Use of freshwater macroalgae Spirogyra sp. for the treatment of municipal wastewaters and biomass production for biofuel applications

Biomass and Bioenergy xxx (2017) 1e11

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Research paper

Use of freshwater macroalgae Spirogyra sp. for the treatment of municipal wastewaters and biomass production for biofuel applications Shijian Ge, Max Madill, Pascale Champagne* Department of Civil Engineering, Queen's University, Kingston K7L 3N6, ON, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 September 2016 Received in revised form 14 February 2017 Accepted 14 March 2017 Available online xxx

Freshwater macroalgae has competitive advantages compared to microalgae and marine macroalgae, such as lower separation and drying cost requirements and an abundance of available freshwater media. Municipal wastewater containing large quantities of nutrients (particularly nitrogen and phosphorus) is a valuable and underutilized resource. In this study, the cultivation of the naturally isolated filamentous freshwater macroalgae Spirogyra sp. was investigated in three different types of municipal wastewater including primary (PW), secondary (SW) and centrate (CW) wastewaters. Two different types of reactors including closed column photobioreactors and open rectangular aquarium reactors were operated under no and low air flow rates of less than (18 ± 2) cm
3$min
1, respectively. The SW, PW diluted with water to a 20 % volume fraction and CW diluted with water to a 2 % volume fraction appeared to promote ashfree biomass productivities of (2.17-6.68) g$m
2$d
1 and specific growth rates of (16.4e29.7) %$d
1. Nitrogen and phosphorus removal efficiencies ranged from (50.6e90.6) % and (60.4e99.1) %, respectively. Based on ultimate analysis, the biomass produced a higher heating value of (12.4e17.1) MJ$kg
1, and also showed relatively consistent protein ((16.7e19.5) % of the dry mass fraction), carbohydrate ((41.5e55.0) %) and lipid ((2.8e10.0) %) contents. These results indicate the feasibility of using Spirogyra sp. to recover nutrients from multiple municipal wastewater sources with the simultaneous production of biomass that contains value-added biochemical components for bioenergy and biofuel applications. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Algae Municipal wastewater Centrate Nutrient removal Biofuel Biomass

1. Introduction With growing global socio-economic expansion, an urgent need exists for the development of efficient and sustainable methods to address both wastewater pollution from municipal and industrial activities, as well as greenhouse gas emissions [1]. Compared to physio-chemical processes, biological processes such as activated sludge treatment are widely used for municipal wastewater treatment [2]. Most conventional biological wastewater treatments consist of three stages: primary treatment to remove solids, secondary treatment for the microbial degradation of organic substances and nutrients, and tertiary or facultative treatment as a final polishing stage for nutrient removal and/or disinfection prior to the release of effluent into the environment. During this process, carbon and nitrogen present in wastewater are converted into

* Corresponding author. E-mail address: [email protected] (P. Champagne).

carbon dioxide (CO2) and nitrogen gas, and phosphate is removed as a sludge, which requires additional disposal. Recently, new sustainable treatment options have emerged targeting the recovery and beneficial use of the nutrients and carbon present in wastewater as an alternative to conventional high-energy aerated biological treatment methods [3]. One such method is the cultivation of algae in wastewater, where algal-based treatment technologies can allow for efficient nutrient and energy recoveries while absorbing CO2 and producing an oxygenated and treated effluent. Furthermore, algal-based wastewater treatments eliminate the need for multiple anaerobic/anoxic/aerobic tanks and chemical additions common in more conventional operations. Algae exhibit high areal productivities, rapid growth rates and their cultivation in wastewater does not compete with land-based food crops [4]. The resulting high-value algal biomass can be converted to a broad range of third generation biofuels such as biodiesel, bioethanol and biomethane [5]. To date, much of the research into algal bioremediation of wastewater and the production of biofuels has focused on

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Please cite this article in press as: S. Ge, et al., Use of freshwater macroalgae Spirogyra sp. for the treatment of municipal wastewaters and biomass production for biofuel applications, Biomass and Bioenergy (2017), http://dx.doi.org/10.1016/j.biombioe.2017.03.014

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S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11

microalgae [6e8]. Comparatively, macroalgae also exhibit high areal productivities and their ability to form dense floating mats on the water surface may offer significant reductions in cost and energy during the biomass harvesting and dewatering steps compared to suspended microalgae [9]. In addition, macroalgal biomass has shown potential for the production of a variety of liquid and solid biofuels, using similar conversion pathways as with microalgae [10e12]. Current macroalgae-to-liquid fuel conversion pathways include the biochemical conversion of carbohydrates to bioethanol [13] and biobutanol [14], lipid extraction and esterification of fatty acids for biodiesel production [15], and thermochemical conversions such as hydrothermal liquefaction (HTL) to produce a liquid biocrude [12]. Comparatively, HTL provides an attractive advantage as it utilizes the whole organic fraction of the biomass, converting proteins and carbohydrates, in part, and the entire lipid fraction to oil [16e18]. HTL can also process wet biomass, which eliminates the need for dewatering and drying, thereby, reducing costs [16,19]. To date, the majority of current research conducted on the production of alternative third generation biofuels from macroalgae has focused on marine seaweeds, with a much smaller focus on freshwater filamentous macroalgae [20,21]. Although marine macroalgae could represent an important bio-feedstock resource, their need for a saline growing environment limits their potential use in the bioremediation of most municipal and industrial wastewater streams, which are primarily freshwater based. Therefore, freshwater macroalgae may have strong potentials for bioremediation applications and biofuel production as they are able to utilize these freshwater waste streams for growth. Freshwater macroalgae are ubiquitous around the world in natural waterways as well as in engineered wastewater treatment systems. Species such as Oedogonium, Cladophora and Spirogyra have been isolated from municipal and aquaculture wastewaters [10,20] and from naturally occurring water bodies, irrigation channels and wetland areas [3,22e24]. The presence of freshwater macroalgae in wastewater streams, and their competitive dominance over other species, demonstrates their natural capacity to thrive in wastewater and therefore their potential as a bioremediation strategy [25]. Several studies have demonstrated the nutrient removal capabilities of freshwater macroalgae, however, in wastewaters with relatively low nutrient concentrations. For example, Kangas et al. [26] recorded 0.1 g$m
2$d
1 for nitrogen and 0.011 g$m
2$d
1 for phosphate removal rates using algal turf scrubber (ATS) raceways and agricultural drainage wastewater. Similarly, Cole et al. [27] reported maximum nutrient removal rates of 0.564 g$m
2 $d
1 for nitrogen and 0.236 g$m
2$d
1 for phosphate from aquaculture wastewater using Oedogonium sp. Freshwater macroalgal biomass has also shown strong potential as a biofuel feedstock, for both direct combustion and its conversion into a variety of liquid fuels [10,20,21]. Based on its nutrient attenuation capacity and biofuels potential, the application of freshwater macroalgae should be further expanded into the bioremediation of municipal wastewaters with high nutrient concentrations for the production of algal biofuel feedstocks. The goal of this study was to demonstrate the potential of using a naturally isolated freshwater filamentous macroalgae for the bioremediation of high-level municipal wastewaters and biomass production. Both short-term batch, and long-term semi-continuous growth experiments were conducted using cylindrical photobioreactors (PBRs) and flat plate aquariums, respectively, with a variety of primary, secondary and centrate wastewater media. Nutrient removal efficiencies, treatment efficiencies and removal rates were used as indicators to assess the potential of the macroalgae for wastewater treatment. Biomass productivities, specific growth rates and biochemical composition of the final biomass

