**4.3 Recycling nutrients**

Optimal microalgal growth relies on continuous and adequate supply of nutrients (nitrogen, phosphorous, carbon, potassium, trace elements and water) and sunlight. Nutrient input can be in the form of fertilizer and waste-water streams. Nutrient supply in the form of fertilizers can incur significant cost to the cultivation and is also a competition to fertilizer for agriculture [65]. Therefore, it is important to minimize nutrient losses during cultivation. One way is through stoichiometrically balanced nutrient management to minimize nutrient losses during cultivation [76] and other ways are by recycling of spent medium (water recycle) and nutrient recycling post biomass conversion process.

#### *4.3.1 Water recycling*

During growth not, all the nutrients are used completely, and these unused nutrients will be lost if the water is not recycled post harvesting. Water recycling is important not just for nutrient recycling but also from an economics perspective. Water reuse reduces the need to acquire new water for cultivation, thus reducing the water foot print for cultivation and lowering energy usage in pumping water from source to site [77]. There is a finite possibility that water recycling can affect subsequent growth performance of the algae if the recycled water quality does not meet required standards. Primary factors influencing recycled water quality can be increased salinity of the water, use of chemical based harvesting system, accumulation of extracellular metabolites (protein, carbohydrate, fatty acids, nitrogen rich small organic molecules, cell wall debris and other particulate matter) which may be directly inhibit algal growth or increase the dissolved organic carbon (DOC) leading to increased bacterial load and gradual accumulation of toxic metabolites [77–79]. However, multiple studies, both at small and large scale have successfully demonstrated recycling of water without negatively affecting algal growth. Recycled water obtained after electro-flocculant, bio-flocculant, nannochitosan, filtration, and centrifugation based harvesting methods had shown no negative effect on the growth of tested algal species [80–84]. Flocculation-based methods have been predicted to be better for water recycling than other methods because they do not lyse the cells and help in reducing dissolved organic matter during harvesting [78]. Farooq et al. (2015) compared chemical flocculation (FeCl3 or alum) of *Chlorella vulgaris* against centrifugation and showed that recycled media obtained after centrifugation or flocculation with FeCl3 had positive effect on growth and lipid productivity. However, recycled medium obtained through treatment with alum even in low dose (<5 ppm) inhibited the growth of *C. vulgaris* due to the toxic effect of residual Al in the recycled water [85]. Similar results were obtained in case of *Scenedesmus* sp., where growth was affected in recycled medium, when alum (1 mM) was used to harvest the cells [84]. Likewise, in another study strain dependent growth inhibition was observed due to accumulated DOC in recycled water. Growth of *Navicula* sp. and *Chlorella* sp. were comparable to fresh medium, while growth of *Staurosira* sp. was completely inhibited in reused medium [79]. It is important to note that stage at which the culture is harvested also affects DOC concentration. Water recycled from exponentially growing cells was found to be more supportive of growth than cells in late log phase or stationary phase, conditions that lead to the maximum accumulation of growth inhibitory substances secreted by algae. As DOC accumulation is more during late log and stationary phases due to the release of secondary metabolites into extracellular space, it is better to avoid recycling water from cultures harvested from these phases [78]. Pretreatment of water before recycling can be considered to improve water quality for long term cultivation with recycled medium. Filtration, high speed centrifugation and sterilization methods have been studied for pretreatment, but their commercial scale application is questionable [77]. In one study, activated carbon was used to process recycled medium to remove humic and fulvic acid like growth inhibitors. This step moderately improved growth of *Nannochloropsis oceanica* in recycled water [86]. Recently, advanced oxidation process has been evaluated for pretreatment of recycled water. It was observed that UV/peroxydisulfate and UV/ H2O2 processes are quite effective in addressing organic matter load in the water. Oxidation method could degrade and converts inhibitory substances into nutrient source for algal growth. This method helps in utilization of DOC in recycled water rather than its removal [87].

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*Recent Advances in Algal Biomass Production DOI: http://dx.doi.org/10.5772/intechopen.94218*

*4.3.2 Nutrient recycling from HTL aqueous phase*

overall nutrient input.

Thus, recycling of spent medium is commercially viable and practically feasible option, which not only helps in saving loss of unused nutrients but also reduces the

Hydrothermal liquification (HTL) is a potential technology to convert wet algal biomass into bio-oil with biochar and aqueous phase (AP) as byproducts. AP is substantial portion because high moisture containing (~10–20% algal slurry) biomass is used as feedstock in HTL [88]. AP is nutritionally rich, containing organic carbon

