1 Introduction

Arid climates that occur in the Middle East North Africa (MENA) region, and particularly the Gulf Cooperation Council states, have one of the highest food security risks globally [1]. The region is plagued with severe water stress, poor soil fertility, and increased irrigation requirements due to high ambient temperature, making agricultural practices expensive and challenging [2]. The agricultural challenges of the region have made fisheries a historical food source of current importance, which is reflected by the high per capita seafood consumption in countries like the UAE (51 kg/annum), Oman (36.7 kg/annum) and Qatar (16.5 kg/annum) [3, 4]. However, the gradual shift from fishing to aquaculture has created an increasing demand for aquaculture feed. Over the past three decades, there has been a 527% increment in aquaculture as opposed to a 14% increment in traditional fisheries [5]. Aquaculture was recently reported to account for 52% of global human seafood consumption [6]. The MENA region has also reported a similar trend [4]. Mainstream aquafeed protein sources like forage fish and soybean have been considered unsustainable due to the increasing overexploitation of the former and the scarce resources available, such as freshwater and arable land, to produce the latter [7]. Some alternative aquafeed protein sources explored are fish waste, food waste, insects, and microbial protein [7]. Of the substitute protein sources explored so far, microbial protein, also known as single-cell protein (SCP), has been widely appraised [8].

SCPs are microbial biomass mostly consisting of unicellular organisms like yeast, bacteria, and microalgae. They are broadly credited with having a high protein content and a high-quality amino acid profile [9, 10]. The comprehensive appraisal for SCP as a sustainable means of aquafeed production is chiefly because of its low energy inputs, high productivity, and potential circular economy approach using waste streams as feedstock. Protein-rich biomass has been generated by treating waste streams, such as lignocellulosic material, brewery wastewater, orange wastewater, fruit waste, and other forms of agro-waste [8, 11, 12]. Moreover, among the microbes examined for SCP production, purple non-sulphur bacteria (PNSB) have received recent attention. This is primarily due to the organism’s metabolic versatility under diverse culture conditions and its ability to efficiently valorise nutrients by accumulating value products like high-quality protein, carotenoids, coenzyme-Q10, 5-aminolevulinic acid and polyhydroxyalkanoates [13, 14]. Also, PNSB reportedly has a comparatively higher cell protein content than SCP sources like fungi, microalgae, and yeast, with one study reporting over 90% protein content [15].

So far, most studies examining PNSB’s resource recovery abilities have utilised agricultural waste streams because of their rich nutrient and organic carbon nature [8]. However, the paucity of such waste streams in arid climes creates the need to examine other more readily available alternatives. With the region being a hub for oil and natural gas, fuel synthesis process water (FSPW) from gas-to-liquid (GTL) plants stands to be a potential source for carbon substrate. The relative abundance, physical characteristics, and chemical composition of the FSPW enhances its suitability as an ideal feedstock for resource recovery in the MENA area, and being the result of a synthetic catalytic reaction, has a consistent composition with low toxicity [16]. The region houses 40% of GTL plants globally, including the world’s largest plant in Qatar. With over a million cubic meters of FSPW generated on a monthly basis [17], there is an abundant feedstock for potential resource recovery processes. Such wastewater generation magnitude enhances large-scale SCP production’s economic feasibility [18]. Using such readily available wastewater as the carbon substrate significantly reduces the cost burden of the resource recovery process and guarantees continued operation, if a large-scale operation is achieved.

In addition, the physical properties of the wastewater make it highly favorable for SCP recovery compared to several other waste streams. This is in contrast to various SCP feedstocks that have been reported to require physical pretreatment (heating, milling, filtration, and ultrasonication), chemical pretreatment (acid, ionic liquid, and alkaline wet oxidation) and biological pretreatment (enzyme addition) [19]. Another study that employed animal manure substrate reported the need for extensive pretreatments like drying, crushing, reverse osmosis, incubation, and centrifugation [20]. These processes add significant cost, energy and time to the microbial protein production process. However, FSPW is clear, with negligible total dissolved solids and a total suspended solid of less than 30 mg/L, with no pretreatment requirements. Besides the reduced processing cost compared to other wastewater, large-scale production of SCP via liquid fermentation has also been reported to be more economically beneficial than SCP from solid-state fermentation [18]. FSPW also has an exceptionally high organic load, with chemical oxygen demand (COD) concentrations as high as 30 g/L reported [21]. The wastewater contains soluble organics like volatile fatty acids (VFAs), alcohols, and ketones that can be degraded directly [22, 23]. Hence this study examines the potential to upcycle biomass from the anaerobic treatment of FSPW with PNSB to microbial protein for aquaculture feed. A preliminary quality assessment was also performed to determine the safety of the wastewater and biomass.