were employed as indicators for biofuel feedstock potential of the macroalgae. 2. Materials and method 2.1. Macroalgae identification and stock cultures Freshwater filamentous macroalgae was obtained using a fiber net from the first waste stabilization pond at the Amherstview Water Pollution Control Plant, located in South-Eastern Ontario Canada [28]. The collected macroalgae was washed with distilled water, then the green filamentous species were manually separated and transferred into three flat-plate aquariums (35 cm  40 cm  50 cm) filled with three types of real wastewaters including primary wastewater (PW), secondary wastewater (SW) and a volume fraction of 2% dilute centrate (2-CW), collected from Ravensview WWTP located in Kingston, Ontario, Canada [29]. A relatively pure culture of filamentous macroalgae was then obtained through repeated growth and separations cycles. Spirogyra sp. was identified according to the following morphological characteristics: vegetative cells (85e230) mm long and (40e60) mm wide; 1e5 chloroplasts spirally arranged; ellipsoidal zygospores [30,31]. The aquariums were equipped with an Orphek Atlantik Aquarium LED lighting platform, which provided appropriate light spectra for macroalgal growth ranging from (380e440) nm and (650e670) nm with a light intensity of (50e85) mmol$m
2$s
1. Air pumps (Tetra Whisper, Canada) equipped with membrane filters provided aeration and mixing conditions and the wastewater media were changed every 2 weeks. 2.2. Experimental design 2.2.1. Effect of mixing rate During the cultivation of stock cultures, a relationship between Spirogyra sp. growth and different mixing rates was observed. It was hypothesized that the growth of Spirogyra sp. would likely be sensitive to mixing rates and that degradation or cell death would occur with strong mixing. Hence the effects of three different mixing conditions (Air flow rates: 0, (18 ± 2) and (206 ± 14) cm
3$min
1 at 296.15 K and 101.3 kPa) on the biomass productivity and nutrient removal efficiency of Spirogyra sp. were studied by adjusting the flow rates from air pumps which provided aeration for the PBRs through in-line filters and air diffusers. Experiments were conducted using 2.0 L column-type UTEX photobioreactors (PBRs) in an algal growth chamber (Caron model 6321e1, U.S.). The PBR platforms have been discussed in detail elsewhere [6]. For each treatment, 3 replicate PBRs were employed with 0.8 L of autoclave sterilized (20 min, 120  C, 103.4 kPa) secondary wastewater (SW) and 0.5 g (fresh weight, FW) of macroalgae (equivalent to 0.625 g$L
1). A controlled cultivation environment was maintained in the growth chamber at 23  C and 2% CO2 concentration, and with white fluorescent lighting on a continuous lighting regime (mean light intensity of (120 ± 15) mmol$m
2$s
1). Within the closed growth chamber, CO2 was fed into the ambient internal environment and each PBR in the chamber was aerated with an internally placed air pump. Biomass and wastewater samples were collected every 2e3 days during the 13-day experimental period. Biomass was collected and then filtered using vacuum filtration with a 0.45 mm pore size membrane filter (Whatman® membrane filters nylon) for 3 minutes to remove excess surface water. Liquid samples (15 cm
3) were filtered using a 0.22 mm pore size glass fiber filter (Fisher Whatman puradisc-25 mm) to remove microalgal cells and other particles prior to analysis.

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S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11

2.2.2. Different wastewater types in PBRs After the mixing experiments, wastewaters were screened for the growth of Spirogyra sp. The same PBR experimental setups were used in the growth chamber as noted above. During a period of 2 weeks, Spirogyra sp. was exposed to one of five different primary, secondary or centrate based wastewater mediums: (1) PW and PW diluted with water to a 20 % volume fraction (20-PW), (2) SW and (3) CW diluted with water to 1 % and 2 % volume fractions (1-CW, 2CW), after their autoclave sterilization (20 min at 120  C, 103.4 kPa). Respective wastewater mediums noted above without macroalgae were employed as control treatments. For each treatment, 3 replicates were employed, where each PBR was operated with a working volume of 0.8 L, an air flow rate of (18 ± 2) cm
3$min
1 and an initial biomass inoculum of approximately 0.625 g$L
1. The cultivation conditions in the growth chamber remained consistent for all trials as previously noted. All PBRs were illuminated with continuous white fluorescent lighting with a light intensity of (75e120) mmol$m
2$s
1. The volumes (0.8 L) of the PBRs were kept constant over the experimental period with the addition of DI (deionized) water every sampling day. Sampling procedures were performed as outlined in the mixing experiment 2.2.1.

