**4. Enhanced Biological Phosphorus Removal (EBPR)**

Biological nutrient process (BNP) refers to modified activated sludge processes where contaminants, biochemical oxygen demand (BOD), nitrogen and phosphorus can be simulta‐ neously removed from the bulk wastewater and accumulated to the waste sludge [98]. In this method, phosphorus removal is usually realized by enhanced biological phosphorus removal process (EBPR), a process that recirculates activated sludge in alternating anaerobic and aerobic conditions and enriches or selects the microbial strains synthesizing polyphosphate (polyphosphate accumulating organisms, PAO). It has been applied to municipal wastewater treatment, but is also acknowledged that in large-scale applications the process experiences reactor upset and failure from time to time due to environmental or biological reasons such as nitrate overloading, high rainfall, and microbial competition [99]. Selecting and maintaining suitable operational conditions to avoid reactor upset can be important to the process. One limitation for EBPR is the insufficient carbon source in wastewater, or relatively low soluble COD-to-phosphorus ratio so PAOs does not sequester enough PHAs or energy for phosphorus accumulation. Manure before or even after anaerobic digestion usually contains a high level of organic compounds and a large portion of it is readily biodegradable such as acetate, propionate and butyrate. So manure can at least be a good substrate for providing carbon source for PAOs; in other words, EBPR can be an alternative method for phosphorus removal from liquid manure [100]. Some of the previous studies using animal manure in fact achieved good results in phosphorus removal via EBPR. Note that EBPR process usually is not the final step closing the phosphorus removal but a pretreatment method, although phosphorus is concentrated in sludge. The reason is that poly-phosphate in the sludge tends to be hydrolyzed, released and leaked to liquid phase. There are several other processes that can be used to further treat the EBPR sludge and phosphorus-accumulated media, e.g., coagulation and crystallization.

### **4.1. Microbiology and mechanisms**

EBPR has been used in wastewater treatment plants for a long period of time, and most fundamental studies were based on activated/recycled sludge from these treatment plants. Briefly, the anaerobic/aerobic cycling selects and enriches PAOs, and PAOs take up phosphate from wastewater at the aerobic stage (process diagram illustrated in Figure 2). In the PAOs well developed sludge, acetate and other short-chain fatty acids are taken up at the anaerobic stage by PAO cells and transferred to acetyl-CoA with ATP consumption from poly-P hydrolysis. This process is accompanied with release of cations such as K+ and Mg2+ as well as H2PO4 - to wastewater. After several steps of transformation and polymerization, acetyl-CoA is transformed to poly-β-hydroxyalkanoates (PHAs), mainly poly-β-hydroxybutyrate (PHB) and poly-β-hydroxyvalerate (PHV). The reducing power for acetyl-CoA transformation at anaerobic condition comes from NADH in the degradation of internal carbohydrate, via full [102] or split TCA cycles [103], but also found via the EMP pathway (a glycolysis pathway) from consumption of internal carbohydrates mainly glycogen [104]. As said before, hydrolysis of poly-P may also contribute energy source as ATP at the anaerobic stage, for acetate transport across cell membranes. Multiple of these pathways may co-exist depending on sludge sources and environmental conditions, which needs clarification by future studies [99]. At aerobic condition, the degradation of PHAs leads to the generation of acetyl-CoA and propionyl-CoA, both of which enter TCA cycle as carbon and energy source for biomass growth, phosphate uptake and poly-P generation, and glycogen generation.

Another important microbial composition of activated sludge is glycogen-accumulating organisms (GAOs). GAOs consume external carbon source at anaerobic stage, but at aerobic stage they do not uptake phosphate from environment and there is no poly-P accumulation occurring; instead, carbon and energy from PHAs hydrolysis is mostly used for biomass growth and glycogen synthesis. Therefore, there is a substrate competition between PAOs and GAOs for acetate and other short-chain fatty acids utilization. This relationship dominates the performance of phosphate removal in EBPR process [99], and is affected by environmental

**Figure 2.** Schematic diagrams of the anaerobic and aerobic PAO metabolism in EBPR process [101].

conditions for selective advantages between two groups, such as types and concentrations of carbon sources, organic loading rate, nutrients level and their ratio to carbon sources, pH, temperature, etc. Generally speaking, at the ambient pH range (6 to 8), a relatively higher pH is found advantages for phosphate removal and for PAOs selection over GAOs. At the ambient temperature range (5 to 35 o C), a lower temperature is favored by PAOs over GAOs for phosphate removal. Relative abundance of PAOs over GAOs may be increased by a relative lower dissolved oxygen levels (1.5 to 3 mg/L) at the aerobic stage [99].

