Aquaponic growth of basil (Ocimum basilicum) with African catfish (Clarias gariepinus) in standard substrate combined with a Humicacid Fiber-Substrate (HFS)

A major challenge in agriculture, horticulture and aquaponics practices is the reduction of mineral fertilisers and peat to reduce CO2 emissions and increase sustainability. This study used a three-phase-natural fertiliser, the Humicacid Fiber-Substrate (HFS), made from natural regenerative organic and mineral-fractions (Humus-Mineral-Complex), to reduce the peat content in plant pots for aquaponics farming. Basil (Ocimum basilicum) growth was compared with i) 100% standard media substrate ("Einheitserde", white peat 80%, clay 20%), and ii) 85% "Einheitserde" and 15% of HFS under irrigation with aquaculture process waters from an extensive and intensive production of African catfish (Clarias gariepinus) under coupled aquaponic conditions. The substitution with 15% HFS and use of intensive fish water resulted in comparable plant growth to a fertiliser solution as control, and in higher leaf width and leaf green weight and lower root dry weight compared with the standard media substrate "Einheitserde". Basil leaf chlorophyll content from the aquaponics was higher compared with local market plants. This suggests the possible substitution of the peat substrate "Einheitserde" with at least 15% HFS to reduce the natural peat fraction. Further studies on crop-specific substrates are needed to reduce peat in aquaponics farming plant cultivation.

The reduction of commercial mineral fertilisers is one of the most important advantages of aquaponics and depends on the fish feed-based nutrient input 1 , which is mainly influenced by the fish species and variations in fish stocking density 2 .Also, hydroponic components, such as "grow pipes" or "aeroponic systems", influence the plant growth [3][4][5][6] .Aquaponics (s.l.) farming as pot cultivation is a relatively new technique for e.g.spearmint (Mentha spicata) 7 .The potting substrate provides supplementary nutrients when applying aquaponics horticultural techniques 2 .Thus, the supplementary "nutrient substrate" should best originate from the circular economy in the form of agricultural by-products to ensure highest ecological benefits 8 .The reuse of nutrients from agricultural biomass or "lost material" 9 can contribute to a more sustainable production ("nutrient cascade").Phosphorus and nitrogen reuse from agricultural by-products for crop production directly reduces the demand for fertiliser from mineral and fossil raw materials and returns the nutrients back into the agricultural economy 10 .
Substrates for hydroponic crop production mainly contain peat moss (Sphagnum magellanicum) 11 .The standard garden pot substrate, according to Fruhstorfer (German: "Fruhstorfer Erde" or "Einheitserde"), is a mixture of decomposed raised bog peat (white peat, with more than 94% organic matter) and clay 12,13 .As a substrate starting material, bog peat has positive properties, such as a low pH value, a low content of plant-available nutrients, low microbial activity, and a good water and air supply; it can be fertilised individually according to the plant needs 12 .However, in order to replace peat, alternative growth substrates are needed that allow similar growth performance.Soil conditioners consist of organic and inorganic residual materials such as greens, wood, and compost (municipal, forestry, agricultural) as well as by-products from the processing industry e.g., burned clay, slaked lime or wine waste, and have been used as adsorbents for heavy metals, for increasing the nutrient contents, and for improving soil properties [14][15][16][17] .A soil regenerator (soil conditioner) was patented in the early 1990's under the title "Biologically pure, three-phase natural fertilizer and process for producing the same" (WO1993010061A1, Switzerland, 1992) 18 , consisted of the three fractions a) crushed igneous rock, b) brown coal and c) mussel lime and optional different seeds.In a more recent design (green stuff), local plant material was combined with the mineral components.The resulted was a homogeneous industry made rock/plant/humus fiber substrate (humus equivalent) of uniform quality with all necessary micro-and macroelements, comparable to natural black peat, and was already successfully applied in the desert of Jordan 19 .
African catfish (C.gariepinus) is a popular food fish in Mecklenburg-Western Pomerania (Northern Germany), and the production of six aquaculture farms reached a yield of 883 t in 2020, representing 76% of the total aquaculture production in this federal state 20 .This species was newly introduced around 2010 to aquaponics, resulting in good feed conversion ratios (FCRs), ranging from 0.94 to 0.96 when cultivated with spearmint (Mentha spicata) 7 , and 1.23-1.39when cultivated with water spinach (Ipomoea aquatica) 21 .Good FCR values of C. gariepinus, ranging from 0.97 to 1.12 were also achieved when iron (FeSO 4 ) was added to the process water under aquaponic conditions 22 .These studies demonstrated C. gariepinus as a useful fish species for coupled aquaponics.Basil (Ocimum basilicum, Lamiaceae) was identified as a high-value, fast-growing herb, and was successfully applied in aquaponics with Nile tilapia (Oreochromis niloticus) 23 , African catfish (Clarias gariepinus) 4,5 , carp (Cyprinus carpio) 24 , and crayfish (Procambarus spp.) 25 .
In the present study, the cultivation of basil (O.basilicum) was evaluated in combination with African catfish (C.gariepinus) as aquaponics farming sensu lato (s.l.) according to Palm et al. in horticulture 2 .The growth of basil was compared in two trials with (i) a proportion of 100% standard growth media substrate "Einheitserde" (E; Abbreviations) as a potting substrate, and (ii) using a soil conditioner, the Humicacid Fiber-Substrate (HFS) with a content of 15% in the pots and 85% "Einheitserde".The pot cultures were irrigated with aquaculture wastewater from extensive (EAU) and intensive (IAU) African catfish production (Fig. 4) and compared with a commercially available hydroponic fertiliser at a standardised EC of approx.2000 µS/cm as a control (C) with 100% standard growth media substrate "Einheitserde" and an addition of dried commercial start fertiliser.The influence of HFS on the growth performance of basil and the change in the nutrient composition of the substrates is discussed.