2 Materials and methods

2.1 Raw wastewater

FSPW was obtained from a local GTL plant. The plant produces around 40,000 m3 of wastewater daily [17]. The process water, a byproduct of the natural gas liquefaction process, was collected from the plant in a 1 m3 intermediate bulk container totes, and later transferred to high-density polyethylene jerry cans without pretreatment. The wastewater was stored at 4 °C upon arriving at the laboratory, after which wastewater characterization was performed. The FSPW is shown in Fig. A1, demonstrating the transparent solid-free nature of this liquid, allowing for direct application without pretreatment requirements. FSPW’s purity is owed to its means of generation, which is via the Fischer-Tropsch (FT) process. FSPW is a by-product of the FT process, which converts syngas into petroleum products via a systematic polymerization process at high pressure and temperature [24]. The reaction is usually accelerated by applying catalysts like iron and cobalt [25]. The process can be summarised by Eq. (1) below as reported by Evans and Smith [25].

$$n\textrm{CO}+2n{\textrm{H}}_2+\textrm{heat}=n\ \left(\textrm{C}{\textrm{H}}_2\right)+n{\textrm{H}}_2\textrm{O}$$
(1)

2.2 Inoculum

A non-axenic culture dominated by PNSB was utilized. The PNSB originated from microbial mats extracted from a local mangrove ecosystem as described by George et al. [26]. This culture was then developed on FSPW for over a year prior to the experiments. The dominant PNSB in the culture was Rhodopseudomonas sp. (>60%). The cells were incubated in a sub-culture of nutrient-enriched FSPW for 48 h before the experiment. A mixed culture is preferred due to the economic impracticability of utilizing an axenic culture at a large scale in an outdoor setting. The high selectivity of PNSB under light and limited oxygen conditions enhances the feasibility of their dominance in a culture solution.

2.3 Experimental design

Preliminary assessment of the PNSB-dominated mixed culture’s (0.05 g/L) ability to thrive and sufficiently degrade FSPW was initially conducted via batch cultivation in a 2 L Duran glass bottle. The culture media consisted of diluted FSPW (1:5 dilution with ultrapure water), NH4Cl (80 mg/L), KH2PO4 (70 mg/L), NaHCO3 (500 mg/L), ATCC trace mineral supplements (which consists of EDTA, MgSO4, MnSO4, NaCl, FeSO4, Co(NO3)2, CaCl2, ZnSO4, CuSO4, AlK(SO4)2, H3BO3, Na2MoO4, Na2SeO3, Na2WO4, and NiCl2), and ATCC vitamin supplements (which consists of folic acid, pyridoxine hydrochloride, riboflavin, biotin, thiamine, nicotinic acid, calcium pantothenate, vitamin B12, p-aminobenzoic acid, thioctic acid, and monopotassium phosphate). The culture bottle was subjected to continuous light via two LED strip lights (20 W) and continuous mixing via a magnetic stirring bar at 250 rpm using a magnetic stirrer (VELP Scientifica, CG-1994-V). The cultivation period lasted 16 days, with a pH between 7.45 and 7.84 and was conducted at ambient temperature (24.4±0.1 °C). The LED strip lights had an intensity of 79 W/m2 each, with most of the irradiance within the visible light spectrum (400–500 nm: 31.1%; 501–600 nm: 22.9%; 601–700 nm: 25.3%; 701–800 nm: 12.1%; 801–900 nm: 0.7%; 901–1100 nm: 1.9%).

Upon obtaining favorable results from the preliminary assessment, biological triplicates of a non-axenic culture dominated with PNSB (0.05 g/L) were prepared using 1-L cylindrical benchtop photobioreactors (Multifors, Infors), with a working volume of 0.7 L. By volume, the feedstock consisted mainly of undiluted FSPW (84%) as carbon substrate. The other integral ingredients were in similar concentrations as the preliminary assessment, and their respective volumes were: ATCC vitamin supplement solution (10 ml/L), ATCC mineral supplement solution (10 ml/L), NH4Cl (45 ml/L), KH2PO4 (45 ml/L), and NaHCO3 (53 ml/L). The batch experiments were conducted for 11 days under continuous light (LED) at an intensity of 21.8 W/m2 with most peaks within the visible light spectrum. The range of spectral irradiance were 400–500 nm: 15.9%; 501–600 nm: 37.5%; 601–700 nm: 40.5%; 701–800 nm: 4.8%; 801–900 nm: 0.4%; 901–1100 nm: 0.9%). The bioreactors were kept at a constant temperature of 30 °C. The fermenters were not sparged to remove oxygen before inoculation. The starting dissolved oxygen saturation was 100% but rapidly dropped below 2.0% after a couple of hours. A dissolved oxygen saturation level of ≤1.5% was maintained for the rest of the batch culture period. The cultures’ pH was automatically regulated between 7.4 and 7.7 using 0.1 N HCl and 0.1N NaOH. The fermenters also operated at a stirring speed of 300 rpm. Samples were periodically collected to monitor biomass growth and organic degradation.