Phosphate uptake is also possible by denitrifying PAOs which use nitrate as electron acceptor in the anoxic condition, corresponding to oxygen in the aerobic condition. This pathway is promising because it removes both nitrogen and phosphate and it reduces sludge production due to less energy release compared to aerobic condition [99]. There is no exclusive relationship between the concepts of general PAOs and denitrifying PAOs. Multiple commercial processes have been established for the combined biological nitrogen and phosphate removal in wastewater treatment plants, such as A2O, modified-UCT, five-stage Bardenpho, and DE‐ PHANOX with either pre- or post-denitrification [99].

### **4.2. EBPR with animal manure as substrate**

suitable operational conditions to avoid reactor upset can be important to the process. One limitation for EBPR is the insufficient carbon source in wastewater, or relatively low soluble COD-to-phosphorus ratio so PAOs does not sequester enough PHAs or energy for phosphorus accumulation. Manure before or even after anaerobic digestion usually contains a high level of organic compounds and a large portion of it is readily biodegradable such as acetate, propionate and butyrate. So manure can at least be a good substrate for providing carbon source for PAOs; in other words, EBPR can be an alternative method for phosphorus removal from liquid manure [100]. Some of the previous studies using animal manure in fact achieved good results in phosphorus removal via EBPR. Note that EBPR process usually is not the final step closing the phosphorus removal but a pretreatment method, although phosphorus is concentrated in sludge. The reason is that poly-phosphate in the sludge tends to be hydrolyzed, released and leaked to liquid phase. There are several other processes that can be used to further treat the EBPR sludge and phosphorus-accumulated media, e.g., coagulation and

EBPR has been used in wastewater treatment plants for a long period of time, and most fundamental studies were based on activated/recycled sludge from these treatment plants. Briefly, the anaerobic/aerobic cycling selects and enriches PAOs, and PAOs take up phosphate from wastewater at the aerobic stage (process diagram illustrated in Figure 2). In the PAOs well developed sludge, acetate and other short-chain fatty acids are taken up at the anaerobic stage by PAO cells and transferred to acetyl-CoA with ATP consumption from poly-P


Another important microbial composition of activated sludge is glycogen-accumulating organisms (GAOs). GAOs consume external carbon source at anaerobic stage, but at aerobic stage they do not uptake phosphate from environment and there is no poly-P accumulation occurring; instead, carbon and energy from PHAs hydrolysis is mostly used for biomass growth and glycogen synthesis. Therefore, there is a substrate competition between PAOs and GAOs for acetate and other short-chain fatty acids utilization. This relationship dominates the performance of phosphate removal in EBPR process [99], and is affected by environmental

and Mg2+ as well as

hydrolysis. This process is accompanied with release of cations such as K+

uptake and poly-P generation, and glycogen generation.

crystallization.

530 Biofuels - Status and Perspective

H2PO4

**4.1. Microbiology and mechanisms**

At the anaerobic stage, VFAs are substrates for PHAs regeneration inside PAOs cells, and the accumulated PHAs can be degraded for biomass growth and phosphate uptake at the aerobic stage. However, municipal wastewater contains only a small amount of VFAs, which reduces the suitability of EBPR process. Acetate may be added to wastewater but it increases overall chemical cost. So supplementing agro-food wastewater can be a good option, because it is considered waste and cost-effective. EBPR potential test, which evaluates the phosphorus release from activated sludge in certain medium composition in batch mode, was developed for assessing the feasibility of implementing EBPR for a substrate [105, 106]. Based on this test, it was found that tomato processing and milk bottling industry wastewater had short-term enhancement for EBPR, while wastewater from cheese indus‐ try, slaughter houses, beet sugar processing, and winery processing didn't show improve‐ ment [107].

