**3. Results and Discussion**

### *3.1. Nutrient Distribution in Feedstocks and Between HTL Products*

Table 1 illustrates the difference between PSS and *SPR* in terms of composition. The ash content, which reflected the inorganic matter of *SPR* (5.8 wt%), was similar to those reported by other studies [22,38,44,45] and significantly lower than the ash content of PSS (19.3 wt%). Its value in the literature ranged from 7.5 wt% [14,15] to over 30 wt% [46,47]. This variation was linked to the strong dependencies of the composition of PSS on the sewage sludge source and treatment techniques of wastewater. The nutrient content in the *SPR* mainly reflected the elemental concentration of the cultivation medium that was used [48]. Thus, the analytes of potassium (1.7 wt%), phosphorus (1.1 wt%), sodium (0.4 wt%), and magnesium (0.3 wt%) constituted the main inorganics of *SPR*, which (with the exception of magnesium) was in accordance with previous studies [26]. By contrast, calcium (4.0 wt%), phosphorus (2.1 wt%), iron (0.6 wt%), and aluminum (0.4 wt%) constituted the major inorganic composition of PSS [49]. The higher protein content in *SPR* [50] resulted in higher nitrogen and sulfur contents compared to those in PSS.


**Table 1.** Nutrient distribution in feedstocks and between hydrothermal liquefaction (HTL) products (mean value (MV) ± standard deviation (SD) of two replicates).

a on dry basis; b analyzed as received; c from [38]; d not analyzed.

Di fferences in the composition and structure of feedstocks resulted in disparate distributions of nutrients in the HTL products (Table 1). The inorganics that were found in the oil phase can have an impact on upgrading to fuel [51–53]. For example, phosphor- and iron-containing species could settle on the upgrading catalyst, thereby blocking access to the catalyst interior and thus deactivating it. The pollution of the HTL oil phase with nitrogen also appears to inhibit the upgrading step [54]. The low level of sulfur in the HTL oil phase could have a negative e ffect on the activity of conventional sulfided upgrading catalysts, e.g., CoMo catalyst [52,55]. In the *SPR* oil phase, iron was present in abundant concentrations (0.1 wt%). These results concur with previous studies of HTL of microalgae [44,56,57]. Iron-associated proteins that were present in microalgae biomass [58] appeared to be stable to decomposition during HTL and can be recovered in bio-crude as iron-porphyrins [51]. The demineralization regarding iron was di fficult [59] and required more detailed investigation. The PSS oil phase was contaminated primarily by the elements of calcium (4.5 wt%) and phosphorus (2.8 wt%). The presence of inorganic elements in HTL-PSS-OP may be related to solid particles [60]. To investigate this hypothesis, the PSS oil phase was dissolved in ethanol, and the measurement of the particle size distribution was conducted with dynamic image analysis after destroying agglomerates in an ultrasonic bath. It was detected that a noticeable number of particles with a mean diameter of 38 μm were suspended in the PSS oil phase (see Figure S1). In contrast, by the same method, a number of solid particles in HTL-*SPR*-OP were negligible. The transport of solid particles in the oil phase is not ye<sup>t</sup> known. Their content could be reduced by, for example, an in-line filtration system during HTL, which was not in operation during the present study [38,61].