2.4 Analytical methods

The cultures’ pH, temperature, and dissolved oxygen levels were constantly monitored using Hamilton probes and sensors incorporated in the bioreactors’ systems. In addition, the optical density of the samples at 420 nm and 620 nm (OD420 and OD620) were measured using a UV-3600 plus UV-Vis-NIR spectrophotometer (Shimadzu). The specific growth rate was measured by determining the slope of the log plot of optical density vs time. Samples for COD, total organic carbon (TOC), inorganic carbon (IC), total nitrogen (TN), VFAs, and anions were first centrifuged at 6,000 g (Sorvall Lynx 6000, ThermoScientific), after which the supernatant was sieved through 0.2 μm sterile syringe filters (polyethersulfone, VWR). COD was measured via the closed micro-digestion method, using Hach high-range COD vials (0 to 1500 mg/L). TOC, IC, and TN were determined using a TOC analyzer (TOC-L series with TNM-1, Shimadzu), which employs a 680 °C combustion catalytic oxidation method. Total carbon and IC were obtained directly, while TOC was determined by subtraction. Measurements of VFAs and anions were measured using ion chromatography (940 Professional IC Vario, Metrohm). Light intensity and spectrum were measured using the UV-vis-NIR spectroradiometer Black-Comet-HR-Series (StellarNet Inc.).

The biomass’s total and volatile suspended solids (TSS, VSS) were measured using standard methods [27]. VSS for each day was estimated by absorbance measurements at 420 nm using Eq. (2), which was developed from measurements taken with R2 of 0.999.

$$\textrm{VSS}=14.343\times {\textrm{Abs}}^2+91.88\times \textrm{Abs}-27.126$$
(2)

Biomass morphological characteristics via imaging and elemental mapping were determined using a scanning electron microscope (SEM), model Quanta 650 FEG-ESEM (FEI) and using a transmission electron microscope (TEM), model Talos F200C TEM (Thermo Scientific), respectively. Suspended biomass samples were collected during the stationary phase. For SEM analysis, the liquid sample was directly placed in the sample holder using the environmental SEM feature. For TEM analysis, a 10 μL drop of suspended biomass was deposited onto a 400-mesh lacey carbon-Cu grid (CF400-CU, Electron Microscopy Sciences) and visualized directly. 16S metagenomics was performed by obtaining 5 ml culture on day 5 of the preliminary study, after which DNA extraction was performed as described by the DNeasy PowerBiofilm kit (Qiagen) protocol. Subsequently, DNA amplification and sequencing were performed as described by George et al. [26], while bioinformatics was performed using QIIME 2 via cross-referencing with the SILVA database. Biomass ash content was determined by accounting for biomass residual after oven drying at 550 °C. Biomass-based analysis was performed after harvesting the centrifuged biomass at 10,000 g and freeze-drying for 24 h. Freeze-dried biomass was then homogenized with a mortar and pestle, sealed with parafilm, and stored in a desiccator until further use. Following pretreatment via ultrasonic-assisted alkali extraction, the biomass protein was quantified using the Modified Lowry method. Subsequently, a 100 μg protein aliquot was hydrolyzed via exhaustive enzymatic hydrolysis under sterile oxygen-free condition using a PAL sample autoprocessor (CTC Analytics, Zwingen, Switzerland). The hydrolyzed protein was the utilized for amino acid characterization by LC-MS/MS using an AcquityTM UPLC system with a Quattro Premier tandem mass spectrometer as described by Rabbani and Thornlley [28]. In addition, biomass lipid and carbohydrate contents were determined using Bligh and Dyer and Anthrone methods, respectively [29, 30]. Coenzyme Q-10 was determined spectrophotometrically using the MyBioSource Human Coenzyme Q10 ELISA kit, with absorbance reading taken at 450 nm using a Tecan Spark plate reader. Pigment analysis was also performed spectrophotometrically as described by Lu et al. [12]. Trace elements and heavy metal concentration of the wastewater and microbial biomass were assessed using acid microwave digestion and inductively coupled plasma–optical emission spectrometry (ICP-OES; Agilent 5110). The limit of detection (LOD) for all characterized metals was 0.01 mg/L and 0.00001 mg/kg. Biomass digestion was performed using high-performance microwave digestion (Ethos Up, Milestone) by firstly treating 200 mg of sample biomass with 9 mL HNO3 and 2 mL H2O2 and subsequently digesting the sample at 200 °C for a 30-min duration following a steady ramp rate of 13 °C per min. After digestion, the sample was diluted and filtered through 0.2 μm sterile syringe filters (VWR) for metal content determination. The ICP-OES was operated with the radial view, and calibration for all metals of interest at their respective elemental wavelengths was performed at a range of 0 to 5 ppm. Moreover, extensive characterization for organic compounds in FSPW was conducted using gas chromatography/mass spectrometry via the headspace trap sampler (Agilent, 7820B GC system with 5977A MS) as described in the EPA methods 8260 B and 8260 C [31]. Over 130 volatile and semi-volatile organic compounds were characterized. Finally, freeze-dried biomass was qualitatively characterized using a Py-GC-MS setup (CDS 6200 pyrobrobe, Shimadzu GCMS-QP2020 NX, Kyoto, Japan) according to the protocol described by Sabah et al. [32].