3

PBRs with different wastewater, where C0 and Ct are the nutrient concentrations on the first and final day (mg$L
1), t is the experimental time (d), m is the mass of biomass (g) on day t at the end of the growth cycle, and k is the removal rate coefficient (d
1).

lnCt ¼
kt þ lnC0

(4)
2


1

Ash free biomass productivities (AFBP, g$m $d ) and specific growth rates (% d
1) were calculated using Equations (5) and (6), where m0 and mt are the biomass weight on Day 0 and t (g), FW/ DW is the fresh to dry weight ratio, a is the ash content based on a dry weight (%), S is the surface area of the PBRs or the aquarium reactor (m2).

 ðm
m Þð1
a=100Þ  t 0 BP g,m
2 ,d
1 ¼ ðFW=DWÞSt

(5)

  lnðm =m Þ t 0 SGR %,d
1 ¼  100 t

(6)

2.3. Ultimate and proximate analysis for bioenergy potential 2.2.3. Long-term demonstration in aquarium To determine the effect of different reactor designs and demonstrate the long-term growth characteristics of Spirogyra sp., specifically in terms of biomass productivity and nutrient removal efficiency, a 6-week trial was run using two flat-plate aquariums (35 cm  40 cm  50 cm) to mimic an open pond cultivation system, with 4.0 L of sterilized SW and 2-CW for each aquarium, respectively. An initial biomass inoculum of 10 g (FW) was used for each aquarium and air flow was applied at a rate of (10e15) cm
3$min
1. During the experimental period, visual checks and microscopic observations were conducted daily to check for contaminations, such as microalgae invasions, in order to insure the purity of the cell culture. Lighting was supplied by two Orphek Atlantik Aquarium LED lighting platforms with light intensities of (50e85) mmol$m
2$s
1. Aeration was provided using air pumps (Tetra Whisper, Canada) and air stones. The experiment was set up in the laboratory with an ambient temperature of (20e25)  C. Biomass and liquid samples (15 cm
3) were collected every week in accordance with sampling procedures outlined in the mixing experiment 2.3.1. DI water was added during each sampling to maintain a working volume of 4.0 L for each aquarium. The wastewater media were changed every 2 weeks, or once any of the 3
þ nitrate (NO
3 -N), phosphate (PO4 -P) and ammonium (NH4 -N)
1 concentrations were lower than 1.0 mg$L . 2.2.4. Nutrient removal and biomass growth characterization Three indicators were used to evaluate wastewater treatment performance, including nutrient removal efficiencies (RE, %), treatment efficiencies (TE, % d
1, on a dry mass basis) and removal rates (RR, mg $g
1$L
1$d
1, on a dry biomass basis) as defined by Equations (1)e(3), respectively.

RE ð%Þ ¼

C0
Ct  100% C0

(1)

 C
C  t  100% TE %,d
1 ¼ 0 C0 ,d

(2)

 C
C  t RR mg,g
1 ,L
1 ,d
1 ¼ 0 m,t

(3)

In addition, the simplified pseudo-first order as shown in Equation (4) was used to determine nutrient removal kinetics in

Biomass from the different treatments noted above was collected in the late exponential phase and oven-dried at 60  C for 24 h. The dry biomass, after being powdered manually with a mortar and pestle, and sifted through a piece of muslin mesh (approximately 10 mm), was used for elemental content quantification using a PerkinElmer 2400 Series II CHNS/O Elemental Analyzer (Massachusetts, USA). The ash content was quantified through the combustion of approximately 0.5 g samples at 550  C in a muffle furnace until a constant weight was reached. Oxygen content was determined by difference, by subtracting mass fractions of carbon, hydrogen, nitrogen, sulfur and ash contents from 100%. The higher heating value (HHV) was calculated based on the elemental compositions to evaluate the energy storage in the biomass feedstock, according to equation (7) [32], where C, H, S, O, N, and a are the carbon, hydrogen, sulfur, oxygen, nitrogen, and ash mass as percent dry weight. The estimation for all parameters were conducted in triplicate.

  HHV MJ,kg
1 ¼ 0:3491C þ 1:1783H þ 0:1005S
0:1034O
0:0151N
0:0211a (7)

2.4. Biochemical composition measurement Biomass samples were dried, powdered and sifted following the same procedure as noted above. The protein, carbohydrate and lipid extraction procedures have been described previously [33]. The protein concentration was quantified by Bio-Rad DC protein assay (Cat. 500e0111, Bio-Rad Laboratories, Hercules, U.S.) using bovine serum albumin as the standard [6]. The carbohydrate content was measured with the Anthrone colorimetric method using glucose as the standard [6]. The total lipids were extracted with the mixture of chloroform and methanol at a volume ratio of 2.0 and quantified gravimetrically [34]. All protein, carbohydrate and lipid contents were determined by 3 replicate measurements of the same sample. 2.5. Nutrient analysis Temperature and pH were monitored using a microprocessor

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S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11