Results
Trial I: Basil production with standard growth media substrate (Einheitserde) Growth of O. basilicum in 100% standard growth media substrate ("Einheitserde") showed the highest values in the control and comparable levels in the EAU and IAU (Table 1).Plant height was significantly higher in the control (61.9 ± 8.5 cm), followed by the IAU (48.5 ± 5.0 cm) and EAU (45.6 ± 4.3 cm).Root length was highest in the control (18.9 ± 2.6 cm) and not significantly different to the intensive aquaculture process water (17.9 ± 2.0 cm), which was comparable to the extensive process water (16.9 ± 2.6 cm).In contrast, root dry weight was significantly different between the groups with the highest value in the control (1.1 ± 0.2 g), followed by the IAU (0.5 ± 0.1 g) and EAU (0.4 ± 0.1 g).Leaf number was highest in the control (71.5 ± 12.0) and similar in the IAU (39.7 ± 5.8) and EAU (36.3 ± 5.8).Leaf length was significantly different between the groups with the order

Comparison of basil growth with standard growth media substrate (Einheitserde, E, Trial I) and Humicacid Fiber-Substrate (HFS, Trial II)
Growth of O. basilicum was different between pots filled with the commercial substrates (S1, S2), the standard growth media substrate (100% "Einheitserde", E), and pots filled with 15% of the soil conditioner Humicacid Fiber-Substrate (HFS, Fig. 1a-c).The highest growth of basil was found in plants cultured in the control groups irrigated with commercial fertiliser and the original commercial pot substrates (S1, S2; Fig. 1a), followed by plants irrigated with intensive aquaculture effluents (HFS, Fig. 1c), and the lowest basil growth was found in plants watered with extensive fish process water (Fig. 3b).The growth performance of O. basilicum grown in pots with 15% Humicacid Fiber-Substrate and irrigated with intensive fish water was more comparable to the standard growth media substrate with 10 similar parameters out of 13 (Fig. 1c): plant height, green weight, dry weight, shoot length, shoot green weight, shoot dry weight, root length, root green weight, leaf number, and leaf length.Leaf width and leaf green weight were higher in plants cultured with HFS (Trial II), and only root dry weight was significantly higher in basil grown in the standard substrate (Trial I).Basil and HFS-Substrate (Trial II) irrigated with extensive aquaculture process water was only comparable in three parameters (root length, root green weight, root dry weight; Fig. 1b), whereas the standard substrate (E, Trial I) resulted in higher growth with other parameters.

Nutrient compositions of the pot substrates
Nutrient amounts of plant available nutrients (PAN, Table 3) in the control group substrates (S1, S2) were, in general, higher for NO 3 -N, P, K, B, Zn and Fe-EDTA compared to the EAU and IAU in the groups with the standard growth media substrate (E, Trial I) and Humicacid Fiber-Substrate (HFS, Trial II).In pots irrigated with intensive aquaculture effluents, the NO 3 -N levels were substantially higher by 39.9-fold in the standard substrate (E + IAU, Trial I), and by 38.4-fold in the HFS-substrate (HFS + IAU, Trial II) compared to the substrates irrigated with extensive fish process (EAU) water of the same trial.In PAN with intensive fish water and between trials (E + IAU, HFS + IAU), levels were comparable of P, K, B and Mo, and higher in the Einheitserde substrate (Trial I) of organic substance (7.7%), and NO 3 -N (23%), whereas higher values were found in HFS + IAU substrate (Trial II) of Zn (46%), NH 4 -N (41.7%), and Fe-EDTA (28.7%).Gross nutrient composition (GNC, Fig. 2) in HFS + IAU substrate was higher than E + IAU substrate in P with 25%, in Mg of 15%, and in Fe of 4.4%, whereas K was 1.4% higher in Einheitserde substrate with intensive process water (E + IAU), and N was comparable between trials.

