light: dark cycle of 12:12 The ratio of 3:1 showed comparable total biomass productivity with 1:0 ratio (One-way ANOVA, p > 0.05), being 136 ± 10 mg L -1 d -1 and 144 ± 10 mg L -1 d -1 , respectively However, 3:1 ratio exhibited significant higher total biomass productivity than other ratios i.e., 9:1 (88 ± 20 mg L -1 d -1 ) and 1:1 (58 ± 20 mg L -1 d -1 ) (One-way ANOVA, p < 0.05) The findings suggested that ratio 1:0 and ratio 3:1 were the optimal one for total biomass production The pure microalgae culture (ratio 1:0) could produce high biomass when feeding wastewater for cultivation [61]±[63] As previously reported microalgae biomass has been utilized for biofuel production [5], [6] Therefore, for the co-culture system, the 3:1 ratio might be an alternative way to attain beneficial biomass production, with high microalgae biomass attained 0.95 g/L To come up with the conclusion which inoculum ratios was optimal in this study, we further investigated performance of those ratios for pollutant remediation in section 4.1.2.1 and 4.1.2.2

Regarding DO, it is an indicator for microalgae biomass growth ( Morales et al., 2018; Kazbar et al., 2019) and thus it was recorded for all the studied ratios (Fig 4.2a,b) For 0:1 ratio (sludge only), DO concentration stayed below 0.5 mg L -1 since day 1 for the whole studied period (Fig 4.2a) This happened because the stirred condition of the experiment favored anoxic condition and consequently dropped DO level significantly [15] Such condition prefers to slow-growing microorganism, which resulted in longer acclimatization and consequently restrained microorganism growing (Nguyen et al., 2016) Meanwhile, the single microalgae system (ratio 1:0) performed differently and such DO concentration rose two-fold from 4 mg L -1 to 8 mg L -1 , indicating substantial microalgae growth Notably, compared to 1:0 ratio, ratios in co-culture systems (i.e., 9:1, 3:1, and 1:1 wt/wt) exhibited lower DO concentration at the light phase Furthermore, DO concentration is expected to be lower at the dark phase, being 0.5 mg L -1 , as observed in Fig 4.2b This meant that bacteria consortium in activated sludge consumed oxygen which released by photosynthesis of microalgae in both light and dark phases Thus, co-culture system which having higher initial fraction of activated sludge (3:1 and 1:1 ratios) possessed lower DO level (Fig 4.2a) These facts can be regarded as mutualism of microalgae and bacteria under the photoautotrophic condition (Guo and Tong, 2019) Given the aforementioned conditions, DO concentration started decreasing gradually from day

5 th and this is attributed to the reduction of microalgae biomass (Fig S-5b, d) during the death phase occurred Meanwhile, DO of 1:1 ratio decreased to approximately 0 mg L -1 on day 3 rd This can probably be subjected to oxygen released from photosynthesis of microalgae being consumed quickly by aerobic bacteria [67] Then,

DO increased gradually and remained unchanged from day 6 th to 11 th The result was consistent with the augment of microalgae fraction in those ratios (Fig 4.1d)

Figure 4.2 DO concentration and pH change at different microalgae-activated sludge inoculation ratios: (a) DO in the light phase, (b) DO in the dark phase, and

Apart from biomass production and DO, pH variation of the microalgae: sludge ratios was presented in Fig 4.2 Since the initial stage, pH value of around 7.7 was set for all culture systems For both 1:0 and 3:1 ratios, pH increased more than 1 unit in log phase, subsequently decreased slightly in death phase pH increased due to the intensive CO2 consumption from the medium by microalgae If the released CO2 from the respiration of bacteria is not sufficient for microalgae photosynthesis, the balance between CO2 from the air and CO2 uptake by microalgae tended to occur and render pH stability [15] This fact was consistent with the case of 1:1 ratio which pH was stable from 7.6 to 8.0 The pH change might be attributed to either autotrophic microalgae or and nitrifying bacteria existed in a reactor As reported, photosynthesis of microalgae increased pH whilst the nitrification process of bacteria decreased pH [67] In a co-culture system, pH is a dependent factor on biomass growth of microalgae, alkalinity and DO concentration of the media themselves [67] Notably, pH in activated system alone dropped from 7.7 to 6.5 at ratio of 0:1 This happened due to the occurrence of nitrification given it utilized oxygen, produced H + and thus reduced pH [40], [68]