meter with corresponding probes (Fisher Scientific™ accumet™
Excel XL60). Ammonia (NHþ 4 -N), nitrate (NO3 -N) and phosphate 3
(PO4 -P) concentrations were analyzed using a Hach model DR 2800 spectrophotometer according to APHA Standard Methods [35]. Total phosphorus (TP) was measured using the Hach PhoVer 3 Method no. 8190 with acid-persulfate digestion. Total nitrogen (TN) was measured using the Hach TNT Persulfate Digestion Method no. 10072. Chemical oxygen demand (COD) was analyzed with the same spectrophotometer according to APHA Standard Methods [35]. 2.6. Statistical analysis The results presented are mean values ± standard deviation from independent replicate experiments. The differences in ash contents, RE, TE, RR, AFBP, SGR, HHV values as well as biochemical composition in biomass between test groups were analyzed for significance using one-way ANOVA with a post-hoc Tukey's test at a significant level of 0.05. 3. Results 3.1. Wastewater quality Table 1 shows the range in composition of the three types of wastewaters. The NHþ 4 -N was predominant in the TN of both PW (77.9%) and CW (98.0%) while NO
3 -N was the main nitrogen source in SW (86.9%). The TN concentrations in CW and PW were approximately 80- and 5-fold higher than that in SW respectively. Similar trends were observed in both TP and COD concentrations for the three wastewaters. Most of the nitrogen and phosphorus
were present in dissolved inorganic forms (NHþ 4 -N, NO3 -N and PO3
-P), which are available for algal uptake. The mass ratio of TN 4 to TP in all three wastewaters ranged between 18.8 and 25.3. Although these values are higher than the ideal Redfield N:P ratio of 16:1 (species-specific, from 8:1 to 45:1) for microalgal growth, further confirmation for macroalgal cultivation should be carried out as the optimal ratios for macroalgae may differ from the specified Redfield ratio. Further analysis was conducted by Townsend et al. [36], who concluded that the Redfield ratio was not applicable to macroalgae, and was probably inappropriate to assess macroalgal nutrient deficiency. 3.2. Effect of mixing rates on macroalgal growth Both AFBPs and SGRs varied significantly between high ((206 ± 14) cm
3$min
1) and low/none ((18 ± 2) cm
3$min
1 or 0 cm
3$min
1) air flow rate conditions (Fig. 1(a)). The low mixing rate resulted in the most productive growth condition with the maximum AFBP of 5.98 ± 0.90 g$m
2$d
1, which was 9.5-fold greater than that under high air flow rates. However, no Table 1 Characteristic of the wastewater used for macroalgal cultivation. Parameters

WWs for macroalgal cultivation PW

SW

CW

pH COD (mg$L
1)
1 NHþ 4 -N (mg$L )
1 NO
-N (mg$L ) 3
1 PO34 -P (mg$L ) TP (mg$L
1) TN (mg$L
1) N/P

7.32 ± 0.65 51.0 ± 2.5 40.3 ± 1.4 2.2 ± 0.4 1.14 ± 0.35 2.68 ± 0.01 51.7 ± 3.1 19.3 ± 1.1

7.05 ± 0.46 14.0 ± 1.8 1.1 ± 1.0 9.3 ± 0.4 0.12 ± 0.03 1.47 ± 0.06 10.7 ± 2.1 18.8 ± 1.6

8.13 ± 0.74 512.5 ± 2.8 787.5 ± 3.2 17.9 ± 0.1 29.2 ± 2.32 31.7 ± 0.02 803.3 ± 1.3 25.3 ± 0.1

Fig. 1. Comparisons of (a) ash-free biomass productivity (AFBP, g$m
2$d
1) and specific growth rate (SGR, %$d
1) and (b) nitrate and phosphate removals in PBRs where Spirogyra sp. were cultivated on SW under high ((206 ± 14) cm
3$min
1), low ((18 ± 2) cm
3$min
1) and no (0 cm
3$min
1) air flow rates, respectively. Different small letters on the bars indicate significant difference (p < 0.05).

difference in AFBP was observed between low and no air flow rate conditions. It is worth noting that, under high air flow rates the fracture of filamentous cells consistently occurred throughout the experiment along with the growth of new cells, which led to lower AFBPs. The cell fractures also made striking differences in the SGR of Spirogyra sp. under high and low air flow rates, which were not observed between low and no mixing treatments. Cultures with low or no mixing had higher nutrient uptakes and removals relative to the high mixing treatment. For example, NO
3N ((91.1 ± 0.6) %) and PO3
4 -P ((51.2 ± 18.8) %) removal efficiencies with low mixing were more than 4-fold and 2-fold those observed with high mixing. Higher removals of NO
3 -N ((91.1 ± 0.6) %) and PO3
4 -P were observed with higher AFBPs and SGRs achieved. Similarly, no differences in removal efficiencies were observed between treatments of low and no mixings. Based on these results, a low air flow rate ((18 ± 2) cm
3$min
1) was utilized in the following experiments. 3.3. Biomass production on different wastewaters in PBRs As can be seen in Fig. 2(a), Spirogyra sp. grew reasonably well in SW and 20-PW and biomass concentrations continuously increased up to (9.12 ± 2.21) and (7.08 ± 2.26) g$L
1 on Day 13 (one-way ANOVA with post-hoc Tukey's test, p < 0.05), respectively. However, 1-CW, 2-CW and PW were found to not be suitable for the growth of Spirogyra sp., as gradual cell fractures to complete degradation was observed. The AFBPs for the SW and 20-PW cultures were (2.90 ± 0.76) and (2.17 ± 0.76) g$m
2$d
1, respectively, but without significant differences (one-way ANOVA with post-hoc Tukey's test, p > 0.05), while negative values were obtained for the other three cultures (Fig. 2(b) and (c)). The specific growth rates were well correlated with the productivities, being (19.6 ± 1.9) %$d
1 for SW and (16.4 ± 2.0) %$d
1 for 20-PW. The PW was found to be the medium that exhibited the fastest cell degradation ((-40.9 ± 0.3) %$ d
1). During the growth period, pH remained relatively stable without obvious increases in the SW and 20-PW cultures likely due to the 2% CO2 supplied to the growth chamber. The pH levels were noted to decrease at the end of the cultivation period in the other three cultures. This was likely attributed to the continuous aeration with CO2, which was not utilized by the dead cells. Nutrient and organic removal profiles for the different WW cultures are illustrated in Fig. 3. For nitrogen (Fig. 3(a) and (b)), the

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5

Fig. 2. Comparisons of, (a) biomass concentration (g$L
1), (b) ash-free biomass productivity (AFBP, g$m
2$d
1), (c) specific growth rate (SGR, %$d
1) and (d) pH where Spirogyra sp. was cultivated on different types of WWs in PBRs (n ¼ 3).


3
Fig. 3. Nutrient ((a) NHþ 4 eN, (b) NO3 -N and (c) PO4 -P) and organics ((d) COD) removal profiles in PBRs where Spirogyra sp. were cultivated on different types of WWs except the PW culture where no growth was observed. Control groups only with respective WWs but without macroalgae were not shown.