SPAD values of basil leaves
The relative chlorophyll content of O. basilicum leaves, measured as SPAD readings (%), showed comparable and significantly higher values in the control group plants (S1, S2) irrigated with commercial fertiliser (+ commercial dried starter fertiliser) and cultured in 100% standard growth media "Einheitserde" (Fig. 3) compared to plants grown in both substrates with aquaculture irrigations (EAU, IAU) and substrates (Humicacid Fiber-Substrate: HFS; Einheitserde: E).The SPAD levels were not significantly different between aquaponic groups (EAU, IAU); however, the samples of market plants (M) showed lower chlorophyll contents in M I (ALDI-Nord) and M II (Netto Marken-Discount Stiftung & Co. KG), whereas a sample from M III (Netto ApS & Co. KG, Salling Group A/S) was not significantly different in SPAD compared to HFS + EAU group and E + IAU group.

Growth performance of O. basilicum
Basil growth was generally moderate (Tables 1, 2).O. basilicum can reach substantial heights of 75-95 cm under natural conditions and a longer growth period 26 ; however, comparable heights in aquaponics were reported from deep-water culture (DWC) of 39.9 cm with production of Nile tilapia (Oreochromis niloticus) 27 and in a decoupled system ranging from 46.78 to 55.75 cm under intensive production of C. gariepinus after 36 days 5 .Substantially better basil heights of 94.8-101.8cm were found in decoupled aquaponics with 6-8 true leaves and a 4-cm shoot axis height at the transplanting stage 4,5 , in contrast to the earlier transplantation with lower heights (1.67 cm) and one pair of fully expanded leaves in the present study.In general, the number of leaves was reduced in all groups (Tables 1,2) as significantly higher leaf numbers of 493.7-518.0 were reported in decoupled aquaponics after 41 days 4 .Consequently, the intensive process water resulted in similar basil growth as observed from other aquaponic systems.This contrasts the low leaf number and reduced leaf dimensions in the EAU combined with HFS substrate, indicating low nutrient contents 28 compared to the IAU (Table 2).Thus, the extensive aquaculture process water effluents with a portion of 15% Humicacid Fiber-Substrate was not able to replace the standard growth media and resulted in reduced plant growth due to nutrient deficiencies.Future studies should extend the transplanting time to a minimum of three weeks 4,5 with four to five leaves 29 to achieve optimal growth.