3 Results and discussion

3.1 Wastewater characteristics

Chemical characterization of FSPW revealed that the wastewater was acidic and rich in organic carbon, like short-chain volatile fatty acids, with a total COD of over 10,000 mg/L. Table 1 summarizes the physicochemical characteristic of the wastewater which show a varied VFA profile, PNSB has been reported to have a higher productivity when cultured in such a carbon substrate [33]. A study that determined the impact of VFAs on PNSB growth kinetics revealed that cultures with mixed VFAs had higher growth rates (up to 2.5 fold) than cultures with individual VFA [34]. A recent study utilizing a different PNSB strain also reported higher growth rates with mixed VFA. However, the group with a sole VFA (acetate) had a similar growth rate [35]. In this study, the characterized VFAs accounted for about 51% of the total COD. In addition, further characterization revealed that alcohols like methanol and butanol account for the majority (>45%) of the outstanding COD. This is similar to the results obtained in another study that utilized process water from the same source [36]. Table A1 shows organic constituents of GTL process water from other studies, revealing similar organic profiles. The GC characterization of over 130 volatile and semi-volatile compounds revealed FSPW was void of toxic hydrocarbons like benzene, o-xylene, and a host of other benzene derivatives. In fact, the organics detected in the exhaustive screening accounted for less than 0.2% of the total COD. However, seven of the 16 US EPA priority polyaromatic hydrocarbons (PAHs)- acenaphthylene, chrysene, dibenz[a,h]anthracene, fluorene, fluoranthene, naphthalene, and pyrene were detected in low concentrations with a combined concentration of 218 μg/L. Based on the EPA classification, five of the priority PAHs detected are classified as “not classifiable as to human carcinogenicity” (Class D), while chrysene (0.008 μg/L) and dibenz[a,h]anthracene (0.0285 μg/L) are classified as “probable human carcinogenic” (Class B) [37]. An exhaustive list of the result is presented in Table A2. Even though in minute concentrations, the presence of potentially toxic aromatic compounds increases the need for scrutiny associated with possible microbial protein recovery from the feedstock. In this study, the characterized biomass was void of all priority PAHs, indicating that they were either degraded or released with the effluents. Organisms like Rhodopseudomonas palustris have been associated with the degradation of aromatic compounds [38], especially in low concentrations. Thus, extensively screening recovered biomass and effluent for toxicants of interest would be an integral consideration if the biotechnology materializes.

Table 1 FSPW physicochemical characteristics prior to nutrient addition. ±values are standard deviation from duplicates
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Heavy metal analysis of the wastewater revealed that heavy metals of public health concern were either absent or below the Water Environment Partnership Asia (WEPA) and the United States Environmental Protection Agency (USEPA) maximum permissible limits for both drinking water and industrial effluents, as shown in Table A3 [39, 40]. Only minute concentration of trace minerals like Co (0.2 mg/L), Fe (0.7 mg/L), Mn (0.9 mg/L), Zn (0.7 mg/L), and K (8.7 mg/L) was detected, which were likely related to reaction catalyst. This is an essential consideration when deliberating on potential feedstock for SCP production, as certain PNSB strains (Rhodobium marinum NW16 and Rhodobacter sphaeroides KMS24) have been reported to bioremediate heavy metals via biosorption and bioaccumulation [41]. Hence, using sources like domestic and non-agroindustrial wastewater is typically not recommended [42]. Another challenge with domestic wastewater and animal manure use is the presence of faecal pathogens, which are not present in FSPW due to the pure chemical synthesis process that generates it. However, a notable drawback of FSPW’s chemical characteristics is the paucity of essential nutrients like nitrogen and phosphorus, which is typically present in agro-based and domestic wastewater [20]. This challenge can be overcome by utilizing a relatively benign nutrient-rich waste stream as nitrogen and phosphorus sources. A study that examined hydrogen production from organic wastewater using PNSB successfully utilized monosodium glutamate crystallization (Aji-L) waste as a nitrogen source. Interestingly, the study found that Aji-L performed more favorably compared to the commercial nitrogen source (glutamate) as it contained some essential micronutrients [43].

