Scholarly article on topic 'Effects of influent C/N ratios and treatment technologies on integral biogas upgrading and pollutants removal from synthetic domestic sewage'

Effects of influent C/N ratios and treatment technologies on integral biogas upgrading and pollutants removal from synthetic domestic sewage Academic research paper on "Chemical engineering"

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Academic research paper on topic "Effects of influent C/N ratios and treatment technologies on integral biogas upgrading and pollutants removal from synthetic domestic sewage"


OPEN Effects of influent C/N ratios and treatment technologies on integral biogas upgrading and pollutants

Received: 21 February 2017 Accepted: 21 August 2017 Published online: 07 September 2017

removal from synthetic domestic sewage

Jie Xu1, Xue Wang2, Shiqing Sun1#Yongjun Zhao1 & Changwei Hu1

Three different treatment technologies, namely mono-algae culture, algal-bacterial culture, and algal-fungal culture, were applied to remove pollutants form synthetic domestic sewage and to remove CO2 from biogas in a photobioreactor. The effects of different initial influent C/N ratios on microalgal growth rates and pollutants removal efficiencies by the three microalgal cultures were investigated. The best biogas upgrading and synthetic domestic sewage pollutants removal effect was achieved in the algal-fungal system at the influent C/N ratio of 5:1. At the influent C/N ratio of 5:1, the algal-fungal system achieved the highest mean chemical oxygen demand (COD) removal efficiency of 81.92% and total phosphorus (TP) removal efficiency of 81.52%, respectively, while the algal-bacterial system demonstrated the highest mean total nitrogen (TN) removal efficiency of 82.28%. The average CH4 concentration in upgraded biogas and the removal efficiencies of COD, TN, and TP were 93.25 ± 3.84% (v/v), 80.23 ± 3.92%, 75.85 ± 6.61%, and 78.41 ± 3.98%, respectively. These results will provide a reference for wastewater purification ad biogas upgrading with microalgae based technology.

In rural areas of China, one of the main water pollution sources is domestic sewage, which contains abundant carbon, nitrogen, and phosphorus compounds. Recently, more than 90% of the domestic sewage in rural areas was directly discharged into natural waters without appropriate treatment, eventually causing water eutrophica-tion1. Many studies indicated that conventional water treatment processes, including oxidation ditch processes2, anaerobic-anoxic-axic (A2/O)3, University of Cape Town (UCT)4, Bardenpho, and sequencing batch reactor (SBR)5, achieved moderate success in removing pollutants. However, these processes generally entail enormous land requirements, operational costs, complex operations, and large volumes of waste sludge production and are therefore not practicable in rural areas in China.

Conventional activated sludge systems will consume around half of the whole energy to convert chemical oxygen demand (COD) into the greenhouse gas CO2. The pollutants (i.e. carbon, nitrogen and phosphorus sources) in wastewater can be assimilated by microalgal species as nutrients for heterotrophic or mixotrophic growth6. Hence, as an alternative for the purification of domestic sewage, biological wastewater treatment system using microalgae is currently attracting increased interest because of its low construction and maintenance costs, minimal energy consumption, freedom from spatial restriction during operation, as well as high removal efficiency7. Study has shown that 85-88% of COD, 78-83% of total nitrogen (TN), and 73-80% of total phosphorus (TP) could be removed from wastewater by cultivating the algae Chlorella sp.8. Kumar et al. (2010) reported that treating digested piggery effluent with Chlorella vulgaris (C. vulgaris.) achieved removal efficiencies of 100% for TP and 78% for NH4+-N9. In addition, culturing certain species of microalgae with sanitary sewage might enable the harvest of potentially high added value microalgae biomass and the metabolic products, such as proteins and fatty acids10, 11. Therefore, microalgae-based technology is suitable for wastewater treatment because of its high effectiveness and low-cost12, 13.

College of Biological Chemical Science and Engineering, Jiaxing University, Jiaxing, 314001, P.R. China. 2Shanghai Public Green Space Construction Affairs Center, Shanghai, 201100, China. Correspondence and requests for materials should be addressed to Y.Z. (email: or C.H. (email:

In previous studies, significant improvement in the removal of xenobiotics from wastewaters has been observed with algae-bacteria/fungus-algae co-cultivation14' 15. The success of this technology is dependent on a mutually beneficial relationship, that is, by fixing CO2 which is produced by fungi/bacteria, algae can synthesize carbon source in the form of sugars and nutrients for fungi/bacteria through photosynthesis16. Therefore, pretreating wastewater by algal-fungal culture or algal-bacterial culture has an advantage on higher pollutants removal efficiencies comparing with mono-cultivation of algae cells, thereby offering a promising and efficient way to treat wastewater.

Biogas represents a renewable energy source based on its high CH4 content. Raw biogas usually consists of methane (47-65%), carbon dioxide (34-41%), and other trace compositions including hydrogen sulfide, water vapor, etc.17. However, the CH4 content of more than 90% (v/v) in biogas is required for vehicle fuel according to the natural gas standard7. The relatively high content of CO2 in raw biogas will lower its heat content as well as increase its energy demand for compression and transportation. Therefore, CO2 removal is essential for biogas upgrading. Numerous methods, such as physical absorption, chemical conversion, membrane separation, pressure swing adsorption, and cryogenic separation, have been used to upgrade biogas17. However, these methods require large amounts of energy, auxiliary materials, and chemicals, as well as generate wastes that require further treatment18. Moreover, the CO2 separated from the raw biogas using the above-mentioned methods is usually released directly into the atmosphere and result in greenhouse gas emission. Among the various strategies for biogas upgrading by CO2 removal, the biological sequestration of CO2 using photosynthetic microalgae has been receiving considerable attention because of the relatively high CO2 fixation capability of microalgae11. There are a limited number of studies on the integration of digestate treatment procedures and biogas upgrading via microal-gae culture systems8. Accordingly, using microalgal growth to remove CO2 from crude biogas is an economic and efficient method for biogas upgrading, which makes biogas upgrading by microalgal culturing an economically convenient technique when used in conjunction with wastewater treatment8, 19.