4.1.2 Effect of inoculation ratio on the performance of PBRs

As shown in Fig 4.3a, TN concentration of 1:0 ratio decreased 50-fold from 200 mg L -1 to 4 mg L -1 , meant 95% TN have been removed in 6 d of operation For 0:1 ratio, TN concentration was removed slightly of 14 % in 12 d, which was due to a minor reduction of NH4 +-N concentration (Fig S6-a) As mentioned, DO concentration of 0:1 ratio stayed below 0.5 mg L -1 , which did not favor nitrification occurred This consequenced low NO2-N and NO3-N concentration in 0:1 ratio (Fig S-6c,e) After 5 d, TN removal of the co-culture ratios differed i.e., 9:1 (67%), 3:1 (86%), and 1:1 (42%), and such results indicated a co-culture system having higher microalgae fraction provided sufficient TN removal As a result from Fig 3e, under co-culture ratios of 1:0, 9:1, 3:1 wt/wt, such TN removal rates were significant higher compared to the other ratios (i.e., 1:1 and 0:1) (One-way ANOVA, p < 0.05) Notably, for the initial 4 d, although the TN removal rates obtained 40 mg L -1 d -1 , 36 mg L -1 d -1 , and 35 mg L -1 d -1 for the inoculum ratios of 1:0, and 9:1, 3:1 respectively, overall removal rates between these ratios were no significant difference based on statistical analysis (One-Way ANOVA, P > 0.05)

As reported in literature, the following conditions were needed to obtain TN removal rate of 4.06 mg L -1 d -1 : alone C vulgaris cultivation (similar to 1:0 ratio of this study), low COD: N ratio of 0.125 [69] Likewise, 2:1 ratio of co-culture system improved TN removal rate to 19 mg L -1 d -1 [1] Such findings indicated the pivotal role of microalgae for TN assimilation rate in most ratios (e.g., 1:0, 9:1, 3:1) However, to select a proper ratio, an evaluation for phosphorus removal need to be considered, and such results are discussed later

Figure 4.3 Nutrient removal in the PBRs operated under different inoculum ratios

(a) TN concentration as a function of time, (b) Mechanisms of nitrogen removal, (c)

TP concentration as a function of time

To obtain an insightful evaluation for optimal microalgae:sludge ratio and understand nitrogen transformation, NH4 +-N, NO2 N and NO3 N concentrations were measured for the whole course of experimental assay (Fig S-6a, c, e) In general, concentrations of NO2 N and NO3 N were very low for the first 2 d Later on, the concentrations started increasing gradually for all co-cultures and this might contribute to nitrification occurred The maximum concentrations of NO2 N and

NO3 N were obtained in 3:1 ratio of the co-culture system For 0:1 ratio, low NH4 +-

N assimilation was denoted as well as the concentrations of NO2 N and NO3 N appeared very minor (Fig S-6a) which was due to insufficient oxygen supply i.e.,

DO < 0.5 mg/L (Fig 4.2a, b) Through literature, it has been indicated that adequate nitrification requires DO level higher than 2.5 mg/L [66] In contrast, NH4 +-N concentration of 1:0 ratio decreased eight-fold from 200 mg L -1 to 25 mg L -1 in 4 d (Fig S-6a) indicated that assimilation was the main pathway for nitrogen remediation (Fig 4.3b)

The symbiosis of microalgae and bacteria occurred in the co-culture systems; thereby the released oxygen from photosynthesis of microalgae was consumed by bacteria towards the nitrification process However, NO3 N concentration was not high, and this fact was caused by either NO3 N denitrification or NO3 N assimilation of microalgae As reported microalgae also used NO3 N as a nutrient source for cell built-up upon exhausted NH4 +-N source [70] The mechanism of nitrogen removal could be described using results in Fig 4.3b Generally, assimilation process contributed dominantly for overall nitrogen removal Table 4.2 exhibits the results of