3.2 Preliminary study with diluted FSPW

Results from preliminary treatment revealed that the FSPW was a suitable carbon substrate for the PNSB-rich mixed culture. After the 16-day culturing period, over 98% of the COD had been degraded. Around 70% of the organics were consumed by the fifth day, after which the COD removal efficiency decreased significantly over the next eight days until nitrogen was added to the culture. The reduced organic removal efficiency rate was most probably attributed to NH4-N depletion as nitrogen is a growth limiting nutrient, but is likely compounded by the complete VFA degradation within the period. Traditionally, it is expected that most PNSBs prefer to degrade only short-chain carbon substrate via a photoheterotrophic mode of metabolism; therefore, degrading more complex organics could be challenging [44]. A study that compared the feasibility of valorizing VFAs and sugar manufacturing wastewater (COD = 8 g/L) to hydrogen and lipids using Rhodobacter sp. reported maximum substrate degradation and higher productivity with VFAs compared to sugar manufacturing wastewater, the latter achieving only 65% degradation [43]. The reduced efficiency was attributed to the presence of more complex carbons like sugars. This is probably why COD degradation is fastest within the first five days in this study, as the easily degradable carbon substrates are expected to be consumed. Hence, studies have explored the possibility of degrading recalcitrant organics to VFAs via anaerobic fermentation before utilizing them as carbon substrates for photoheterotrophic biomass production [34, 45]. Nitrogen concentrations were closely monitored in the extensive experimentation to verify its growth limiting effect. Figure 1 shows the trends of COD removal. In addition, a peak OD620nm of 2.58 was obtained on day 13, while the average VSS obtained was 740±14 mg/L. The biomass yield of the setup was about 0.5 gVSSproduced/gCODconsumed, which is consistent with other studies that utilized non-axenic PNSB cultures for wastewater treatment [13, 46]. A biomass yield close to unity has been reported in studies that cultured PNSB in organic acid substrates, under continuous light-anaerobic conditions [34, 47]. However, in more complex carbon substrates, lower yields are obtained due to increased fermentation activity, PNSB H2 production in nutrient deficient conditions, and competition with non-PNSBs [13]. In a similar study utilizing FSPW from the same source, high biomass yields were obtained when highly preferential VFAs like acetate, propionate and valerate were available [48]. However, upon complete degradation, the biomass yield reduced significantly. An additional factor identified was the reduced photon energy available at the trial end due to light attenuation [48].

Fig. 1
figure 1

COD removal efficiency of the preliminary batch cultivation experiment of diluted FSPW

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Results from the preliminary assessment confirm PNSB growth and enrichment even when cultured under a light source mostly within the visible light spectrum. Metagenomics analysis revealed that Rhodopseudomonas sp was the most abundant organism detected (70.3%), followed by common chemoheterotrophs like Acinetobacter (9.1%) and Paludibacteraceae (4.7%) commonly found in mixed culture studies [49, 50]. Figure 2 shows the microbial community data for microbes with at least 1% abundance. High PNSB growth and selectivity have also been reported in another study that examined PNSB’s light-harvesting ability under several light sources within the visible light spectrum [51]. This is primarily because PNSB’s carotenoids are typically present in the light-harvesting complex 2, which can absorb photons in a wide range of visible spectra (450 to 550 nm). Continuous light-anaerobic conditions favor the growth and selectivity of PNSB over other phototrophs [13], particularly over oxygenic phototrophs like algae. PNSB dominance was further supported by the cell morphological characteristics during the stationary phase via SEM and TEM analysis. Most of the cells captured had a distinctive cylindrical rod-like shape, as seen in several studies that examined axenic cultures of Rhodopseudomonas sp. [52,53,54]. This was anticipated as Rhodopseudomonas was the dominant microbe in the starting inoculum. Furthermore, the SEM and TEM images (Figs. 3 and 4) show that the cells accumulated granules, suspected to be polyphosphates, as seen in other wastewater treatment biomass characterization studies [55]. Moreover, PNSB has been reported to accumulate polyphosphates in the stationary phase [56]. Typically, excess light energy is stored as polyphosphate and released in energy-deficient conditions, while biopolymers are accumulated when carbon is available and depleted in carbon-deficient conditions. Further analysis via elemental mapping and annular dark-field imaging revealed that the percentage weight of phosphorus in the cells was 4.1% and that the granules were most likely comprised of potassium and magnesium polyphosphates. The annular dark-field imaging of carbon, nitrogen, oxygen, potassium, magnesium, and phosphorus can be found in Fig. 5. This shows that beyond the recovery of nutritionally desirable constituents, which is the primary objective of this study, PNSB can also be utilized for direct phosphorus recovery in other types of wastewaters containing significant phosphorus concentrations.