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S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11


predominant nitrogen forms of NHþ 4 -N in 20-PW and NO3 -N in SW were almost completely (over 99 %) removed by the end of the
1
1 trials. However, a higher NHþ 4 -N removal rate (2.96 mg$L $d ) was obtained in the first two days in 20-PW compared to 0.66 mg$L
1$d
1 for NO
3 -N in SW. As shown in Table 2, the highest Pseudo-first order nitrogen removal rate k (0.338 d
1), RE (90.6 %), TE (12.9 %$d
1) and RR (0.16 mg$g
1$L
1$d
1) were obtained in the 20-PW culture, with the lowest half-life value of 2.0 days. In addition, nitrogen removals were also noted in 1-CW (RE 38.0 %) and 2-CW (RE 40.2 %) cultures (which were not observed in controls with only CW wastewaters and without macroalgae, data not shown) even though the biomass concentrations consistently decreased. However, no nitrogen removal was observed in PW cultures as the macroalgal cells fractured and completely degraded within the first two days (Fig. 2(a) and (c)). Phosphorus removals observed for the four wastewaters exhibited similar trends to those observed for nitrogen (Table 2). Competitive phosphorus removal efficiencies (>98.3 %) were obtained in three wastewater cultures (20-PW, 1-CW and 2-CW) with high initial phosphorus concentrations ((0.33e0.85) mg$L
1) except the SW (0.08 mg$L
1). Finally, COD concentrations fluctuated and removals were not obvious in all cultures.

3.4. Long-term demonstration in aquarium Fig. 4 shows the characteristics of algal growth and nutrient removal in the aquariums throughout the cultivation period.

Spirogyra sp. acclimated and grew well in SW in the aquarium as was observed in the closed column PBRs. The AFBP ranged from 2.92 to 4.03 g$m
2$d
1 with corresponding SGRs between (15.3e21.1) %$d
1. An improved quality of effluent wastewater was delivered at the end of each cycle (2 weeks) with a nitrogen removal efficiency of (94.0e97.3) %. For the centrate wastewater, it appeared that the open aquarium was a better growth environment for Spirogyra sp. in comparison to the PBR. Relatively steady and stable growth was achieved after a short acclimation in the first week, although lower values of both AFBP ((0.11e0.40) g$m
2$d
1) and SGR ((2.0e5.3) %$d
1) were observed. Final nutrient removal efficiencies of (50.6e60.0) % for nitrogen and (60.4e78.4) % for phosphorus were also noted.

3.5. Ultimate and proximate analysis for bioenergy potential The different cultivation conditions and reactor types were found to affect biomass composition as quantified by ultimate analysis (Table 3). Ash contents in the biomass grown in the SW and 2-CW aquariums were significantly higher at (13.8 ± 0.8) % and (7.0 ± 1.2) %, respectively (one-way ANOVA with post-hoc Tukey's test, p < 0.05), compared to the SW and 20-PW PBR reactors at (2.7 ± 1.0) % and (2.2 ± 0.2) %, respectively. Final ultimate analysis showed very similar weight percentages of N, H, S and O between the four different cultivation conditions and reactor types (one-way ANOVA with post-hoc Tukey's test, p > 0.05). Carbon contents however were approximately 10% higher in the PBR reactors

Table 2 Kinetic parameters (rate constants and half-life), and nutrient removal parameters of Spirogyra sp. after exposure to different wastewaters types in PBRs. Wastewater

K (d
1)

a

SW 20-PW 1-CW 2-CW

0.13 0.34 0.07 0.04

Pb

SW 20-PW 1-CW 2-CW

e 0.84 ± 0.04 0.58 ± 0.02 0.42 ± 0.03

Nutrient N

a b

± ± ± ±

0.03 0.04 0.01 0.01

TE (%$d
1)

RR (mg$g
1$L
1$d
1)

5.4 3.7 1.3 2.9

6.3 ± 0.6 12.9 ± 0.4 5.4 ± 0.2 3.1 ± 0.3

0.09 ± 0.01 0.16 ± 0.01 e e

e 99.1 ± 9.2 98.3 ± 6.1 99.8 ± 1.5

e 14.2 ± 0.4 14.0 ± 0.3 7.7 ± 0.1

e 0.007 ± 0.01 e e

t1/2(d)

RE (%)

5.2 ± 0.2 2.0 ± 0.1 10.5 ± 0.9 17.5 ± 2.5

82.2 90.6 38.0 40.2

e 0.8 ± 0.1 1.2 ± 0.1 1.7 ± 0.1

± ± ± ±


Only the inorganic nitrogen of NHþ 4 -N and NO3 -N were considered. Only the inorganic dissolved PO3
-P was considered. 4

Fig. 4. (a) Ash-free biomass productivity (g$m
2$d
1) and specific growth rate (%$d
1), and (b) Nutrient removal efficiencies for nitrogen (N) and phosphorus (P) in the aquarium reactors where Spirogyra sp. was cultivated on SW and 2-CW over 6 weeks. The light intensities in two aquariums ranged (50e85) mmol$m
2$s
1 with 24 h lighting regime, aeration flow rates ((10e15 cm
3$min
1), and at room temperature ((22.5e26.4)  C).

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S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11

7

Table 3 Ash and ultimate analysis (%, on a dry mass basis) and higher heating value (MJ$kg
1) of biomass grown under different cultivation conditions. Biomass was sampled at the end of individual experiment. Oxygen percentages were determined by difference. Values sharing the same superscript letter were not significantly different from each other (oneway ANOVA with post-hoc Tukey's test, p < 0.05). WWs SW-PBR 20-PW-PBR SW-Aquarium 2-CW-Aquarium

Ash (%)