Microalgal biomass production exhibits potentials for both pollutants reduction and CO2 removal. However, microalgal growth is also affected by organic matter and nutrient concentrations in wastewater as well as the presence of other heterotrophic microorganisms20. Fungi and bacteria can also have strong degradation abilities for certain wastewater contaminants and can be associated with microalgae to form immobilization systems of algal-fungal culture or algal-bacterial culture with multiple functions. The advantages of these associations in wastewater treatment are: (i) improved collection of biomass from wastewaters, (ii) easily recycled and manipulated consortia, (iii) improved features of microalgae such as thermal stability and productivity, and (iv) harvest-able bioresource from proliferated microalgae biomass19, 21, 22. The use of biogas and sewage as raw materials can not only output high-grade biogas through the microalgae photosynthesis to assimilate carbon dioxide, but also can purify sewage by accumulating C, N, and P in sewage with microalgae. When compared with the traditional biogas upgrading and sewage purification technology, environmental friendly and cost-effective are the biggest advantages. Some researches on biogas upgrading or sewage purification resulted in methane loss and energy consumption, or lead to the increase of effluent18. Microalgae-based technology was therefore developed for simultaneously treating wastewater and upgrading biogas in recently years. Combination of wastewater treatment and biogas upgrading is indispensable because of its highly economically convenient comparing with wastewater treatment by using microalgae only23. Besides, the high cost generated from microalgae biomass harvesting is one of the major bottlenecks for the industrialization of algae-based technology because of the small microalgal size, negative surface charge, and low biomass concentration of microalgae24. Co-culture of microalgae with fungi/bacteria can well solve this problem. The bio-flocculation based microalgae assisted with fungi/bacteria is regarded as one of the best ways to realize the large-scale separation and microalgae recovery25. The energy consumption and the cost of the subsequent enrichment process have been significantly reduced14. At present, the few studies on couple biogas upgrading with wastewater treatment in photobioreactors provide little information on the influence of operational conditions on strain composition. Influent C/N ratio is the main factor affecting biological wastewater treatment processes26. However, thus far, only a few studies have evaluated the effects of different influent C/N ratios on the efficiency of domestic sewage treatment by microalgae27-29. Overlow or overhigh C/N ratios in the influent may result in low growth rate of microalgae and low removal efficiencies of nitrogen and phosphate in wastewater. In addition, the C/N ratio is also vital to the growth of microalgae when cocultivation with fungi or bacteria.

This study proposes an integrated approach for synthetic domestic sewage treatment and biogas upgrading through three algal culture methods (mono-algal culture, algal-fungal culture, and algal-bacterial culture). The purposes of this study were to (1) evaluate the effects of three different microalgae culture methods on sewage purification and biogas upgrading; and (2) identify the optimal influent C/N ratio to achieve the highest efficiencies of pollutants removal combined with biogas upgrading in synthetic domestic sewage.

Results and Discussion

Physicochemical variations. Changes in values of the sewage pH and DO are shown in Table 1. These changes were similar under all tested influent C/N ratios. The pH ranged from 7.11 to 7.54 in the influent, which were optimum values for the microalgae cultivation and the prevention of ammonia toxicity and phosphate precipitation30. No significant difference (p > 0.05) was observed among the three algae source cultures for pH values in the influent or effluent. However, for all treatments, pH of the influent was significantly higher (p < 0.05) than that in the effluent, which was typically below pH 8.0 (Table 1). The pH values observed in this study were consistent with those found by Papazi et al. (2008), who reported that pH values slightly varied between 6.0 and 7.5 under CO2 30% (v/v) with Chlorella minutissima31. This phenomenon meant that the biogas CO2 content in this research met the requirement for microalgae growth. Therefore, the CO2 consumption of the suspension culture only slightly affected the pH, which was rather related to the removal of organic pollutants. For instance, the

C/N ratio Influent Effluent

pH Do (mg L-1) pH Do (mg L-1)

Mono-algae culture

C2.5N1-COD100 Low COD level 7.42 ± 0.23 7.94 ± 2.03 6.45 ± 0.31 6.37 ± 1.92

C5N1-COD200 Medium COD level 7.31 ± 0.19 7.42 ± 1.85 6.11 ± 0.16 6.52 ± 1.41

C10N1-COD400 High COD level 7.19 ± 0.11 2.91 ± 1.07 6.32 ± 0.14 1.95 ± 1.13

C10N1-TN20 Low TN level 7.28 ± 0.25 8.11 ± 1.94 7.03 ± 0.11 6.14 ± 1.02

C5N1-TN40 Medium TN level 7.51 ± 0.28 7.33 ± 1.56 7.12 ± 0.19 6.43 ± 1.27

C2.5N1-TN80 High TN level 7.26 ± 0.13 6.98 ± 1.39 6.39 ± 0.24 5.78 ± 0.82

Algal-fungal culture

C2.5N1-C0D100 Low COD level 7.44 ± 0.17 8.12 ± 2.11 6.87 ± 0.31 6.09 ± 1.17

C5N1-C0D200 Medium COD level 7.11 ± 0.26 7.63 ± 1.55 6.93 ± 0.15 6.28 ± 1.56

C10N1-C0D400 High COD level 7.52 ± 0.15 2.54 ± 1.18 7.04 ± 0.19 2.17 ± 1.08

C10N1-TN20 Low TN level 7.47 ± 0.19 6.91 ± 1.94 6.39 ± 0.21 6.32 ± 1.26

C5N1-TN40 Medium TN level 7.33 ± 0.21 7.72 ± 1.83 6.57 ± 0.16 6.71 ± 1.34

C2.5N1-TN80 High TN level 7.35 ± 0.32 6.53 ± 0.97 6.41 ± 0.18 6.03 ± 1.27

Algal-bacterial culture

C2.5N1-C0D100 Low COD level 7.54 ± 0.27 8.51 ± 1.84 6.58 ± 0.16 6.92 ± 1.57

C5N1-C0D200 Medium COD level 7.41 ± 0.18 7.93 ± 0.92 6.77 ± 0.18 6.31 ± 1.25

C10N1-C0D400 High COD level 7.48 ± 0.29 2.39 ± 1.46 6.32 ± 0.14 1.84 ± 0.93

C10N1-TN20 Low TN level 7.34 ± 0.15 6.77 ± 1.01 6.21 ± 0.17 6.04 ± 1.16

C5N1-TN40 Medium TN level 7.53 ± 0.24 7.02 ± 0.87 6.49 ± 0.16 6.23 ± 1.28

C2.5N1-TN80 High TN level 7.41 ± 0.12 6.15 ± 0.59 6.63 ± 0.26 5.93 ± 1.29

Table 1. Means ± standard deviations of the physico-chemical parameters of influent and effluent for domestic sewage of three different methods of treatments at different influent C/N ratio.

reduced pH partly depends on the extent of nitrification in the photobioreactor, since ammonium volatilization processes play a negligible role in TN removal13.