The spatial requirement on condition variation (anaerobic/aerobic) in conventional flow systems for EBPR can be transformed to temporal variation by using sequencing batch reactors (SBRs) [108, 109]. This approach is widely adopted for treating liquid manure (liquid swine and dairy manure) [110, 111] because it saves space and operating cost over the conventional alternate flow systems, thus suitable for use in animal farms. SBRs consist several phases in its operation, typically including feeding, anaerobic, aerobic, settling, and withdrawal. Operating conditions such as medium composition, ORP, HRT, SRT, length of a cycle, length of each phase, pH, and temperature will have an impact on SBR perform‐ ance. Surplus phosphorus uptake from swine manure and poly-P accumulation in bio‐ mass was observed in liquid swine manure treatment in SBRs, with 93% of COD removal, 88%-93% of total nitrogen removal and 95% of phosphate removal [112]. With nondiluted swine wastewater that initially contained 1500 mg-N/L of ammonium and 144 mg-P/L of phosphate, a removal efficiency of 99.7% for nitrogen and 97.3% for phosphate was achieved in SBR operated with 3 cycles/day at temperature of 30 <sup>o</sup> C, SRT of 1 day and HRT of 11 days [110]. Subjecting the digested manure to EBPR may encounter lack of VFAs at the anaerobic stage of EBPR, so non-digested liquid swine manure can be supplemented to AD effluent as additional carbon source. This combined substrate achieved good nutrient removal by EBPR in SBR, 99.8% of nitrogen and 97.8% of phosphate removal from an initial content of 900 mg/L of ammonia and 90 mg/L of phosphate [111]. A 16 L SBR was constructed for treating liquid dairy manure by EBPR [113], and with 6-fold dilution, 98% of phosphate removal (from 33 mg/L to 0.5 mg/L of phosphate) was obtained, while the removal was dropped to 70% with 5-fold dilution. It was suggested by the authors that the higher strength dairy manure contained inhibitory components to EBPR, and high content of acetate was thought as the major reason but was not conclusive. Another study fed EBPR SBR with dairy industry wastewater, and phosphate concentration was decreased from 29.8 - 43.6 mg/L to less than 1 mg/L, demonstrating a highly effective phosphate removal [114]. The EBPR sludge showed a high proportion (31.4% to 38.3%) of *Rhodociclus*-related bacteria, an indicator of PAOs population, in the total microbial cell counts, and phosphorus accounted for 5.4% to 10.1% of the MLVSS [114]. Another SBR study reported 59% of phosphate removal with an initial phosphate level of 37.4 mg/L in dairy manure, generat‐ ing sludge with 2.6% of phosphorus in the MLVSS [115].

### **5. Phytoremediation**

Phytoremediation could be a low-cost clean up technology for wastewater treatment and also for the P removal, as the green algae and plants offer the finest eco-friendly option for environment remediation. The major hitch with the phytoremediation is the slow growth rate of the plant species and its survival capacity in the non-ideal environment. But, algae and aquatic plants offer a realistic time frame for the nutrient recovery from eutrophic water and other contaminated waters rather than the P removal from the soils, and also require less than one-tenth of the area to recover phosphorus compared to terrestrial crops [116]. The charac‐ teristic advantages of the system has made the algal ponds and macrophyte wetlands more popular for environmental applications including herbicides [117], heavy metals [118] and antibiotics [119] removal, and is already been explored widely for varieties of wastewater types including animal manure [120]. Anaerobic treatment of manure results in digestate which requires further treatment before discharge, especially for the removal of P and N. Growing microalgae or macrophytes on the anaerobic effluent could be a commendable option consid‐ ering the valuable byproducts and process efficiency.

### **5.1. Algae for phosphorous recovery**

try, slaughter houses, beet sugar processing, and winery processing didn't show improve‐

The spatial requirement on condition variation (anaerobic/aerobic) in conventional flow systems for EBPR can be transformed to temporal variation by using sequencing batch reactors (SBRs) [108, 109]. This approach is widely adopted for treating liquid manure (liquid swine and dairy manure) [110, 111] because it saves space and operating cost over the conventional alternate flow systems, thus suitable for use in animal farms. SBRs consist several phases in its operation, typically including feeding, anaerobic, aerobic, settling, and withdrawal. Operating conditions such as medium composition, ORP, HRT, SRT, length of a cycle, length of each phase, pH, and temperature will have an impact on SBR perform‐ ance. Surplus phosphorus uptake from swine manure and poly-P accumulation in bio‐ mass was observed in liquid swine manure treatment in SBRs, with 93% of COD removal, 88%-93% of total nitrogen removal and 95% of phosphate removal [112]. With nondiluted swine wastewater that initially contained 1500 mg-N/L of ammonium and 144 mg-P/L of phosphate, a removal efficiency of 99.7% for nitrogen and 97.3% for phosphate was