Fig. 2
figure 2

Microbial community dynamics of preliminary assessment showing relative abundance

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Fig. 3
figure 3

A SEM analysis (secondary electrons); B SEM analysis (backscattered electrons) of the FSPW culture during stationary phase

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Fig. 4
figure 4

TEM analysis of the FSPW culture during stationary phase

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Fig. 5
figure 5

TEM annular dark-field imaging of selected elements in PNSB showing the granules consistency with O, K, Mg, and P

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3.3 Extensive experimentation with undiluted FSPW

3.3.1 Pollutant degradation

The oxygen saturation of the culture was at its peak at the start of the trial (93.9±5.5%); however, dissolved oxygen (DO) levels reduced significantly through the passage of time. By the 14th hour, DO levels reached 1.1±0.1% and remained constant until the end of the trial. This created an illuminated-micro-aerobic environment beneficial for PNSB photoheterotrophic growth. The removal of oxygen in mixed cultures is typically facilitated by aerobic or/and facultative anaerobic microbes. However, once the oxygen level is depleted, PNSB outcompetes other microbes due to their selectivity under light and oxygen-limited conditions [8, 13]. Confirmation of PNSB dominance (Rhodopseudomonas sp. >50%) was obtained from community diversity analysis (authors’ unpublished data). Figure A2 shows the trend of DO removal.

The average COD removal efficiency over 11 days was 78±0.3%, of which around 63% was consumed within the first 72 h. On the 8th day, a peak average OD420nm of 6.268±0.096 was obtained, with average biomass concentration of 1.11±0.037 gvss/L and a specific growth rate (μmax) of 0.589 d−1. Figures 6 A and 7 B show the biomass growth at OD420nm and biomass concentration through culture duration. A similar range of specific growth rate (0.48–0.71 d−1) has been reported in a study that examined the impact of VFAs, light, and oxygen on Rhodobacter sphaeroides [35]. Another study utilizing naturally occurring chemoheterotrophic bacteria in FSPW reported a significantly lower COD removal rate of around 260 mg L−d− compared to the rate of about 710 mg. L−d− obtained in this study by the photoheterotroph-dominated community [36]. This demonstrates that PNSB’s productivity and metabolic versatility have the potential to be a strong performer for the biological treatment of FSPW. The comparatively higher biomass concentration obtained in the undiluted culture compared to the diluted culture is in line with another study that reported a positive correlation between PNSB biomass concentration and the organic strength of the wastewater [33] and is beneficial for industrial application. The average biomass yield was 0.14±0.005 gVSSproduced/gCODconsumed, which was significantly lower than the yield obtained in the preliminary study. The substantially lower biomass yield obtained is most probably attributed to the microaerobic condition of the reactors. Oxygen has been reported to suppress photoheterotrophic metabolism and favor chemoheterotrophy, resulting in lower biomass yields [47]. Besides, in non-sterile cultures, there is increased competition with aerobic heterotrophs. A similar level of biomass yield has been reported in mixed-culture studies under continuous light-microaerobic conditions [57].

Fig. 6
figure 6

A Biomass growth at OD420nm and OD620nm; B biomass concentration in VSSmg/L; C trend of TOC, IC, and TN removal

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The nitrogen concentration, measured by TN analysis, was depleted within 72 h, indicating that nitrogen was a limiting nutrient when considered along with the COD degradation profile. The complete organic degradation, aided by nitrogen replenishment in the preliminary study, supports this stance. The COD removal rate within the first 72 h prior to nitrogen depletion (2100 mg L−d−) was significantly higher than the removal rate at the trial end (710 mg L−d−). Similar trends have also been reported in past studies, where increased nitrogen concentrations significantly enhance biomass concentration, protein content, and COD removal [44]. Furthermore, the high nitrogen removal efficiency (NH4+-N) at a rate of 29.5 mg/L/day observed in this study outperforms the removal rates from similar studies. A recent study that examined nitrogen metabolism in photosynthetic bacteria reported an NH4+-N removal efficiency of around 22 mg/L/d after three days under similar conditions [58]. This gives further credence to the suitability of FSPW for PNSB-based biomass production. Figure 6 C shows the trends of TOC, TN, and IC removal. TOC decreased throughout the test but at a lower rate after TN was nearly depleted. IC also decreased slightly throughout the test.