A proportion of biomass phosphorus (as phosphate) was recovered in both the HTL solid and liquid phases. The HTL solid phase was the main reserve of phosphate from the PSS, as in Marrone's study [15], while the phosphate from the *SPR* was found in both the HTL solid and liquid phases (Table 1). Moreover, the content of phosphorus in HTL-*SPR*-LP was in the range of previous studies [26,32]. The distribution of phosphate can relate to the presence of other dissolved multivalent metal ions, such as Ca2+, Mg<sup>2</sup>+, Al3+, and Fe2+/3<sup>+</sup> [6,62], in the reaction solution that tended to precipitate in the form of phosphate salts under a subcritical condition [63–65]. Their high concentration can shift the phosphate from the liquid phase to the solid phase. Therefore, the considerable concentration of metal ions (primarily Ca2+) in PSS compared to *SPR* could be responsible for the recovery of phosphate in the HTL solid phase. While the multivalent ions demonstrated a tendency to precipitate under the studied hydrothermal conditions—which was reflected in the fact that calcium, magnesium, iron, and aluminum were mainly recovered in the HTL solid phase—the monovalent ions (K<sup>+</sup>, Na<sup>+</sup>) remained dissolved in the HTL liquid phase. A similar tendency has been described in references [66,67]. The nitrogen that was recovered in the HTL liquid phase, which resulted from the degradation of proteins under hydrothermal conditions [15,26], was clearly higher for HTL-*SPR*-LP than for HTL-PSS-LP. The di fferent levels of nitrogen (as ammonium) in the HTL liquid phase between PSS and *SPR* resulted in di fferent pH levels [68]. The pH of the HTL liquid phase from the processing of *SPR* became basic (pH 8.5), and that of PSS became neutral (pH 7.0), which in turn induced alteration of the mineral solubility.

Figure 2 presents the estimated elemental balance for the experimental HTL pilot plant runs of PSS and *SPR*. By taking into account the mean yield of the oil phase (24.5 wt% and 32.9 wt% on a dry basis [38]), the dry matter content of feedstock slurries, the flow rate of slurries, and the total duration of runs, the elemental balance was calculated from the average elemental content in the HTL products that were obtained after the reactor runs (Table 1). The density of slurries was calculated with consideration to the percentage of solids in the slurry and by a solid density of 500 kg·m<sup>−</sup><sup>3</sup> [38]. It is important to note that the yields of gas, liquid, and solid HTL byproducts were not directly measured at this stage of the development of the HTL pilot plant. The yields for the gas and solid phases, which were determined on the basis of the dry matter of feedstock, were taken from previous studies at the batch system as average values. The yields for the HTL solid and gas phases were assumed to be 10 wt% and 14 wt% [47,69] for PSS and 5 wt% and 20 wt% [9,44,47,50] for *SPR*. The amount of the HTL liquid phase after the run was calculated as the di fference between the mass of feedstock slurry and

the mass of oil, solid, and gas phases that were produced in each run. The percentage of the respective elements for the HTL runs of PSS and *SPR* was calculated from the division of the mean amount of the element in the corresponding phase by the mean amount of this element in the biomass feedstock. The SD of the percentage was calculated from the SD of the elemental content in the biomass feedstock and the HTL product (Table 1); the SD of the yield of the HTL product was not taken into account due to lack of information.

**Figure 2.** Estimated elemental balance for HTL runs of PSS (**A**) and *SPR* (**B**) (MV ± SD; error bars are not shown in the figure).

Such assumptions might lead to an over- or underestimation of the elemental mass balance. In general, a lower balance closure was observed for the HTL run of PSS than for the HTL run of *SPR*. The mass balance for HTL of PSS (Figure 2) indicated an overestimation of good soluble elements, such as potassium and sodium, and underestimation of poor soluble elements, such as phosphorus, calcium, iron, and aluminum. The causes for this over- or underestimation are unclear but might originate from an overestimation of the amount of the HTL liquid phase or an overestimation of the elemental content by, for instance, evaporation of some HTL liquid phase during sampling (for K and Na) or from fouling and deposition in the reactor (for P, Ca, Fe, and Al). The especially high overestimation of magnesium in PSS is questionable and might be due to contamination of the pilot plant from previous runs. The calculated mass balance for the HTL run of *SPR* (Figure 2) is more or less closed. Only the balance for calcium exceeded 100%, which may be linked to the dissolution of Ca deposits in the reactor. In addition, the sampling of a multi-phase system is always challenging since the ratio of the phases might not be correct in each sample. Despite the over- and underestimation, several general tendencies regarding mass balance can be identified. The preliminary mass balance can assist in selecting a promising strategy for nutrient extraction.