C (%) a

2.7 ± 1.0 2.2 ± 0.2a 13.8 ± 0.8b 7.0 ± 1.2c

41.0 40.9 31.1 35.5

N (%) ± ± ± ±

a

0.6 2.0a 0.8b 0.6b

4.8 4.5 4.3 4.4

± ± ± ±

H (%) a

0.2 0.2a 0.3a 0.1a

compared to the aquarium reactors. Due to the higher carbon and lower ash contents, the PBR reactors had slightly higher HHV values, hence indicating a higher biofuel potential [20]. Overall, the range of HHVs obtained in this study ((13.8e17.3) MJ$kg
1) was comparable to the HHVs reported for traditional, terrestrial biomass ((10.7e28.2) MJ$kg
1) [21]. 3.6. Biochemical composition The biochemical analysis of Spirogyra sp. grown in SW, 20-PW and 2-CW wastewaters in both PBR and aquarium showed relatively consistent protein, carbohydrate and lipid contents, expressed as the percentage of the dry weight of samples (Table 4). Protein content ranged from (16.7e19.5) % and showed no variation between the cultivation conditions (one-way ANOVA with post-hoc Tukey's test, p > 0.05). Carbohydrates were the main organic component (over 40 %) for all conditions, but the contents were independent of cultivation conditions (one-way ANOVA with posthoc Tukey's test, p > 0.05). By comparison, lipid contents in Spirogyra sp. were low between (2.8e10.0) % with the highest obtained value (10.0 %) for the biomass grown on 2-CW in aquarium (oneway ANOVA with post-hoc Tukey's test, p < 0.05). 4. Discussion 4.1. The critical effects of the hydrodynamic environment on biomass growth The results demonstrated that high mixing rates limited the biomass productivity, specific growth rate and nutrient removal of Spirogyra sp. grown in PBRs. Furthermore, the high mixing rates degraded the initial inoculum, dispersing it into fine filaments that later settled to the bottom of the reactors. This was also observed when a higher inoculum loading of 2.0 g, instead of 0.5 g, with longer and more developed filaments was investigated (data not shown). Reactors with low mixing and no mixing showed consistently higher AFBPs, SGRs and nutrient REs. These two mixing rates showed very little differences in growth despite the prediction that a low mixing rate would increase the distribution and availability of nutrients. The outcomes of this experiment are inconsistent with the findings reported in other studies. Increased turbulence in macroalgal cultures enhanced productivity and growth through the

Table 4 Biochemical composition (%, on a dry mass basis) of the biomass resulting from different cultivation conditions. Biomass was sampled at the end of individual experiment. Values sharing the same superscript letter were not significantly different (one-way ANOVA with post-hoc Tukey's test, p < 0.05). WWs

Protein (%)

SW-PBR 20-PW-PBR SW-Aquarium 2-CW-Aquarium

19.5 17.7 17.0 16.7

± ± ± ±

3.3a 0.6a 1.2a 0.6a

Carbohydrate (%) 41.5 48.4 43.1 55.0

± ± ± ±

9.6a 1.8a 0.5a 5.1a

Lipid (%) 2.8 ± 0.5a 3.1 ± 0.4a 3.5 ± 0.3a 10.0 ± 0.1b

6.6 6.2 6.7 6.7

± ± ± ±

S (%) a

0.2 0.2a 0.3a 0.2a

0.09 0.12 0.10 0.11

HHV (MJ$kg
1)

O (%) ± ± ± ±

a

0.01 0.01a 0.04a 0.01a

44.8 46.1 44.1 46.3

± ± ± ±

a

0.9 2.3a 0.1b 0.2c

17.3 16.7 13.8 15.3

± ± ± ±

0.1a 0.7a 0.1b 0.2c

mixing and increased availability of nutrients and dissolved CO2 [37,38]. For example, Lawton et al. [20] demonstrated higher SGRs and AFDW productivities from Cladophora, Spirogyra and Oedogonium with high and low aeration compared to no aeration. However, in their study 20 L plastic buckets were used as reactors, a much larger working volume than the 0.8 L used in this experiment. It was therefore believed that the damaging effects of the high-mixing rates in this experiment were mainly a result of the small working volume and the narrow water column of the 2.0 L PBRs used. Overall, the differences observed in the effects of different mixing rates between studies suggested that the optimal growing conditions for a given macroalgae were not predetermined, but vary based on the algal species, reactor type, working volume, nutrient source, temperature and likely cultivation conditions. Biomimicry might be a valuable methodology for determining the optimal growth conditions for a particular macroalgal species. That was, mimicking the natural growth conditions in which a species of algae thrives could enhance biomass productivity, nutrient removal efficiency and possibly favorable changes in biomass composition (lipid, protein and carbohydrate). Spirogyra sp., for example, the freshwater filamentous macroalgae used in this study, thrived in stagnant and nutrient dense waters through the formation of floating mats [20,39]. Therefore, the use of attached growth systems, such as the ATS [40], might be able to mimic the natural mat like formations of Spirogyra sp. and induce higher AFBPs and REs compared to the suspended growth conditions used in this study. The ecology of Spirogyra sp. might also provide an explanation for the results of the mixing experiments. That is, the high mixing rates were damaging to Spirogyra sp. as these conditions are not representative of the natural conditions in which Spirogyra sp. thrives. The ATS system facilitated higher biomass production with freshwater macroalgae [41,42] and should therefore be further explored as a potential technology for the large-scale cultivation of Spirogyra sp. 4.2. Nutrient removals and feasibility of freshwater macroalgaebased wastewater treatment The high SGR and AFBP of Spirogyra sp. in 20-PW and SW led to a successful bioremediation of these wastewaters. 20-PW and SW showed the highest nitrogen removal efficiencies of (90.6 ± 3.7) % and (82.2 ± 5.4) %, respectively. However, the nitrogen composition of these wastewaters was drastically different with ammonia nitrogen (NHþ 4 -N) being the main nitrogen source in 20-PW (77.9 %) and nitrate nitrogen (NO
3 -N) being the prominent nitrogen source in SW (86.9 %). This therefore demonstrated the ability of Spirogyra sp. to efficiently utilize both nitrogen species, possibly with a slight preference for ammonia nitrogen due to the higher RE and RR in 20-PW. The relatively high removal of phosphorus in all wastewaters except SW, which had a very low initial PO3
4 -P concentration of (0.08 mg$L
1), further demonstrated the nutrient attenuating abilities of Spirogyra sp. In addition to N and P removal, COD removal remained relatively constant throughout all trials.