of 11 days [110]. Subjecting the digested manure to EBPR may encounter lack of VFAs at the anaerobic stage of EBPR, so non-digested liquid swine manure can be supplemented to AD effluent as additional carbon source. This combined substrate achieved good nutrient removal by EBPR in SBR, 99.8% of nitrogen and 97.8% of phosphate removal from an initial content of 900 mg/L of ammonia and 90 mg/L of phosphate [111]. A 16 L SBR was constructed for treating liquid dairy manure by EBPR [113], and with 6-fold dilution, 98% of phosphate removal (from 33 mg/L to 0.5 mg/L of phosphate) was obtained, while the removal was dropped to 70% with 5-fold dilution. It was suggested by the authors that the higher strength dairy manure contained inhibitory components to EBPR, and high content of acetate was thought as the major reason but was not conclusive. Another study fed EBPR SBR with dairy industry wastewater, and phosphate concentration was decreased from 29.8 - 43.6 mg/L to less than 1 mg/L, demonstrating a highly effective phosphate removal [114]. The EBPR sludge showed a high proportion (31.4% to 38.3%) of *Rhodociclus*-related bacteria, an indicator of PAOs population, in the total microbial cell counts, and phosphorus accounted for 5.4% to 10.1% of the MLVSS [114]. Another SBR study reported 59% of phosphate removal with an initial phosphate level of 37.4 mg/L in dairy manure, generat‐

Phytoremediation could be a low-cost clean up technology for wastewater treatment and also for the P removal, as the green algae and plants offer the finest eco-friendly option for environment remediation. The major hitch with the phytoremediation is the slow growth rate of the plant species and its survival capacity in the non-ideal environment. But, algae and aquatic plants offer a realistic time frame for the nutrient recovery from eutrophic water and

C, SRT of 1 day and HRT

achieved in SBR operated with 3 cycles/day at temperature of 30 <sup>o</sup>

ing sludge with 2.6% of phosphorus in the MLVSS [115].

**5. Phytoremediation**

ment [107].

532 Biofuels - Status and Perspective

Anaerobically treated swine manure has proved to be good medium for algae growth [120-122]. Microalgae with its rapid growth potential, better adaptation to various ecological habitats and as an important feedstock for third generation biofuel, can be a best suited strain to grow in the waste waters for its valuable biomass and the nutrient recovery. It is reported that the major bottleneck to commercialization of algal fuels is the supply of N and P nutrients [123, 124]. Many recent research focuses on a suitable nutrient management strategy to use wastewater (industrial, municipal, dairy, food wastewater and digested dairy manure) as a nutrient supplement for cultivation of oil-rich green microalgae growth and recycling of nutrients [121, 122, 125-127]. The anaerobic digested effluent after decomposing organic waste to produce biogas has been used to grow algae for nutrient recovery. The effluent has relatively lower carbon levels for algae because of microbial utilization during anaerobic digestion [121]. Pretreatment steps like dilution to avoid inhibition and sterilization to prevent the contami‐ nation may be required for certain algae systems [121]. Raw and anaerobically digested swine manure has been treated widely by (1) suspended algae in (i) high rate pond systems [128-131], (ii) mixed algae systems [132] and (iii) mixed algae-bacterial systems [133] or (2) by immobi‐ lized algae [120], e.g., algal turf scrubber units [134-136]. The harvested algae can be a good high-grade protein supplement for animal feed and also can be used as a slow-release fertilizer [136] which can be directly sprayed as suspension in farm land or stored for future use [137].