The phosphorus balance was closed at 82% and 80% for the HTL run of PSS and *SPR*. The balance suggests that approximately half of the phosphorus from the processed *SPR* was extracted in the HTL liquid phase, while half of the phosphorus from PSS in the HTL solid phase. The smaller fraction of phosphorus from *SPR* was recovered in the HTL solid phase, and from PSS in the bio-crude. The phosphorus distribution determines the possibility of using the HTL solid and liquid phases as an indirect or direct source of phosphates in the case of HTL of PSS and *SPR*, respectively. Nonetheless, there appears to be a strong tendency to recover phosphates with the PSS bio-crude. As mentioned, a substantial load of particles with a mean diameter of 38 μm were impurifying the crude oil. Therefore, the phosphate remains in mineral form and can be filtered from the crude oil. The data for nitrogen also provided an acceptable balance. Approximately four-fifths of the recovered nitrogen was in the HTL liquid phase for the HTL run of PSS, and three-fourths for the HTL run of *SPR.* Thus, the availability of nitrogen for recovery from the liquid phase was increased by HTL. Magnesium, calcium, iron, and aluminum from PSS exhibited the same tendency as the phosphorus distribution. Meanwhile, these metals from *SPR*, with the exception of iron, were recovered primarily in the HTL solid phase. The occurrence of iron in the form of recalcitrant porphyrin in bio-crude has already been discussed. The majority of the potassium and sodium that were initially present in PSS and *SPR* was found in the HTL liquid phase, which was consistent with previous studies on batch systems [66].

## *3.2. Strategy of Phosphate Recovery*

The mass balance that has been presented in Section 3.1 demonstrates that the HTL solid byproduct could be an appropriate point for phosphate recycling and reuse. In view of the aforementioned disadvantages, this work does not further consider the direct use of the HTL liquid. The HTL solid phase from PSS and *SPR* with the agronomic relevant composition (N-P2O5-K2O) of 0.9-22.7-0.4 and 5.3-13.3-1.9 (Table 1), respectively, may represent a material with properties that are relevant to the application as a fertilizer. For this purpose, the phosphate must be released from the HTL solid phase to become available for the crops. The release behavior of phosphate is related to its form.

Figure 3 presents the respective content of the various forms of phosphate in the HTL solid phases from PSS and *SPR,* as well as the percentage of crop-available phosphate. The phosphate was mainly present in IP form; in both cases, the OP amounted to less than 1%. Moreover, IP consisted mainly of AP (97 wt% and 65 wt% for HTL-PSS-SP and HTL-*SPR*-SP, respectively). The phosphate form distribution was in line with those in previous studies of phosphate behavior under hydrothermal conditions [70–73]. The higher percentage of AP in the HTL solid phase from PSS could relate to the higher level of calcium in PSS (Table 1) compared to in *SPR* [73]. The Ca-bound phosphate was the most prevalent phosphate form in HTL-PSS-SP, which was consistent with XRD analysis. Such analysis concluded that calcium phosphate with inclusions of sodium and magnesium ((Ca3.892Na0.087Mg0.021)(Ca5.491Na0.121Mg0.028)(PO4)5.1) was the most abundant phosphate crystalline mineral in the HTL solid residual from PSS (see Figure S2). This finding was consistent with findings of minerals by other researchers after hydrothermal treatment of biomass that is rich in metals [43,74,75]. In turn, the recorded diffraction patterns of HTL-*SPR*-SP corresponded to a dittmarite mineral (NH4MgPO4·H2O) (see Figure S2). In contrast, Roberts et al. [64] have detected hydroxyapatite after HTL of microalgae biomass. The differences may be explained by the lower calcium content in *SPR* and the presence of calcium phosphate in HTL-*SPR*-SP in an amorphous form [74] that cannot be detected by XRD. The CAL-P extraction illustrated that these minerals might not be used directly as fertilizer. The crop-available form of phosphate was only 1 wt% in HTL-PSS-SP and 31 wt% in HTL-*SPR-*SP. Furthermore, previous studies [17,18] have identified enrichment of the HTL solid phase with heavy metals. Thus, a concentration of phosphate in high-value fertilizer, such as struvite was required. Struvite production through the approach of precipitating phosphate that was released from the HTL solid phase [19] does not seem difficult to implement at the industrial scale and is investigated further in the next section.