TN specific uptake rates for all microalgae:sludge ratios Higher microalgae fraction in a co-culture system resulted in higher nitrogen assimilation in biomass (Fig 4.3b) Compared to activated sludge, microalgae played a dominant role in biological assimilation of nitrogen It can be seen that, for ratio 1:0, TN denitrification and assimilation fractions were minor and marked 9.3% and 13.5%, respectively It rendered low TN removal Among co-culture systems, inoculation ratios having higher sludge fraction provided better denitrification pattern As observed in Fig 4.2a, DO concentration in the light phase was over 4 mg L -1 , which hindered the denitrification process It is anticipated that denitrification was possible to occur in the dark phase as a low DO < 0.5 mg L -1 was recorded However, this fact was activated in a weak manner for 9:1 and 3:1 ratios Given these conditions, visualization of the outcomes was made by microscopic images Our results confirmed a spatially adjacent microcosmic structure forming in between sludge and microalgae cells (Fig S-4a, b) Such structure supported bacteria to obtain adequate

O2 released by microalgae, it also potentially hinder bacteria from contacting the anoxic zone in the reactor and inhibited denitrification (Guo and Tong, 2014) As reported from past work [71], while the substrate extent strongly influenced nitrogen formation, their study indicated denitrification was the main contribution in a co- culture system in pig slurry WW having high COD:N ratio of 7.7:1 [71] Another one reported that with the addition of an organic carbon source to attain COD:N ratio of 3.5:1, 80% nitrogen removal was obtained from nitrification-denitrification [72] Therefore, a low COD:N ratio (2.5:1) of WW used in this study might be a final explanation for a low denitrification Overall findings indicate that the COD:N ratio and inoculum ratio posed certain effects on the nitrogen removal mechanism

Table 4.2 Specific uptake rate for different inoculum ratios

Apart from nitrogen, phosphorus played a key role for microalgae growth as it was a vital element for cell metabolism Polyphosphate-accumulating organisms (PAOs) are a vital group in activated sludge which functioned phosphorus removal [12], [13], [16] The data showed that ratios of pure microalgae and co-culture systems remediated TP in a greater extent than single bacteria culture (One-Way ANOVA, P

< 0.05) In general, TP was removed increasingly with the fraction of microalgae in co-culture systems (Fig 4.3-c) For 0:1 ratio, TP was removed insignificantly and this fact can probably be attributed to the poor presence of PAOs in the experimented cultures [73] In microalgae cultivation, TP could be assimilated by biomass which was the key mechanism [15] Therefore, in this study, the highest TP removal attributed to single microalgae culture (1:0 ratio) which remediated 98% TP after 9 d Meanwhile, for the co-culture systems, the removal efficiencies of 9:1, 3:1 and 1:1 ratios were 98%, 93% and 62%, respectively Such results indicated that bacteria did not contribute effectively in phosphorus removal rather than microalgae Another study reported that pH and DO could also impact TP remediation [8] Phosphorus could be precipitated at pH beyond 8 and high DO level In this study, pH was higher than 8 and DO exceeded 4 mg L -1 for 1:0, 9:1, and 3:1 ratios during the growth phase This implied that phosphate precipitation joined to decrease phosphorus concentration; however, this mechanism appeared very minor in the co-culture system [11] Fig S7-b shows that TP removal rates ranged from 1.6 to 7.2 mg L -1 d -

CONCLUSION AND RECOMMENDATION

Conclusion

This study evaluated the effect of inoculation ratios and biomass retention times on co-culture system of microalgae:activated sludge for wastewater treatment