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8

S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11

This might first be attributed to the already low initial COD concentrations in the wastewaters ((10e15) mg$L
1) and secondly due to the relatively sterile conditions in the reactors. Organic compound degradation in wastewater generally occurred through bacterial oxidation and utilized dissolved oxygen, which in part was provided through algal photosynthesis [43]. However, without the presence of microorganisms, the breakdown of COD was minimal. It was predicted that higher COD removals would be observed in unsterilized wastewater, which would most likely be used in fullscale wastewater bioremediation. The successful treatment of municipal wastewaters with freshwater macroalgae, such as Spirogyra sp., depends on maintaining high biomass productivities and corresponding nutrient removal efficiencies. Overall, nutrient removal rates exhibited a hyperbolic relationship with nutrient loadings, where nutrient removal increased until a maximum was reached [41,44]. Therefore, in order to promote efficient nutrient removal, without the washout of untreated nutrients and possible toxic effects, or the limitation of nutrients for macroalgal growth, an optimal nutrient loading rate must be obtained. The effects of different nutrient loadings were demonstrated in this study with the use of raw and diluted PW based media. Raw PW exhibited the highest biomass degradations for all wastewaters tested, possibly due to toxic levels of ammonium, which would represent an over-loading of nutrients for Spirogyra sp. However, when diluted to 20%, 20-PW showed the second highest AFBP and SGR, as well as the highest N and P removal efficiencies. These results suggest that an optimal dilution rate for PW could be found and possibly implemented in a full-scale wastewater treatment system with the recycling of treated effluent for dilutions. Spirogyra sp. grown in PBRs with 1-CW and 2-CW mediums showed biomass degradation and limited nutrient removals. However, positive SGR and AFBP values were obtained using 2-CW in a larger volume aquarium. It is assumed that the complex compositions, namely the higher organic, nutrient and metal concentrations [45], of CWs may have required a significantly higher inoculum-loading rate in order for Spirogyra sp. to survive. However, the bioremediation of CW needs further study to identify the maximum survivable micronutrient and metal concentrations and therefore the proper starting conditions for different freshwater macroalgae. Spirogyra sp. showed potential for the effective treatment of SW and 20-PW wastewaters, reducing nutrient and organic concentrations well below the local discharge limits. However, although effective treatment was observed, the small volume and closedbatch systems used in this study are not representative of the types of treatment systems that would be implemented on a large scale. For the treatment of municipal or industrial wastewaters with freshwater macroalgae to be feasible, large volume treatment systems with optimal and stable growth conditions are necessary. Several studies, as listed in Table 5, have demonstrated the treatment capabilities of engineered freshwater macroalgae-based treatment systems. The use of attached growth, typically utilizing ATS units, showed significant potential for treating large volumes of various wastewater types. Two separate studies by Mulbury et al. [26,46] demonstrated the potential of using freshwater macroalgae in raceways for the treatment of both agricultural and swine manure wastewaters at pilot scale. The use of ATS raceways with UV disinfection was also successful in further polishing secondary municipal wastewaters at a large scale of 302 8 m3$d
1 [47]. In summary, similar open ponds or tanks often used in activated sludge-based systems could also be suitable for macroalgae-based wastewater treatment, which would lower costs in terms of the capital configuration for new WWTPs and reduce retrofitting costs for existing WWTPs. Certainly, all operational parameters at an

actual WWTP would need to be re-computed and optimized as these are relatively complex systems, and fluctuations in conditions such as daily wastewater quality, potential contamination and natural sunlight could lead to failure or decreased performance of the macroalgal-wastewater treatment system. The use of polycultures may, however, increase the resistance to environmental and invasion pressures, two major challenges faced with open cultivation methods [48]. 4.3. Potential biomass applications The biomass productivities and biochemical compositions of macroalgae may allow for the development of suitable biomass conversion processes for biofuel and other bio-product applications. Theoretical biocrude estimates from Spirogyra sp. were not included in this study, however, HTL was predicted to produce higher biocrude yields, due to the contribution of carbohydrates ((41.5e55.0) %) and proteins ((16.7e19.5) %), compared to the yields of biodiesel which would depend solely on lipid contents ((2.8e10) %). However, several challenges currently existed in obtaining high HTL biocrude yields from macroalgae. Primarily, carbohydrates did not liquefy as well as lipids and proteins [18] and therefore the high carbohydrate contents in macroalgae hindered biocrude yields. This was due to the breakdown of large fractions of carbohydrates into polar water-soluble organics that did not contribute to biocrude yields [49,50]. However, the use of the alkali catalyst Na2CO3 was shown to enhance oil formation during the HTL of carbohydrates [51]. The presence of inorganic salts in the macroalgae biomass posed another challenge as these salts tended to promote gasification under HTL conditions and, therefore, reduced biocrude yields [52e54]. To achieve the maximum value from a wastewater-derived macroalgal feedstock, a sequential treatment, or biorefinery approach, might be the most feasible. Utilizing HTL in a biorefinery approach would involve the addition of effective pre- and posttreatment steps to convert all major biochemical components into multiple value-added co-products. For example, the preextraction of protein ((16.7e19.5) % in this study) produced a secondary value-added product suitable for animal consumption [55] and also reduced the nitrogen content in the finished biocrude, which would be beneficial if further refining was envisioned. Nicolas et al. [17] showed that the extraction of protein prior to HTL increased the conversion rate of the macroalgae up to 77 %. Another biorefinery approach demonstrated by Ron Pate et al. [56] first used biochemical pretreatments and fermentations to target the high carbohydrate and protein fractions in macroalgae turf, while producing liquid fuels (ethanol and isobutanol) at high yields. HTL of the preprocessed residue (after carbohydrate and protein fermentations) yielded 22 % biocrude with a reduced nitrogen content of 0.89%, compared to that without pretreatment. Thus, based on a biorefinery consideration, HTL with preliminary treatments such as protein extraction or carbohydrate and protein fermentations would be recommended for Spirogyra sp. as conversion pathways that should be further investigated. 4.4. Limitation and future perspectives Significant limitations currently existed in the technical development of freshwater macroalgae-based municipal wastewater treatment with biofuel production. Although macroalgae have been comparatively less studied than microalgae, their physiological traits, growth characteristics and low harvesting and dewatering costs may make their further exploration worthwhile. This and a number of other studies have demonstrated that municipal wastewater streams (PW, SW and CW) can support high biomass