This study supports the ability of PNSB to thrive in a high-strength wastewater. PNSB’s versatility in similar high-strength wastewater has been previously reported in several studies [48, 59, 60] and is one reason they are purported as an ideal organism for integration with wastewater treatment and resource recovery [8, 10]. Furthermore, in this study, PNSB’s versatility was tested at an influent C/N ratio of 115:1, as opposed to the widely acceptable range of 2:1 to 20:1 for other activated sludge, microalgae, and purple phototrophic bacteria studies [61,62,63]. Results from this study provide further credence to PNSB’s adaptability. In fact, in one study, Rhodobacter sphaeroides’ versatility in pollutant removal and bioconversion was reported across C/N ratios ranging from 400 to 0.1, resulting in at least 60% COD removal and 40–60% microbial protein content [64]. This has been one of the drawbacks of microalgae-based wastewater treatment which has been more widely explored for potential resource recovery potential [65]. High COD concentrations (C:N>20) have been reported to inhibit algal growth, thereby making wastewater pretreatment an integral part of the process [66, 67]. However, in this instance, wastewater was directly applied to the biotechnology, resulting in respectable treatment efficiencies and biomass concentration. This is particularly advantageous for organic-rich wastewaters innately lacking essential nutrients like nitrogen. Industries involved in winery, textile processing, vinegar production, pineapple processing, and pharmaceuticals are among those that produce nutrient deficient wastewaters [68, 69]. Augmenting feedstock at recommended C/N ratios could potentially increase operating cost significantly. Therefore, being able to achieve pollutant removal rate and biomass concentration at high C/N ratios enhances the feasibility of utilizing such waste streams for integrated treatment and resource recovery purposes. Overall, the biomass concentration and organic and nitrogen removal rates obtained in this study exceed values reported in several algae-based wastewater treatment studies and are comparable to other purple phototrophic bacteria-based studies [8, 66]. For example, removal rates of around 1,000 mgCOD.L−1d−1 and 38 mgNH4-N.L−1d−1were obtained in studies that cultured PNSB in pharmaceutical and brewery wastewater using cultures of R. sphaeroides and R. capsulate [12, 70].

3.3.2 Biomass characterization

Proximate analysis revealed that the biomass consisted of 35±0.6% protein, 31.6±1.9% lipid, 15.9±0.6% carbohydrate and 7.4±2.9% fiber. Similar protein content has been reported in aquafeed protein sources like soybean, squid liver paste, and commercial aquafeed [71, 72]. Figure 7 is a descriptive comparison between PNSB biomass in this study and soybean flour from another study. The detection of 35% dry cell protein content under baseline experimentation is promising, considering this result was achieved under nitrogen-limited conditions at trial end and that several studies have reported that PNSB protein content tends to increase significantly under optimized conditions [19, 20]. The COD:N:P uptake ratio was 408:5:1 as opposed to the widely reported 100:7:1 [13]. Comparative protein content was obtained in similar studies under nitrogen deficient conditions [60, 74]. This is promising for converting carbon-rich (nitrogen-limited) feedstocks to protein feed, such as FSPW, that may not usually be considered for such application. However, further studies are needed to enhance protein content and quality.

Fig. 7
figure 7

Proximate analysis of PNSB biomass (this study) and soybean flour (from Useh et al. [73])

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Amino acid characterization revealed the biomass protein consisted of all the nine essential amino acids for humans (tryptophan, threonine, phenylalanine, leucine, isoleucine, histidine, valine, methionine, and lysine). All other non-essential amino acids were also present. Other amino acids like glycine and arginine which specifically are considered as essential for aquaculture and poultry feed were also present in substantial quantities [75, 76]. This is highly desirable especially when compared to plant-based protein sources which have been reported to be deficient in essential amino acids like lysine and tryptophan and sulphur containing amino acids like methionine and cysteine [77]. Thus, such amino acids are typically manufactured via chemical synthesis and often added as supplements to plant-based animal feeds [78]. For example, in a recent study that examined the amino acid profile of soybean grain, essential amino acids like methionine and tryptophan were absent [79], thereby making feed mixing and supplementation important [76]. As seen in Table 2, the amino acid profile of the biomass protein was comparative to R. faecalis biomass and soyabean grains [20, 79]. The amino acid profile was also within the dietary requirement of feeds for penaeid shrimp, channel catfish, and broilers (poultry) [81,82,83]. Nevertheless, it is important for future studies to investigate the protein digestibility of the biomass, which has impacts on amino acid bioavailability in test organisms [84]. For instance, studies have suggested plant protein sources have a poorer digestibility than animal protein sources in humans [84].