For task 1: Defind optimal inoculum ratio of microalgae:activated-sludge under photobioreactor: x A certain symbiosis between microalgae and activated sludge in the co-culture PBRs of 3:1 and 1:1 ratio; x The 3:1 ratio of microalgae:activated sludge was proposed to attain simultaneously organic/nutrient removal and biomass production for low COD:N wastewater This condition performed sufficient removal of TN (86%), TP (79%) and COD (99%) and total biomass concentration of 1.12 g L -1 ; x Microalgae played a pivotal role in nutrient assimilation while activated sludge contributed to TN assimilation, denitrification and COD removal;

For task 2: To evaluate treatment performance (N, P, COD removal), and biomass production and fouling behavior of MPBR under various BRTs: x The BRTs had significant effects on pollutants removal and fouling rate in MPBR; x Prolonging the BRT was beneficial for the utilization of nitrogen while maximum phosphorus removal was achieved at shorten BRT due to withdraw biomass that contained PAOs; x The highest COD removal rate belonged to BRT of 7 days (128.65 mg L -1 d -1 ) that indicated proper one to promote cooperation between the microalgae and activated sludge for organic matter removal; x Fouling rate increased when operating at the shorted BRT, mainly due to smaller floc size of biomass.

Recommendation

x Nowadays, the great potential of wastewater treatment using co-cultures is known by researchers If the co-culture system is implemented in a continuous mode, it should be noted that influence of the factors such as wastewater (WW) compositions COD:N and N:P ratios), microalgae/ bacteria strain, the light:dark cycle, light intensity, mixing/aeration condition on pollutant removal and biomass production need to be considered for evaluation x The fundamentals underlying the formation of granules is still not fully understood Aggregation of microalgae and bacteria in a co-culture and the granulation process are still new research topics Therefore, there is no shortage of opportunities for further research x Furthermore, the influence of the wastewater composition fed to the co-culture should be addressed in future research Different COD:N and N:P ratio, as well as different organic loading rate definitely have an influence on the biomass aggregation abilities and its COD and nutrients removal efficiency x Finally, the formation of granules and their settleability characteristics under outdoor conditions would be an interesting topic for further research As the ultimate goal is to apply this treatment to wastewater from several industries, its feasibility in outdoor conditions have to be investigated

1 Nguyen, T-T., Nguyen, T-T., An Binh, Q., Bui, X-T., Hao Ngo, H., Vo, H-N., Andrew Lin, K-Y., Vo, T-D., Guo, W., Lin, C., Breider, F., ³Co-culture of microalgae-activated sludge for wastewater treatment and biomass production: Exploring their role under different inoculation ratios´, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech.2020.123754

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Co-culture of microalgae-activated sludge for wastewater treatment and biomass production: Exploring their role under different inoculation ratios

Thi-Thuy-Duong Nguyen a,b,1 , Thanh-Tin Nguyen c,1 , Quach An Binh d , Xuan-Thanh Bui a,b, ⁎ ,

Huu Hao Ngo e , Hoang Nhat Phong Vo e , Kun-Yi Andrew Lin f , Thi-Dieu-Hien Vo g , Wenshan Guo e ,

Chitsan Lin h , Florian Breider i a Key Laboratory of Advanced Waste Treatment Technology, Vietnam National University Ho Chi Minh (VNU-HCM), Linh Trung Ward, Thu Duc District, Ho Chi Minh City

700000, Viet Nam b Faculty of Environment and Natural Resources, Ho Chi Minh City University of Technology (HCMUT), Ho Chi Minh City 700000, Viet Nam c Institute of Research and Development, Duy Tan University, 03 Quang Trung, Da Nang 550000, Viet Nam d Faculty of Applied Sciences-Health, Dong Nai Technology University, Dong Nai 810000, Viet Nam e Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Sydney, NSW 2007, Australia f Department of Environmental Engineering & Innovation and Development Center of Sustainable Agriculture & Research Center of Sustainable Energy and Nanotechnology,

National Chung Hsing University, 250 Kuo-Kuang Road, Taichung, Taiwan g Faculty of Environmental and Food Engineering, Nguyen Tat Thanh University, Ho Chi Minh City, Viet Nam h National Kaohsiung University of Science and Technology, Kaohsiung 81157, Taiwan i ENAC, IIE, Central Environmental Laboratory (CEL), Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 2, 1015 Lausanne, Switzerland