+

, nitrate

as short chain organic acids, like acetic and propionic acid, nitrogen as NH4

and other nitrogen containing compounds, phosphorous as orthrophosphates and other macro and micro nutrients [89]. This makes AP a potential nutrients source for microalgae when recycled back into cultivation, which are otherwise lost. It is also reported that even harmful algal blooms are also good feedstock for HTL and AP produced is promising nutrient source for microalgae cultivation [90]. AP also has growth inhibitory compounds like phenols, amides, pyrazines, indole, metal ions like Ni etc., which either must be removed or diluted to the extent that they are no more growth inhibitory [89, 91]. Composition of AP is quite variable and depends on algal feedstock used for HTL, processing parameters, biomass loading and use of AP separation method from bio-oil. For instance, high protein content in feedstock leads to higher organic carbon and nitrogen content in AP [92]. Likewise, increasing resident time in HTL process also has shown to result in increased total nitrogen in the AP. Since, the concentration of nutrients and toxic compounds is often high in AP, substantial dilution of AP is needed to bring concentration of nutrients in the usable range and dilute growth inhibitory toxic elements. There are multiple studies reported where AP is used as sole nutrient source for algal cultivation or a supplement with systematic heavy dilutions made either with water or combination of water and standard nutrient medium. Outcome of these studies is quite variable and was dependent on AP composition and strain being used for cultivation. When AP was used as sole nutrient source, growth of the tested algae was relatively compromised. For instance, AP obtained from *Spirulina* HTL was used as sole nutrient source for cultivating *Chlorella minutissima,* where AP consisted ~16,200 mg/L N and 795 mg/L P along with other nutrients. Biomass productivity obtained was 0.035 g/L/d at 0.2% AP (500X dilution), which was significantly less than BG11 control, having 0.07 g/L/d productivity [91]. Likewise, APs obtained from HTL of *Chlorella vulgaris, Scenedesmus dimorphous* or *Spirulina platensis* as feedstocks were also evaluated as sole nutrient source at various dilutions to grow these stains. Growth of *Chlorella* and *Scenedesmus* was less in comparison to standard medium even at 400X dilution, however, *Spirulina* showed comparable growth in AP and standard medium [93]. Alba et al. (2013), presented comparative account of AP diluted with water versus standard medium for cultivation of *Desmodesmus* sp*.* A substantial reduction in growth was observed when AP was diluted with water, however, when mixture of water and AP was enriched with standard

medium, growth comparative to standard medium was observed. This study clearly indicates that it is not just N and P content that is important for growth but balancing AP in such a way that other macro and micro nutrients are also not limiting is essential for successful use of AP for cultivation [94]. Similar results were obtained in other studies, where AP diluent was enriched with desired nutrients [95–100]. Interestingly, Lopez Barreiro et al. (2015) observed that growth in AP diluted with standard medium was strain dependent. *Nannochloropsis gaditana* and *Chlorella vulgaris* could grow well in AP diluted with standard medium, however, *Phaeodactylum*  *Biotechnological Applications of Biomass*

During growth not, all the nutrients are used completely, and these unused nutrients will be lost if the water is not recycled post harvesting. Water recycling is important not just for nutrient recycling but also from an economics perspective. Water reuse reduces the need to acquire new water for cultivation, thus reducing the water foot print for cultivation and lowering energy usage in pumping water from source to site [77]. There is a finite possibility that water recycling can affect subsequent growth performance of the algae if the recycled water quality does not meet required standards. Primary factors influencing recycled water quality can be increased salinity of the water, use of chemical based harvesting system, accumulation of extracellular metabolites (protein, carbohydrate, fatty acids, nitrogen rich small organic molecules, cell wall debris and other particulate matter) which may be directly inhibit algal growth or increase the dissolved organic carbon (DOC) leading to increased bacterial load and gradual accumulation of toxic metabolites [77–79]. However, multiple studies, both at small and large scale have successfully demonstrated recycling of water without negatively affecting algal growth. Recycled water obtained after electro-flocculant, bio-flocculant, nannochitosan, filtration, and centrifugation based harvesting methods had shown no negative effect on the growth of tested algal species [80–84]. Flocculation-based methods have been predicted to be better for water recycling than other methods because they do not lyse the cells and help in reducing dissolved organic matter during harvesting [78]. Farooq et al. (2015) compared chemical flocculation (FeCl3 or alum) of *Chlorella vulgaris* against centrifugation and showed that recycled media obtained after centrifugation or flocculation with FeCl3 had positive effect on growth and lipid productivity. However, recycled medium obtained through treatment with alum even in low dose (<5 ppm) inhibited the growth of *C. vulgaris* due to the toxic effect of residual Al in the recycled water [85]. Similar results were obtained in case of *Scenedesmus* sp., where growth was affected in recycled medium, when alum (1 mM) was used to harvest the cells [84]. Likewise, in another study strain dependent growth inhibition was observed due to accumulated DOC in recycled water. Growth of *Navicula* sp. and *Chlorella* sp. were comparable to fresh medium, while growth of *Staurosira* sp. was completely inhibited in reused medium [79]. It is important to note that stage at which the culture is harvested also affects DOC concentration. Water recycled from exponentially growing cells was found to be more supportive of growth than cells in late log phase or stationary phase, conditions that lead to the maximum accumulation of growth inhibitory substances secreted by algae. As DOC accumulation is more during late log and stationary phases due to the release of secondary metabolites into extracellular space, it is better to avoid recycling water from cultures harvested from these phases [78]. Pretreatment of water before recycling can be considered to improve water quality for long term cultivation with recycled medium. Filtration, high speed centrifugation and sterilization methods have been studied for pretreatment, but their commercial scale application is questionable [77]. In one study, activated carbon was used to process recycled medium to remove humic and fulvic acid like growth inhibitors. This step moderately improved growth of *Nannochloropsis oceanica* in recycled water [86]. Recently, advanced oxidation process has been evaluated for pretreatment of recycled water. It was observed that UV/peroxydisulfate and UV/ H2O2 processes are quite effective in addressing organic matter load in the water. Oxidation method could degrade and converts inhibitory substances into nutrient source for algal growth. This method helps in utilization of DOC in recycled water

*4.3.1 Water recycling*

**464**

rather than its removal [87].

Thus, recycling of spent medium is commercially viable and practically feasible option, which not only helps in saving loss of unused nutrients but also reduces the overall nutrient input.