The DO of the wastewater slightly decreased during the experimental period. The biggest decrease varied from 8.12 ± 2.11 mg L-1 to 6.09 ± 1.17 mg L-1 in the C2.5N1-COD100 treatment by algal-fungal culture. However, this variation ranged below the excessive DO level (35 mg L-1, thereby would not inhibit microbial growth32. Carvalho et al. (2006) have reported that in a closed photobioreactor (similar to the photobioreactor used in this study), O2 in the influent might inhibit microalgal growth because of photorespiration33. This phenomenon did not occur in this research because of relatively high initial CO2 concentrations (28.43 ± 3.04%, v/v) in the photobioreactor and high organic carbon values in the influent34.

Growth of the three selected cultures at various influent C/N ratios. Biomass productivity is a key parameter to analyze the potential of the three selected cultures to remove CO2. It generally varies with operational factors, such as light intensity, pH, working volume of the photobioreactor, and initial CO2 concentration in the simulated biogas20. Studies have found that biomass growth is coupled not only with higher N/C and P/C ratios, but also with lower N/P ratios in many heterotrophic organisms, including bacteria29.

Table 2 shows the microalgal mean daily productivity, sewage pollutant removal efficiencies and CH4 content in biogas under three cultures and various C/N ratios. The behavior of the three selected cultures varied even under identical environmental conditions and media. In both the mono-algae and the algal-fungal culture, biomass grew faster in the C5N1-COD200 treatment than in the other treatments (p < 0.05). In the present study, biomass productivity of the algal-fungal culture was higher than the other two cultures. The highest biomass productivity (0.44 g L-1 d-1, Table 2) for algal-fungal culture was found in C5N1-COD200, although there was no differences among other C/N ratios (p < 0.05).

Pollutants removal efficiencies. Pollutants uptake by the three selected cultures significantly contributed to COD, TN and TP removal from the wastewater. Figures 1-3 indicated the effects of various influent C/N ratios and culture methods on COD, TN, and TP removal efficiencies using microalgal-based technologies. All the cultures studied efficiently removed pollutants (COD, TN, and TP) from the synthetic domestic sewage. Pollutant removal efficiencies fluctuated for the three algae source culture during the operational period, and the variation tendencies of pollutant removal were similar under different influent C/N ratios. The COD removal efficiencies varied between 50.06% and 89.47% for the microalgal monoculture (Fig. 1a), between 52.36% and 93.87% for the microalgal-fungal co-culture (Fig. 1b), and between 50.35% and 93.78% for the algal-bacterial co-culture (Fig. 1c) under different COD or TN level treatments. The TN removal efficiency variations for the microalgal monoculture ranged from 50.26% to 90.69% (Fig. 2a), for the microalgal-fungal co-culture from 54.25% to 94.28 % (Fig. 2b), and for the algal-bacterial co-culture from 49.23% to 93.97% (Fig. 2c). The TP removal efficiencies for the microalgal monoculture varied between 42.17% and 89.57% (Fig. 3a), for the microalgal-fungal co-culture between 49.36% and 93.77% (Fig. 3b), and for the algal-bacterial co-culture between 47.14% and 92.54% (Fig. 3c). It is noteworthy that after the first day of the assays, pollutant concentrations (in terms of COD, TN, and TP)

C/N ratio Effluent Upgraded biogas Biomass productivity

COD Removal (%) TN Removal (%) TP Removal (%) Concentration of CH4 (%,v/v) Mean daily productivity (g L-1 d-1)

Mono-algae culture

C2.5N1-COD100 Low C0D level 69.34b ± 2.18 69.83b ± 2.18 62.91c ± 3.24 86.33b ± 2.93 0.18b ± 0.02

C5N1-COD200 Medium C0D level 72.09b ± 3.26 71.89",b ± 3.26 69.34b ± 3.11 92.54" ± 4.57 0.24" ± 0.02

C10N1-COD400 High C0D level 67.07b ± 3.43 68.45b ± 3.43 63.95c ± 2.96 88.21",b ± 3.09 0.16b ± 0.01

C10N1-TN20 Low TN level 72.58b ± 2.97 75.16" ± 2.97 70.48",b ± 3.17 87.95b ± 3.38 0.19b ± 0.02

C5N1-TN40 Medium TN level 77.64" ± 4.35 77.06",b ± 4.35 73.06" ± 4.35 93.62" ± 3.47 0.25" ± 0.03

C2.5N1-TN80 High TN level 68.75b ± 3.82 75.38" ± 3.82 68.72b ± 2.76 85.47b ± 3.92 0.17b ± 0.01

Algal-fungal culture

C2.5N1-C0D100 Low C0D level 78.65" ± 4.02 75.08b ± 5.12 75.94b ± 4.09 90.58"b ± 3.46 0.35b ± 0.04

C5N1-C0D200 Medium C0D level 81.92" ± 4.57 79.35" ± 4.26 79.88" ± 3.87 93.04" ± 2.11 0.44" ± 0.05

C10N1-C0D400 High C0D level 79.11" ± 2.43 75.54b ± 4.08 77.81",b ± 4.16 89.37b ± 3.92 0.29c ± 0.02

C10N1-TN20 Low TN level 79.13" ± 3.77 78.42" ± 3.17 78.47b ± 3.57 91.04",b ± 3.76 0.39b ± 0.03

C5N1-TN40 Medium TN level 80.64" ± 3.92 81.66" ± 6.61 81.52" ± 3.98 93.25" ± 3.84 0.43" ± 0.04