Different species of freshwater micaralgae have been tested for nutrient removal from municipal wastewater and manure. The nutrient removal capacity of a *Chlorella sp.* from a highly concentrated municipal wastewater stream generated from activated sludge thickening process (raw and autoclaved medium) was tested by Li and coworkers [125]. After 14-day batch culture, algae could remove ammonia, total nitrogen, total phosphorus, and chemical oxygen demand (COD) by 93.9%, 89.1%, 80.9%, and 90.8%, from raw medium respectively. It was concluded that the system could be successfully scaled up, and continuously operated at 50% daily harvesting rate, providing a net biomass productivity of 0.92 g-algae/(L day) [6]. The immobilized and free cell cultures of two nanoplanktonic algal species, *Scenedesmus intermedius Chod.* and *Nannochloris sp.* isolated from different sources of pig manure was used to study the growth rate, phosphorus and nitrogen uptake from anaerobically treated manure [120]. P and N uptake rates for *S. intermedius* were 0.014 and 0.012 mg P h−1 and 0.022 and 0.009 mg N h−1 for free and immobilized cells respectively; and rates for *Nannochloris sp.* were 0.006 and 0.009 mg P h−1 and 0.011 and 0.006 mg N h−1 for free and immobilized cells. It was observed that the isolated species were more efficient in nutrient recovery than the commercially available strains [120], as the isolated strains were better acclimatized to the prevailing conditions. The anaerobically digested swine manure from a farm digester was used to culture *Chlorophyceae*, *Chlorella sp.*, *Scenedesmus obliquus,* and a cyanobacterium, *Phormidium bohneri*, to evaluate the inorganic nitrogen and orthophosphate removal efficiency. *Chlorella sp.* per‐ formed well in batch cultures wheres *P. bohneri* in semi-continuous conditions [138]. Benthic freshwater algae was also used to recover nutrients from dairy manure [134] in algae growth chambers operated in semi-batch mode by continuously recycling wastewater and adding manure inputs daily. It was found that, when compared to a conventional corn/rye rotation, such benthic algae production rates would require 26% of the land area requirements for equivalent N uptake rates and 23% of the land area requirements on a P uptake basis [134]. Besides microalgae species, some filamentous fungal species also showed some potential to combine with AD to remove and recover the phosphorus [139].

Harvesting microalgae from treated wastewater is cost intensive, therefore becoming the key to remove and recover the phosphorus. The attached algae cultures for the nutrient removal from the manure waste water was evaluated [140]. It was found that, depending on different culture conditions, the attached algal culture removed 61–79% total N and 62–93% total P from dairy manure. Overall, the attached algal culture removed 62–90% of total phosphorus, 62– 87% of soluble phosphorus, and 43–80% of orthophosphate from dairy manure. The economic assessment of algal turf scrubber technology for treatment of dairy manure effluent showed that economic balance would become more favorable if values from algae as a byproduct and nutrient trading credits can be realized [141].

### **5.2. Macrophytes for phosphorous recovery**

Aquatic macrophytes are the conspicuous plants that dominate wetlands, shallow lakes, and streams, playing a vital role in healthy ecosystems. Total nitrogen and total phosphorous removal in treatment wetlands can range from 3–98% to 31–99% respectively [142, 143] with an average removal of about 50% [144]. Studies have showed that vascular aquatic plants have acceptable animal feed qualities, ability to remove nutrients from water, and high production rates [145]. Macrophytes constitute a diverse assemblage of taxonomic groups and are often separated into four categories based on their habit of growth: floating unattached, floating attached, submersed, and emergent [146]. Macroscopic flora includes the aquatic angiosperms (flowering plants), pteridophytes (ferns), and bryophytes (mosses, hornworts, and liverworts). Macrophytes based nutrient removal technology has the merits of (1) high productivity of several large-leaf floating plants; (2) high nutritive value of floating plants relative to many emergent species; and (3) ease of stocking and harvesting [147]. Also, the harvested floating macrophytes biomass can potentially be used for composting, soil amendments, anaerobic digestion with methane production, being processed for animal feed, and could be mixed with separated manure solids to increase the amount of nutrients available for exporting off the farm [148]. The biomass can be a good resource of starch, and utilized for the production of value-added products such as fuel ethanol [149]. For example, *Spirodela polyrrhiza* grown on

anaerobically treated swine wastewater had a starch content of 45.8% (dry weight) and enzymatic hydrolysis of the duckweed biomass yielded a hydrolysate with a reducing sugar content corresponding to 50.9% of the original dry duckweed biomass, yielding 25.8% ethanol (dry weight) after fermentation by yeast [150]. Duckweed (*Lemnaceae*) has been widely used to recover the nutrients from pig effluents, because of its tolerance to high nutrient levels and preferred absorption of ammonium [149, 151], and can also grow all seasons in areas with warm climates and doubles its biomass within two days under the optimal conditions [152]. Screened duckweed strains, *Lemnaceae* that grew well on the anaerobically treated swine wastewater in laboratory and greenhouse experiments were tested for nutrient recovery under field conditions. Under nutrient abundant conditions in waste water, duckweed takes the nutrients for its growth and store the nutrients in its tissue for future nutrient limited conditions for a significant period of time, and the nutrient reserve in duckweed biomass has been found the key to the kinetics of duckweed growth [153]. Plants take up nutrients while growing and if not harvested, decompose in wetlands returning nutrients to the ecosystems.