In this study, mixed culture (microalgae:activated sludge) of a photobioreactor (PBR) were investigated at different inoculation ratios (1:0, 9:1, 3:1, 1:1, 0:1 wt/wt) This work was not only to determine the optimal ratio for pollutant remediation and biomass production but also to explore the role of microorganisms in the co- culture system The results showed high total biomass concentrations were obtained from 1:0 and 3:1 ratio being values of 1.06, 1.12 g L -1 , respectively Microalgae played a dominant role in nitrogen removal via biological assimilation while activated sludge was responsible for improving COD removal Compared with the single culture of microalgae, the symbiosis between microalgae and bacteria occurred at 3:1 and 1:1 ratio facilitated a higher COD removal by 37.5–45.7 % In general, combined assessment based on treatment performance and biomass productivity facilitated to select an optimal ratio of 3:1 for the operation of the co-culture PBR. https://doi.org/10.1016/j.biortech.2020.123754

Received 29 April 2020; Received in revised form 23 June 2020; Accepted 25 June 2020

E-mail address: bxthanh@hcmut.edu.vn (X.-T Bui).

1 These authors contributed equally to this work.

0960-8524/ © 2020 Elsevier Ltd All rights reserved.

Domestic and industrial activities have discharged wastewater

(WW) possessing concerned nitrogen and phosphorus level Those nu- trient compounds are the main cause of eutrophication in the receiving water reservoir because they diminish oxygen concentration for aquatic living and trigger algae bloom Consequently, excessive nitrogen and phosphorus imbalance the function of ecosystem It is a critical need to phase out nutrient elements in WW prior to discharging to avoid eu- trophication and guarantee healthy water quality for community con- sumption Generally, activated sludge processes (ASPs) are favorite ones which have been applied widely to target nutrient in the dis- charged WW stream (Mujtaba and Lee, 2017) Such processes are se- quencing batch, anaerobic, anoxic, oxic reactors and hybrid systems of those given single technology However, ASPs are restrained by high energy consumption which is mandatory to provide oxygen for bacteria respiration and nutrient metabolism process Energy for aeration ac- counts for 60% to 80% total used energy as a whole of wastewater treatment process using ASPs (Clarens et al., 2010) Another one-hybrid system (e.g., anaerobic-anoxic–oxic) is a viable option but goes along the high capital cost It is essential to propose a robust single-stage process for dual purposes of nutrient remediation and energy-efficient.

This process aims to apply for various types of wastewaters as muni- cipal wastewater, winery wastewater, piggery and, fermentation was- tewater (Godos et al., 2009; Mujtaba and Lee, 2017; Qi et al., 2018;

Microalgae have demonstrated their feasibility for wastewater re- mediation thanks to their low cost, easy operation and revenue-raising potential (e.g., bioenergy production and pigment extraction)

(Rittmann, 2008; Mata et al., 2010) They are prominent candidate to develop the above-mentioned single-stage technology Microalgae can assimilate high load of nutrient and start producing biomass in a short time (< 24 h) (Godos et al., 2009; Zhang et al., 2011) Concurrently, photosynthesis of microalgae generates oxygen given a good chance to provide oxygen for ASPs process (Mata et al., 2010) Therefore, mi- croalgae can serve as an “aeration device” and ideally replace the mechanical aeration system and cut off energy cost for aeration in ASPs

(Jia and Yuan, 2018) Based on this concept, microalgae and bacteria can mutually support each other Studies on the symbiosis of micro- algae–bacteria have been accelerating as a mean for domestic and in- dustrial wastewater remediation (Lee and Lei, 2019) Given a single- stage technology, microalgae consumes nitrogen and phosphorus in

WW for biomass production whilst bacteria metabolize organic matters in a greater extent.