C2.5N1-TN80 High TN level 79.35" ± 5.08 75.15b ± 5.05 74.44b ± 3.19 88.83b ± 4.37 0.31bc ± 0.02

Algal-bacterial culture

C2.5N1-C0D100 Low C0D level 74.79b ± 3.56 76.95b ± 3.27 69.08c ± 4.02 88.42"b ± 3.58 0.23"b ± 0.03

C5N1-C0D200 Medium C0D level 77.41",b ± 4.81 79.48" ± 2.99 75.84",b ± 2.98 91.54" ± 2.77 0.26" ± 0.02

C10N1-C0D400 High C0D level 71.60c ± 3.25 75.32b ± 4.81 70.39c ± 4.57 88.53b ± 3.01 0.17c ± 0.02

C10N1-TN20 Low TN level 78.17" ± 5.79 79.83" ± 3.18 74.60",b ± 3.66 90.39",b ± 3.54 0.28" ± 0.03

C5N1-TN40 Medium TN level 80.59" ± 3.99 82.28" ± 3.36 77.02" ± 4.09 91.75" ± 2.46 0.24" ± 0.02

C2.5N1-TN80 High TN level 75.02b ± 4.17 75.11b ± 2.56 74.79b ± 3.43 86.19b ± 3.45 0.21b ± 0.02

Table 2. Mean values ± SD of the removal efficiency of biogas CO2 and pollutants removal of different C/N ratio at three different methods of treatments. Values with different superscript letters in the same column for the same method of treatments indicate significant differences at p = 0.05 according to Duncan's multiple range tests.

suddenly dropped. This initial drop implied a COD removal of up to 50.06%, TN removal of up to 49.23%, and TP removal of up to 42.17% in all treatments and was attributed to the adsorption of the microalgae cell walls and subsequent assimilation. The highest pollutant removal efficiencies were observed on day 6 (Figs 1a-3a), day 8 (Figs 1b-3b), and day 7 (Figs 1c-3c) by mono-algae culture, algal-fungal culture, and algal-bacterial culture, respectively. Afterwards, removal efficiencies decreased until the end of the experimental period. This decrease was probably due to the accumulation of metabolic waste, which ultimately led to cell death20. Therefore it is suggested that the cultivation times of 6, 8, and 7 days for mono-algae culture, algal-fungal culture, and algal-bacterial culture, respectively, are needed to achieve the highest pollutants removal efficiencies.

In carbon addition treatments, the C5N1-COD200 treatment (69.34%) had a higher TP removal efficiency than the C2.5N1-COD100 (62.91%) and C10N1-COD400 treatments (63.95%) (p < 0.05), but no significant difference (p > 0.05) was observed in removal efficiencies for COD and TN among C variation treatments by the mono-algae culture. For the algal-fungal culture, the C5N1-COD200 treatment had a higher TN removal efficiency (79.35%) than C2.5N1-COD100 (75.08%) and C10N1-COD400 (75.54%) (p < 0.05), but the influent C/N ratios showed no statistically significant effects (p > 0.05) on COD removal. For the algal-bacterial culture, the highest TN (79.48%) and TP (69.34%) removal efficiencies were achieved by C5N1-COD200 (p < 0.05), and the C10N1-COD400 treatment had a lower COD removal efficiency (71.60%) than the C2.5N1-COD100 (74.79%) and C5N1-COD200 treatments (77.41%) (p < 0.05).

Under N addition treatments, the C5N1-TN40 treatment had a higher COD removal efficiency (77.64%) than the C10N1-TN20 (72.58%) and C2.5N1-TN80 treatments (68.75%) (p < 0.05), but the N variation treatments (C10N1-TN20, C5N1-TN40, and C2.5N1-TN80) did not significantly affect (p > 0.05) the TN removal of the mono-algae culture. For the algal-fungal culture, the C5N1-TN40 treatment showed a significantly higher (p < 0.05) TP removal efficiency (81.52%) than those of the C10N1-TN20 (78.47%) and C2.5N1-TN80 treatments (74.44%), and the C2.5N1-TN80 treatment had a lower TN removal efficiency (75.15%) than the C10N1-TN20 (78.42%) and C5N1-TN40 treatments (81.66%) (p < 0.05), but no significant difference (p > 0.05) was detected in removal efficiencies of COD among different N treatments. However, in the algal-bacterial culture, the C2.5N1-TN80 treatment had significantly lower (p < 0.05) COD (75.02%) and TN (75.11%) removal efficiencies than the other treatments.

As shown in Table 3, the interaction of treatment methods and influent C/N ratios as well as the treatment methods significantly influenced pollutants removal efficiencies (p < 0.05). Only TN and TP removal efficiency were significantly affected (p < 0.05) by influent C/N ratios. This shows that appropriate selection of treatment methods and influent C/N ratios is a simple and effective strategy to increase pollutants removal efficiencies.

Carbon is a basal element for microalgal growth, contributing to up to about 50% of microalgal biomass35. A previous study accessed the carbon mass balance and found that assimilation into biomass was the main carbon removal pathway20. Dissolved nitrogen and phosphorus in wastewater could be efficiently removed through

Figure 1. COD removal efficiency over time under various influent C/N ratios for three algae source cultures: (a) microalgal monoculture, (b) microalgal-fungal co-culture, and (c) algal-bacterial co-culture.

continuous microalgal growth. Total N was mainly reduced via microalgal assimilation, as algae cells require nitrogen for protein, nucleic acid, and phospholipid synthesis9. Previous work has documented that in wastewater, simple organic nitrogen, including urea and amino acids, can be assimilated by microalgae15. Thus, microal-gae growth is essential for nitrogen removal via uptake, decay, and sedimentation36. On the other hand, partial loss of TN could be attributed to the physical absorption by strain complexes because of their unique structure37. In addition, phosphorus is an important nutrient in algal production as a constituent of phospholipids (for cell membranes) and adenosine triphosphate (to supply energy for cell functions), although it constitutes less than 1% ofthe biomass38. In a similar study, Yan et al. (2014) obtained a phosphorus removal efficiency of 73.89% for Chlorella sp. in biogas slurry8.