Table 2 Amino acid characterization of the PNSB-dominated mixed culture from this study compared with R. faecalis, soya bean grain and the requirements for penaeid shrimp, channel catfish and broiler chickens
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In terms of biomolecules of interest, the biomass contained 0.55±0.03% bacteriochlorophylls, 0.47±0.08% carotenoids and 0.004±(5.4 E-6)% coenzyme Q10. The presence of other valuable products of nutritional benefit to aquatic organisms and humans, such as lipids, carotenoids, and coenzyme Q10, can increase the monetary value of the SCP [85,86,87]. Compared to other studies, the biomass pigment and coenzyme Q10 concentrations are lower [12, 20]. This is probably due to the differences in culture conditions, such as increased light intensity and the use of pure PNSB strains, different feedstock, and light sources. However, when biomass pigment is compared with other mixed culture studies, similar pigment concentrations were obtained [13].

Metal analysis of the biomass also revealed that undesirable heavy metals like arsenic, cadmium, mercury, and lead were not detected. These metals have no nutritional value to livestock and are generally classified as heavy metals of public health concern with very low maximum permissible limits in animal feeds [88]. This supports the feasibility of SCP resource recovery from a relatively benign industrial process water rather than from other more hazardous waste streams. The bioaccumulation of toxic metals has been identified as a major hindrance to upscaling biomass protein recovery from other waste streams [8]. As seen in Table 3, recommended dietary concentrations of trace minerals essential for livestock growth, like zinc (152 mg/kg) and copper (86 mg/kg), were present in the biomass, thereby enhancing the value of the biomass. A deficiency of essential trace minerals like iron, copper, manganese, zinc, selenium, cobalt, and chromium in fish feed has been associated with high morbidity and mortality rate among several fish species [99].

Table 3 Metal content of the dry PNSB biomass in relation to European Commission Standards
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On the other hand, metals like cobalt (33 mg/kg), manganese (519 mg/kg) and chromium (72 mg/kg) were above the limits recommended by the European Commission for animal feed [88]. The former two metals were present in both the FSPW and trace mineral solution, so there is a possibility to reduce them through optimization of the trace element solution. For instance, when estimating the total metal concentration from both FSPW and mineral supplements, under 50% of total feedstock Mn, Co, Cu, Mo, Ni, and Sr concentrations were from FSPW, indicating that these trace minerals can be significantly regulated. Chromium was not detected in the wastewater, trace mineral solution or vitamin solutions and is therefore expected to be a result of cross-contamination. Table A4 shows the metal concentrations of the trace mineral and vitamin supplements before adding to the feedstock. Their relative concentrations signify that concentrations in the final biomass product can be controlled by regulating the nutrients added to the feedstock. Moreover, concentrations in the final feed product can also be controlled by blending biomass with commercial feed having reduced levels of the same metals.

4 Conclusion

Conventional industrial wastewater treatment plants are constructed to remove impurities and reuse treated water. However, incorporating a resource recovery process into wastewater treatment has recently been recognized as one of the hallmarks of environmental sustainability. Hence, this novel preliminary study has shown the potential of upcycling protein-rich biomass from anaerobic treatment of FSPW to biomass that could be considered for animal feed. While the study did not focus on overall treatment optimization, the results indicate that under non-axenic conditions, PNSB was able to efficiently treat high-strength industrial wastewater by achieving 78 to 100% COD removal and complete nitrogen removal at the end of the batch trials. The overall process rates were slow due to batch operation from a small seeding concentration (50 mg/L) and unconventional starting C:N:P ratio (429:4:1). However, the upcycled biomass also had an amino acid profile comparative to soybean and meeting the dietary requirement of livestock like channel catfish, penaeid shrimp, and poultry birds. Considering the agricultural dynamics of the study region, developing this biobased circular economy could potentially strengthen food security. However, further experiments are required to optimize culturing conditions for high-quality protein/amino acid yield and ascertain the safety of the SCP with more extensive testing, including aquaculture studies.