As reported for the co-culture system, nutrient remediation and biomass growth are influenced by several factors, encompassing dif- ferent inoculum ratio, operating condition, wastewater composition and reactor configuration (Zhu et al., 2019) The effects of wastewater matrix and inoculum ratio has been reported in several studies (Ji et al.,

2018) (Huo et al., 2020a) The former reported that 1:3 inoculation ratio (microalgae:bacteria wt/wt from now on) was an appropriate one for both nutrient remediation and biomass production amongst a range of ratio (1:0, 1:1, 1:2, 1:3, 2:1, 3:1 wt/wt) (Ji et al., 2018) The latter study indicated that COD, total nitrogen (TN), and total phosphorus

(TP) in vinegar production wastewater were removed more sig- nificantly by adding either 1% (v/v) or 10% (v/v) of Bacillus firmus and

Beijerinckia species into the real WW-cultivated microalgae (Huo et al.,

2020a) Compared to a single microalgae culture, COD, TN and TP assimilation efficiency in co-culture system increased by 22.1%, 20%, and 8.1%, respectively (Huo et al., 2020a) The ratio of mixed micro- algae-bacteria could affect performance of nutrient remediation How- ever, the co-culture system of microalgae and bacteria still faces in- herent obstacles in term of biomass harvest (Mallick, 2002) The traditional technologies for biomass harvest are coagulation and cen- trifugation/separation The efficiency of those traditional technologies remains insignificant and operation cost is still high (Su et al., 2012). tackled by adding activated sludge Activated sludge performs better settling ability compared to microalgae and exhibits extensive COD removal than the available bacteria in wastewater (Gutzeit et al., 2005). Therefore, several studies have integrated microalgae and ASPs to be co-culture system based on the above-mentioned co-benefits for WW remediation (Su et al., 2012); Mujtaba and Lee, 2017; Zhu et al., 2019). For instance, the 5:1 ratio was found for sufficient nitrogen and phos- phorus removal (91.0% and 93.5% respectively) (Su et al., 2012) In turn, their findings indicated the inoculum ratio did not impact on COD removal (Su et al., 2012).

In another study, an optimal ratio primed to 2:1 to attain the highest efficiency of municipal wastewater treatment (Mujtaba and Lee, 2017). High removal of COD (82.7%), TN (75.5%) and TP (100%) could be obtained under an inoculum ratio of 1:1 (Zhu et al., 2019) It can be seen the optimal microalgae:sludge ratio of the previous studies varied due to the discrepancies of wastewater composition For instance, Su et al (2012) studied high loading concentration of COD (380 mg L -1 ),

TN (50 mg L -1 ) and PO 4 3- (8 mg L -1 ) while (Mujtaba and Lee, 2017) experimented low concentration level of COD (60 mg L -1 ), NH 4 + -N (50 mg L -1 ) and TP (1.3 mg L -1 ) Also, COD: N ratio could affect bacteria and microalgae growth A 4.3:1 ratio could enrich microalgae biomass and nutrient recovery (Zhu et al., 2019) It is important to note that the previous works did not explore thoroughly an optimal ratio for si- multaneous nutrient remediation and biomass production In addition, the role and contribution of microalgae and activated sludge in the co- culture system have not been addressed adequately A minor change in microalgae:activated sludge ratio could influence the whole perfor- mance Therefore, biomass fraction of both microalgae and bacteria needs to be quantified, and thus to elaborate their role on organic and nutrient remediation Given the research gaps indicated above, this study was built to explore the following objectives: i) To determine optimum microalgae-activated sludge inoculum ratio (wt:wt) for si- multaneous nutrient/organic matter remediation and biomass produc- tion; ii) To investigate the mechanism of nutrient remediation and (iii) to validate the role of microalgae and activated sludge incorporated in a symbiotic system.

The microalgae strain used in this study was taken from Aquaculture Research Institute 2- Ministry of Agriculture and Rural Development, Vietnam This strain is Chlorella sp., which is capable of nitrogen and phosphorus uptake as nutrient sources The strain was cultivated and maintained in a sterilized Bold́s Basal Medium (BBM).