Carbon dioxide dissolving in sewage will provides a substrate for the microalgal photosynthesis. The dissolution of carbon dioxide promoted the growth of microalgae and therefore played a key role in nitrogen and phosphorus removal. Accordingly, coculture of microalgal and fungi or bacteria promotes the purification of sewage and biogas upgrading14. The rich carbon dioxide in the biogas slurry promotes the growth of algal bacteria and increases the biomass of algae bacteria, thus increases the absorption of N and P by algae and improes the sewage purification capacity of algae bacteria.

Several researchers have suggested that denitrification rates in a reactor depend largely upon the amounts of nitrate N and organic carbon as well as on environmental conditions such as pH, temperature, and DO concentration39. Here, influent pH levels (7.11-7.54) (Table 1) were within the optimum range for denitrification reported by Paul and Clark (1989)40. In practice, nitrogen removal is highly dependent on pollution load levels. In this study, different influent C/N ratios resulted in different denitrification rates, and high TN removal efficiency occurred only if the C/N ratio was 5:1. Consequently, influent C/N ratio plays a crucial role in wastewater treatment39. Some scholars have suggested that influent C/N ratio is a domain value, rather than a specific value, because when the experimental influent C/N ratio approaches this value, optimal pollutant removal efficiencies can be achieved27. In this research, the balance of carbon and nitrogen nutrient sources in the influent affected the

Figure 2. TN removal efficiency over time under various influent C/N ratios for three algae source cultures: (a) microalgal monoculture, (b) microalgal-fungal co-culture, and (c) algal-bacterial co-culture.

growth of the culture, thereby affecting the nutrient removal efficiencies of the microalgae. On the other hand, the synthetic wastewater used in this research contained carbamide; a high C/N ratio might thus inhibit the removal of ammonia nitrogen due to the competition for DO demand. Therefore, when carbon sources were lacking (C:N = 2.5:1) or nitrogen sources were insufficient (C:N = 10:1), pollutants removal decreased. Considering the combined removal efficiencies for all pollutants, the optimal C/N ratio in this research was 5:1. These results are in agreement with the findings of Yan et al.26, who reported that influent C/N ratio significantly affected nutrient removal efficiency and that the highest removal effect was found with a medium influent C/N ratio.

Influent C/N ratio also affected effluent phosphorus concentrations, and at C/N 5:1, all treatment systems reached their highest TP removal efficiencies. In the nitrogen addition treatments, the highest average removal efficiencies for the three algae source culture occurred at the C/N ratio of 5:1 (Table 2). These results could be explained by the combined carbon and nitrogen effects for removal of TP. Overall, the results showed that appropriate control of carbon and nitrogen input were necessary to achieve the efficient phosphorus removal.

In the present study, among the three selected cultures, the algal-fungal culture showed the highest pollutants removal efficiencies (COD, TN and TP). The sphere structure of the fungus-algae complex was stable and did not easily break into small pieces (data not shown), which might partially explains the high pollutants removal efficiency. According to the references14' 41, when fungi associated with microalgae grew in wastewater, the fungus was pelletized with microalgae cells through bioflocculation and non-bioflocculation. The coagulative mechanism is spore coagulation resulting in accumulation of pellets. The non-coagulative mechanism includes that the spores germinate into hyphae and then intertwines into pellets. In the symbiotic system of algae-fungi/bacteria, on one hand, the extracellular metabolites produced and secreted by the microalgae can be effectively taken up by the surrounding fungi/bacteria for the growth and reproduction. On the other hand, the bacteria in the metabolic process not only can produce the necessary nutrients and growth factors for the growth of microalgae, but also can directly or indirectly regulate the growth environment of microalgae. This forms a mutually beneficial symbiotic relationship42. The intergrowth of microalgae and fungi enhances the specific surface area of

Figure 3. T P removal efficiency over time under various influent C/N ratios for three algae sources cultures: (a) microalgal monoculture, (b) microalgal-fungal co-culture, and (c) algal-bacterial co-culture.

Factor COD RE (%) TN RE (%) TP RE (%) CO2 RE (%)

Influent C/N ratios 0.059 0.027* 0.039* 0.067

Treatment methods 0.034* 0.018* 0.044* 0.025*

Influent C/N ratios x Treatment methods 0.028* 0.032* 0.017* 0.011*

Table 3. P-values of factors and combined effects of factors for each parameter based on analysis of variance. Influent C/N ratios: C variation treatments (C2.5N1-COD100, C5N1-COD200, and C10N1-COD400) and N variation treatments (C10N1-TN20, C5N1-TN40, and C2.5N1-TN80); treatment methods: microalgal monoculture, microalgal-fungal coculture and microalgal-activated sludge coculture (*p < 0.05).

algae-fungi symbionts and thus the nutrient intake capacity43. According to Table 2, coculture of microalgae and fungi resulted in higher nutrient removal as well as CO2 removal except for TN removal. The algal-bacterial culture showed higher TN removal efficiency than algal-fungi culture maybe attribute to the nitrification of bacteria in activated sludge44.

Biogas upgrading. At the end of the experiment, the CH4 contents (v/v) of the biogas and the CO2 removal efficiency (%) were investigated to evaluate differences in biogas upgrade with varying influent C/N ratios for the three selected cultures (Fig. 4a-c). The results showed that the tendencies of CH4 contents and CO2 removal efficiencies were similar to biomass productivities of the three selected culture, as shown in Table 2.

Figure 4. CO2 removal efficiencies and final CH4 contents (v/v) in biogas under six nutrient concentration levels for three algae source cultures: (a) microalgal monoculture, (b) microalgal-fungal co-culture, and (c) algal-bacterial co-culture.