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S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11

9

Table 5 Comparison of freshwater macroalgae-based wastewater treatment system. Reference

[25]

Species

Oedogonium Mixed Species (90 % sp. Oedogonium, Spirogyra, Ulothrix and Vaucheria) PW, SW, CW Pre-chlorination effluent

Wastewater

Nutrient (N&P) concentrations (mg$L
1) Reactor type

Volume (L) Biomass productivity (g$m
2$d
1) Light source and light cycle Nutrient removal

TN:1.12 e27.2 TP:0.23 e5.04 Cylindrical tanks 20 PW: 12.7 e13.8 SW: 6.3 CW: 9.2 Outdoor/ Natural e

[21]

[20]

[46]

TN: 20.4 ± 4.6 TP: 3.5 ± 0.91

Microspora willeana Lagerh, Ulothrix Cladophora, ozonata, Rhizoclonium hieroglyphicum, Spirogyra, Oedogonium Oedogonium Culture water Swine Manure with MAF medium Medium: 13.4 % TN: 1130 N, 1.4 % P TP: 340

Shallow trays

Plastic buckets

Tray type with ATS

5 3.37

20 Oedogonium: 8 g AFDW

205 7.1e9.4

LED, 12:12

e

2  400 W Metal Halide 23:1

e

e

N: 98-40 % P: 76-41 %

pH

8.5e10.8 e (daily range)

Temperature ( C)

25e27

e

8.2 ± 2 with CO2 7.00e7.5 10.5 ± 1.5 without CO2 27.7 ± 1.6

productivities and growth rates of several freshwater macroalgae species (Table 5). However, few studies to date have utilized macroalgae for municipal wastewater treatment at pilot or full scale, although several studies existed for the treatment of agricultural and diary wastewaters [26,57]. Further optimization of freshwater macroalgae-based treatment systems, such as the ATS units, and a greater understanding their growth characteristics will be needed before these systems could be scaled up to accommodate the large volumes required for wastewater treatment. Although sterilization of the wastewater media is an effective strategy for minimizing the risk of contamination in macroalgal cultivation systems, its use on a full-scale would likely be costly and therefore unfeasible. The two basic macroalgal cultivation approaches, closed PBRs and open ponds [58], face different challenges in maintaining desired algal productivities and composition. Closed PBRs, as used in this study, are less prone to invasion despite being significantly more expensive [58,59]. Open cultivation systems are comparatively more economical but are exposed to fluctuating environmental conditions and are at higher risk from both abiotic and biotic contaminants [48]. The viability of open cultivation systems therefore hinges on the development of monocultures or polycultures that are able to remain productive throughout changing environmental conditions and invasion pressures [48]. Furthermore, future studies will be required to determine the viability of unsterilized wastewater as a medium for Spirogyra sp. and other macroalgae cultures, in terms of treatment potentials and biomass productivities, to increase the cost effectiveness and feasibility of macroalgae-based wastewater treatments. The potential applications for macroalgal biomass continue to evolve. From the work of this and other studies, the biochemical compositions of several freshwater species have been characterized. In general, although exact compositions change from species to species, macroalgae differ more considerably from microalgae, specifically in their low lipid and higher carbohydrate contents.

23e26

[27]

[26]

Oedogonium Ulothrix, sp. Spirogyra, Microspora Aquaculture Agricultural system drainage TN: 1.93 e2.75 TP: 0.16 e0.53 1000 L Cylindrical Tanks 853 3.8e23.8

Outdoor/ Natural e

This study Spirogyra sp.

PW, SW, CW

TN < 0.5 TP < 0.1

TN:8.4 e15.1 TP:0.08 e0.82 Raceways with PBR, ATS Aquarium 1200 5

1.8/4 AFDW:2.11 e2.89

Outdoor/ Natural N:125; P: 25 mg$m
2$d
1

Florescence white light N: (38.0 e90.6) % P:(98.3 e99.8) % 6.35e7.38

7.69 e Without CO2 6.78 With CO2 18.9e24.3 e

23e26

Therefore, the more established microalgae conversion techniques, such as biodiesel production through the extraction and transesterification of fatty acids, are relatively ineffective when applied directly to macroalgae. However, the biochemical characteristics, specifically the high carbohydrate contents of macroalgae, may make them more suited for thermochemical conversion like HTL as noted above, as well as traditional alcoholic fermentation or anaerobic digestion. The strategic technological development of these main pathways, with a biorefinery approach consideration, will allow more effective conversions and could lead to a significant increase in the value of macroalgae as a feedstock.

5. Conclusions The results of this study demonstrated the potential for using naturally collected filamentous freshwater macroalgae Spirogyra sp. for the treatment of three different municipal wastewaters and for downstream biomass processing applications. High gas flow rates ((206 ± 14) cm
3$min
1) were demonstrated to be less suitable for growth leading to biomass degradation, while low and no gas flow rates (less than (18 ± 2) cm
3$min
1) maximized the biomass productivities and nutrient removals. In addition, SW offered the best growth conditions in terms of BP and SGRs; however, PW and CW required dilution suggesting that an optimized wastewater dilution ratio should be assessed to allow for suitable nutrient concentrations for Spirogyra sp. growth in these mediums. Biochemical, proximate and ultimate analysis of the final biomass was relatively similar between different cultivation conditions and was comparable to other studies. Due to the high carbohydrate content and considerable protein content of Spirogyra sp., and freshwater macroalgae in general, HTL with protein extraction or protein and carbohydrate fermentation pretreatments are recommended as conversion processes for further study.

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10

S. Ge et al. / Biomass and Bioenergy xxx (2017) 1e11

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Please cite this article in press as: S. Ge, et al., Use of freshwater macroalgae Spirogyra sp. for the treatment of municipal wastewaters and biomass production for biofuel applications, Biomass and Bioenergy (2017), http://dx.doi.org/10.1016/j.biombioe.2017.03.014