During biogas upgrading, CO2 could be effectively removed. In all treatments using algal-bacterial culture, the CO2 removal percentage (%) ranged from 74.71% to 90.26%, which was slightly higher than that observed for mono-algae culture (62.89-91.56%) and algal-fungal culture (65.42-84.98%). The overall CO2 removal percentage followed similar trends and reached the highest values of 91.56 ± 2.36%, 84.98 ± 2.36%, and 90.26 ± 1.76% in the C5N1-TN40 treatment by mono-algae culture, algal-fungal culture, and algal-bacterial culture, respectively. Under the different C and N variation treatments, no significant differences (p > 0.05) in biogas CO2 removal efficiencies were found between C5N1-COD200 and C5N1-TN40 among the three microalgal cultures, but their corresponding removal efficiencies were significantly higher (p < 0.05) than that in C2.5N1-COD100, C10N1-COD400, C10N1-TN20, and C2.5N1-TN80.

During the experiment, biogas upgrading was affected by the initial pollutants concentrations, and the CH4 content (v/v) increased with CO2 contents (v/v) decreasing. The differences in the various treatments for the CH4 contents (v/v) of the biogas mainly resulted from the variations in the CO2 removal, because CH4 and CO2 contents (v/v) in biogas were significantly negatively correlated. The CH4 contents (v/v) enriched by algal-bacterial culture in upgraded biogas in the treatments C2.5N1-COD100, C5N1-COD200, C10N1-COD400, C10N1-TN20, C5N1-TN40, and C2.5N1-TN80 were 90.58 ± 1.25%, 93.04 ± 2.01%, 89.37 ± 1.88%, 91.04 ± 2.16%, 93.25 ± 1.74%, and 88.83 ± 1.69%, respectively;

The CO2 in biogas was utilized for algal photosynthesis. As a result, the CH4 content in biogas was increased. The CH4 content in biogas upgraded by algal-bacterial culture was 88.8%~93.3%, which was higher than those by mono-algae culture and algal-fungal culture. The CH4 contents (v/v) enriched through removing CO2 by algal-bacterial culture reached the standard of the fuel (CH4 > 90%, v/v) in the treatments C5N1-COD200, C10N1-TN20, and C5N1-TN40. Only in C10N1-COD400 and the C2.5N1-TN80, algal-fungal culture, the CH4

C/N ratio Economic efficiency (USD 1)


Mono-algae culture

C2.5N1-COD100 Low COD level 37.34bc ± 2.28 37.42bc ± 2.37 27.28c ± 1.74 29.58c ± 1.94

C5N1-COD200 Medium COD level 39.81b ± 2.74 39.09b ± 2.58 36.44b ± 2.06 37.63" ± 3.18

C10N1-COD400 High COD level 36.16c ± 1.97 36.77c ± 2.61 28.36c ± 1.93 35.44b ± 2.86

C10N1-TN20 Low TN level 39.96b ± 2.45 42.33" ± 2.53 37.24b ± 2.32 33.89b ± 3.05

C5N1-TN40 Medium TN level 43.27" ± 3.18 43.07" ± 3.18 40.31" ± 2.17 38.76" ± 2.89

C2.5N1-TN80 High TN level 35.83c ± 1.84 42.58" ± 2.72 35.57b ± 2.09 28.07c ± 2.77

Algal-fungal culture

C2.5N1-C0D100 Low COD level 44.28b ± 3.22 42.26b ± 2.34 43.19b ± 2.75 31.42c ± 2.35

C5N1-C0D200 Medium COD level 46.16" ± 2.83 45.78" ± 2.57 45.98" ± 2.37 38.17" ± 2.49

C10N1-C0D400 High COD level 45.07"b ± 2.71 42.63b ± 2.28 43.52b ± 2.63 30.61c ± 2.78

C10N1-TN20 Low TN level 45.19",b ± 3.05 43.92",b ± 3.08 44.13",b ± 2.79 37.94" ± 3.37

C5N1-TN40 Medium TN level 45.75",b ± 3.29 45.99" ± 3.11 46.37" ± 3.07 38.23" ± 2.34

C2.5N1-TN80 High TN level 45.41"b ± 2.98 42.18b ± 2.62 42.23b ± 2.66 34.73b ± 3.39

Algal-bacterial culture

C2.5N1-C0D100 Low COD level 41.62bc ± 2.65 42.34b ± 2.37 36.05c ± 2.17 35.59b ± 2.68

C5N1-C0D200 Medium COD level 42.87b ± 3.01 45.58" ± 2.68 42.51" ± 3.79 38.21" ± 3.27

C10N1-C0D400 High COD level 39.25c ± 2.42 42.41b ± 2.26 36.87c ± 2.96 35.79b ± 2.96

C10N1-TN20 Low TN level 43.83",b ± 2.79 45.72" ± 2.89 40.94b ± 2.34 37.32" ± 3.11

C5N1-TN40 Medium TN level 45.32" ± 2.61 46.65" ± 3.42 42.98" ± 3.83 38.63" ± 3.09

C2.5N1-TN80 High TN level 42.12b ± 2.83 42.52b ± 2.63 41.22"b ± 2.62 28.85c ± 2.83

Table 4. The economic efficiency of energy consumption on synthetic domestic sewage purification and biogas upgrading at different C/N ratio with three different culture methods. Values with different superscript letters in the same column for the same method of treatments indicate significant differences at p = 0.05 according to Duncan's multiple range tests.

contents (v/v) did not reach this standard. The highest CH4 content (v/v) in the different C and N variation treatments was found in C5N1-TN40, when using the three cultures.

In this research, the effect of biogas upgrading followed similar trends as biomass productivity (Table 2), mainly because approximately half of the microalgal DW was CO2-derived carbon45. When the microalgae were cultivated in synthetic domestic sewage, CO2 was used for microalgal photosynthesis. The microalgae developed a CO2-concentrating mechanism to adapt to the changes in the CO2 concentration46, 47.

Economic efficiency of the energy consumption. The economic efficiency of energy consumption on synthetic domestic sewage purification and biogas upgrading showed a similar variation (Table 4). The highest economic efficiency of pollutants and CO2 removal with different culture methods can be found at the C/N ratio of 5:1 with the medium TN level. This finding is corresponded to the variation of the microalgal biomass productivity. To be specific, algal-fungal culture achieved the highest economic efficiency of COD and TP removal; algal-bacterial culture achieved the highest economic efficiency of TN and CO2 removal. The ANOVA showed that there was no significant difference in COD, TN and TP removal rates between algal-fungi and algal-bacteria cultures (p > 0.05), and there was no significant difference on energy efficiency CO2 removal by the three cultures (p > 0.05). For these two cocultivations, the energy efficiencies of pollutant removal were higher, but energy efficiencies of CO2 removal were lower of than that of mono-algae culture. Microalgae will grow well in the influent C/N ratio of 5:1 and improve the sewage purification, as a result, the economic efficiency of energy consumption are increased13. For algal-bacteria culture, due to nitrification and denitrification of activated sludge, the economic efficiency of nitrogen removal achieve higher than algal-fungal culture39, 44.


Both of the microalgal culture methods and C/N ratios had significant effects on of synthetic domestic sewage purification and biogas upgrading. The medium level of C/N ratio showed higher pollutants and CO2 removal efficiencies than low and high C/N ratios. Co-culture of C. vulgaris with G. lucidum or activated sludge in photo-bioreactors was more effective than mono-cultivation on removing sewage pollutants and CO2 in biogas simultaneously according to the data in this study. Coculture of microalgae with fungi was the suitable treatment technology for wastewater purification and biogas upgrading under the C/N ratio of 5:1.


Algae sources and culture conditions. The C. vulgaris (FACHB-31) was used for sewage treatment and biogas upgrading based on its high biogas tolerance and rapid growth in high-strength wastewater48. The strain was purchased from the FACHB-Collection, Institute of Hydrobiology, Chinese Academy of Sciences. The BG-11 culture media was prepared for growing the microalgal culture49, with initial pH adjusted to 6.9. The culture

Figure 5. Scheme of the photobioreactor experimental setup.

conditions were as follows: the wavelength spectrum and photosynthetic photon flux density (PPFD) of cool-white LED light were 360~720 nm and 200 |imol m-2 s-1, respectively. The temperature and light period were 25 ± 0.5 °C and 12 h light-12 h dark, respectively. The cultures were intermittently shaken three times a day (8:00 a.m., 2:00 p.m., and 8:00 p.m.).

Algal-fungal culture conditions. Based on the preliminary experiment results, we found that Ganoderma lucidum (G. lucidum, 5.765) achieved high growth rate and high performance of pelletization with C. vulgaris. Therefore, the Ganoderma lucidum stain obtained from China General Microbiological Culture Collection Center was selected for this study. An inoculum was prepared by inoculating 100 mL of a synthetic medium with 25 mycelial discs. The composition of synthetic medium was as follows: glucose, 10 g L-1; NH4NO3, 2.0 g L-1; K2HPO4, 1.0 g L-1; NaH2PO4-H2O, 0.4 g L-1; MgSO4-7H2O, 0.5 g L-1; yeast extract, 2.0 g L-1; pH 6.5. The prepared inoculum was then incubated at 25 ± 1 °C on a rotary shaker at 160 rpm for 7 d. The obtained biomass was washed with sterile distilled water and homogenized with 100 mL of sterile distilled water in a laboratory blender. Subsequently, the obtained cultures were used for the co-cultivation of microalgal cells.

Each 100 mL Microalgal suspension (158.37 ± 14.26 mg L-1) was mixed with 5 mL G. lucidum suspension (82 ± 8 mg L-1) for pelletization. The fungal-algal mixtures were shaken at 160 rpm for 7d under constant PPFD (200 |imol m-2 s-1) at 25 °C. All experiments were performed in triplicate.

Algal-bacterial Culture conditions. The synthetic domestic sewage was inoculated with 1L cultured microalgal strain and 200 mL activated sludge. The total suspended solid (TSS) of microalgae and activated sludge were 0.75 g TSS L-1and 4.14 g TSS, respectively. The activated sludge came from a wastewater treatment plant in Jiaxing, Zhejiang, China. The light intensity and temperature were the same as the algal-fungi culture20.

Photobioreactor. Two interconnected 16.8 L (individual) glass cylinder blocks with a height of 0.6 m and a diameter of 0.2 m were used as a photobioreactor (Fig. 5). The reactors were hermetically sealed with rubber stoppers. The sampling outlet consisted of a plug and rubber gasket. The synthetic domestic sewage was pumped from the right to the left cylinder block of the photobioreactor in one time. The raw biogas was blown into the photoreactors from the raw biogas inlet until the air in the headspace was expelled.

Synthetic domestic sewage and biogas. To facilitate comparison with similar experiments, this study used the synthetic domestic sewage, a modification of the OECD standard sewage44. The concentrations of TN and COD were adjusted, whereas the TP was not. The experiment was divided into two groups. In group 1, low (100 mg L-1), medium (200 mg L-1), and high (400 mg L-1) levels of COD (fixed TN/TP levels at medium strength) were designated as C2.5N1-COD100, C5N1-COD200, and C10N1-COD400. In group 2, low (20 mg L-1), medium (40 mg L-1), and high levels (80 mg L-1) of N (fixed COD/TP levels at medium strength) were designated as C2.5N1-TN80, C5N1-TN40, C10N1-TN2 050. Medium for group 1 was prepared by mixing the following components: 100, 200, and 400 g m-3 glucose, respectively; 80 g m-3 carbamide; 15 g m-3 NaH2PO4; 1.5 g m-3 KH2PO4; 4 g m-3 CaCl2; and 2 g m-3 MgSO4. Medium for group 2 was: 200 g m-3 glucose; 40, 80, and 160 g m-3 carbamide, respectively; 15 g m-3 NaH2PO4; 1.5 g m-3 KH2PO4; 4 g m-3 CaCl2; and 2 g m-3MgSO451. Table 5 shows the characteristics of the synthetic domestic sewage.

Biogas was obtained from a farm biogas plant in JiaYuan Green Meadow. Prior to the experiments, the biogas was pretreated via chemical absorption to reduce the H2S content less than 0.005% (v/v). The raw biogas mainly

C/N ratio Influent concentration (mg L 1)


Mono-algae culture

C2.5N1-C0D100 Low COD level 102.16 ± 3.21 43.12 ± 3.54 5.21 ± 0.42

C5N1-C0D200 Medium COD level 203.77 ± 6.19 41.08 ± 2.75 5.39 ± 0.57

C10N1-C0D400 High COD level 408.35 ± 8.67 44.63 ± 3.18 5.11 ± 0.74

C10N1-TN20 Low TN level 202.49 ± 5.11 20.42 ± 2.06 5.33 ± 0.62

C5N1-TN40 Medium TN level 205.18 ± 4.09 43.71 ± 2.83 5.46 ± 0.83

C2.5N1-TN80 High TN level 206.03 ± 7.16 82.75 ± 4.35 5.08 ± 0.49

Algal-fungal culture

C2.5N1-C0D100 Low COD level 103.83 ± 4.28 42.07 ± 2.98 5.41 ± 0.72

C5N1-C0D200 Medium COD level 204.39 ± 5.93 44.24 ± 3.04 5.22 ± 0.63

C10N1-C0D400 High COD level 403.58 ± 8.47 42.35 ± 3.76 5.35 ± 0.57

C10N1-TN20 Low TN level 206.34 ± 7.53 22.31 ± 2.53 5.05 ± 0.66

C5N1-TN40 Medium TN level 208.37 ± 8.95 41.64 ± 2.32 5.14 ± 0.58

C2.5N1-TN80 High TN level 203.67 ± 7.02 84.09 ± 3.84 5.23 ± 0.67

Algal-bacterial culture

C2.5N1-C0D100 Low COD level 106.25 ± 3.89 40.86 ± 3.21 5.39 ± 0.84

C5N1-C0D200 Medium COD level 208.02 ± 5.92 42.24 ± 2.95 5.04 ± 0.62

C10N1-C0D400 High COD level 407.11 ± 7.81 43.31 ± 3.76 5.28 ± 0.25

C10N1-TN20 Low TN level 205.27 ± 6.47 22.26 ± 2.99 5.19 ± 0.57

C5N1-TN40 Medium TN level 208.81 ± 8.43 41.06 ± 4.33 5.23 ± 0.65

C2.5N1-TN80 High TN level 201.34 ± 7.29 84.32 ± 3.27 5.37 ± 0.53

Table 5. Parameters of influent synthetic sewage and crude biogas in the photobioreactor.

consisted of CH4 (67.59 ± 4.13%, v/v) and CO2 (28.43 ± 3.04%, v/v), trace amounts of other components included H2O (3.54 ± 0.27%, v/v), O2 (0.47 ± 0.03%, v/v), and H2S (<0.005%, v/v).

Experimental procedure. The photobioreactor was filled with 14 L of raw biogas and 2.8 L of synthetic domestic sewage and illuminated on 200 |imol m-2 s-1 by six fluorescent lamps arranged in a circular configuration (20 W, 110 V) around the left cylinder block (Fig. 1). The initial dry weight (DW) ofthe three selected culture was about 90.21 ± 6.39 mg L-1 in all samples to obtain similar initial biomass concentrations. The temperature and light/dark cycle of microalgal for wastewater treatment was the same as the microalgal mono-cultivation. Under different influent C/N ratios, growth rate and pollutants removal efficiencies of the three algal source cultures and their roles in biogas upgrading were determined. Gas samples (100 mL) were drawn daily at the sampling outlet to monitor CO2 and CH4 concentrations. By using a 15-mL syringe, the synthetic domestic sewage was sampled daily at 8:00 a.m. through the sampling port of the photobioreactor, to monitor the COD, TN, and TP concentrations.

All treatments were performed in triplicate. The cultures were sampled and analyzed daily at 8:00 a.m. and mean values were calculated for each time and culture.

Sampling and analyses. The DW of the three algal source cultures was measured according to the reference19. Daily biomass productivity (P, g L-1 d-1) was calculated using Equation (1):

P = (Dj - D0)/(ti - t0) (1)

where Di is the biomass concentration (g L-1) at time ii (d) and D0 is the initial biomass concentration (g L-1) at t0 (d).

The culture filtrates were analyzed for COD, TN, and TP concentrations by using standard methods52. The pH value and dissolved oxygen (DO) was measured using a pH (Orion 250 Aplus ORP Field Kit, USA) and oxygen probes (Model 862 Aplus, USA). Pollutants (COD, TN, and TP) removal efficiency was calculated as follows:

R = (1 - Cj/C0) (2)

where R is the pollutant removal efficiency (%), C0 and Ci are the pollutant concentrations in the initial synthetic domestic sewage and in the filtrates of the cultures (mg L-1), respectively.

The contents (v/v) of CO2, H2S, CH4, and O2 in the biogas were measured using a circulating gas analyzer50. The economic efficiency of the energy consumption for nutrient removal in sewage and CO2 removal in biogas were calculated by Eq. (3)

E = -R-

KTP (3)

where E is the energy consumption for nutrient removal in sewage and CO2 removal in biogas, USD-1; R is the removal efficiency in Eq. (2), %; k stands for the electric power charge per unit of energy consumption, USD kw-1h-1; T is the illumination time according to photoperiod, h; P is the LED electrical power consumption, W. The electric power charge per unit of energy consumption k is around 0.08826 USD kw-1h-1 in local after conversion13.

Statistical analyses. All statistical analyses were performed using the package SPSS (SPSS 2013). A one-way analysis of variance (ANOVA) was used to test the statistical differences of the 6 C/N ratios for the same microal-gae cultures. Duncan's multiple range tests was employed to further test for significant differences among the treatments with different C/N ratios. A two-way ANOVA was used to test for differences among the effects of influent C/N ratios, algae sources, and the interaction between any two of these factors on treatment performance. The threshold for statistical significance was set at p = 0.05. Error bars in the figures showed the standard deviation with n = 3.


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This study was sponsored by the National Natural Science Foundation of China (Project no. 31670511 and 31370520), the Zhejiang Provincial Natural Science Foundation (Project no. LY16C030003 and LQ17E080013), and the Planned Science and Technology Project of Jiaxing (Project no. 2017BY18065).

Author Contributions

Jie Xu, Xue Wang and Shiqing Sun conducted the experiments. Yongjun Zhao and Changwei Hu prepared the figures and wrote the main manuscript text. Shiqing Sun and Yongjun Zhao revised the manuscript. All authors reviewed the manuscript.

Additional Information

Competing Interests: The authors declare that they have no competing interests.

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