Influence of the Humicacid Fiber-Substrate (HFS) on pot nutrient composition
The substitution of 15% standard media substrate ("Einheitserde") by HFS in garden pots of the IAU increased the amount of plant available nutrients of Zn to 46%, of NH 4 -N to 41.7%, and of Fe-EDTA to 28.7%, and in  Fe-EDTA (mg/kg) 1,623.9977.9 807.9 www.nature.com/scientificreports/gross nutrients of P to 25% and Mg to 15%, and in Fe of 4.4%, compared to the 100% standard growth media (Table 3, Fig. 2).The amount of zinc was 1.9-fold higher in the IAU with HFS compared to the standard media substrate (Table 3).Zinc is known to improve chlorophyll formation in plants and could decrease interveinal leaf chlorosis and leaf deformations 30 as was observed in the IAU plants with HFS by visual observation.The level of 5.4 mg/ kg Zn in the HFS trial (IAU) was adequate; however, it should be increased to 10 mg/kg, as it was reported that Zn improved basil biomass production, nutrient uptake of K + and Cu, and the chlorophyll index (SPAD) 31 .Both, the fish feed and the Humicacid Fiber-Substrate were sources of zinc, and the increase in stocking density and the proportion of the soil improver could increase the amount of zinc for the production of high-quality plants, knowing that up to 48% of it could be bound to the sludge 32 .
Plant available NH 4 -N was increased in the IAU Humicacid Fiber-Substrate group 1.7-folds compared to the standard substrate (Table 3), and it originated from organic substances.The addition of humic acid increased cation adsorption and the preference of NH 4 + in a humic-montmorillonite clay mineral complex 33 .NH 4 + is actively involved in plant growth, as studies have shown that the content changed significantly with the growing season, notably in clayey soils 34 .NH 4 + formed pools in clay mineral interlayers, which are resistant to nitrification and can be available for plant growth through gradual release 35,36 .The combination of HFS with a higher proportion of humic acid and the higher proportion of NH 4 -N in the process water from the intensive fish production may have resulted in a fixation of NH 4 + in the clay fraction of the substrate, which was obviously recovered for plant growth.
Plant available Fe-EDTA was 1.4-fold higher in the IAU with Humicacid Fiber-Substrate than in the substrate with "Einheitserde" of IAU (and 2.1-fold greater in EAU with HFS; Table 3) and was described as the most used synthetic chelating agent in fertilisers or as a supplement, e.g., 13% EDTA Fe at 2 mg/L with a three-week interval 37,38 , which is stable at pH 4.0-6.3 39, as was observed in the process waters of the present study (Table 5).The Fe-EDTA origin in this experiment was unclear; however, only the Humicacid Fiber-Substrate might be a source by binding Fe with humic complexes under acidic conditions, as only in these groups the Fe-EDTA content increased.Fe-EDTA can form complexes with free metal cations and is able to prevent plant uptake of metals such as Zn, Cu, and Mn 39 , which was not evident in the case of Zn due to higher amounts in the HFS groups (Table 3).Iron and Fe-EDTA are essential for photosynthesis and a limiting factor in aquaponics, and the increased Fe-EDTA content might have prevented the interveinal chlorosis in basil 30 that was observed in the plants of the HFS groups without chlorosis.The relatively comparable Fe contents in EAU and IAU (GNC) might be due to water leaching effects in the IAU caused by higher sedimenter cleaning intervals as a result of the higher stocking density.
The amount of phosphorus in the intensive group with HFS was about 1.3-folds higher in gross nutrients than in the standard substrate (Fig. 2).Sources of phosphorus included both, the fish feed and addition of HFS, which www.nature.com/scientificreports/increased P in the aquaponic groups.Since the amount of P in the EAU and IAU groups with HFS was almost the same, i.e., independent of the fish stocking density, it can be assumed that the addition of HFS alone increased the amount of P.This is in accordance with the three-weight class production of C. gariepinus, which showed approx.the same phosphorus level inside the process water when the feed input ratio of extensive, semi-intensive and intensive production (feed ratio: 1:2:4) was calculated as 1.0:0.4:0.6 (staggered III production phase) 40 .Thus, an increased amount of phosphorus from adding HFS increased the growth of basil, which in combination with the other nutrients, enhanced the growth performance in the IAU compared with the standard substrate.The amount of magnesium was slightly higher (≈ 1.2-fold) in the IAU with Humicacid Fiber-Substrate compared with the standard media substrate (Fig. 2).Fish feed as the main magnesium source can be excluded, as the Mg nutrient concentration ratios in the process water of African catfish production does not increase proportionally with increasing stocking density (Tables 4,5); magnesium can be bound inside the sludge to a higher extend, up to 16% 32 , and depending on levels of water exchange.This suggests that HFS was an additional source of Mg, and the 15% substitution of this soil conditioner for standard media minimally increased its proportion in the substrate.

Chlorophyll content of basil leaves
SPAD readings (chlorophyll content) were significantly higher in control group plants (S1, S2) due to the use of the commercial fertiliser (Fig. 3).In contrast, SPAD values of basil from aquaponics with HFS and standard substrates were not significantly different (comparable) and higher compared with those taken from supermarkets, except sample MIII (Netto ApS & Co. KG; Fig. 3).SPAD levels in the aquaponics and the market samples were lower than reported for basil irrigated with organic fertiliser (chicken manure) of 35.18%, and the SPAD of the control group plants were closer to basil fertilised with spores of mycorrhizal fungi and bacteria of 40.27% 41 .Lower leaf chlorophyll content was found in basil cultivated in aquaponics with crayfish (Procambarus spp.), with 29.3 SPAD and hydroponics with 28.7 SPAD 25 , and in aquaponics with Nile tilapia (O.niloticus) of 23.2% and hydroponics of 31.7% 27 .O. basilicum cultivated under different daily light intervals showed a lower SPAD value of 25.7% (≈ 7 mol/m 2 d) and a higher SPAD level of 34.1% (≈ 15 mol/m 2 d) in a nutrient-film technique (NFT) hydroponic system 42 .Thus, basil cultivated under aquaponics cultivated in HFS-substrate with effluents from extensive and intensive C. gariepinus aquaculture had a higher leaf chlorophyll content than aquaponics with other aquatic animals and showed a better chlorophyll level than samples from the supermarkets, as an indication of high-quality O. basilicum production that was equivalent to the market samples in terms of leaf colour (SPAD).

Conclusions
The substitution of the standard growth media substrate ("Einheitserde") with 15% of the soil conditioner Humicacid Fiber-Substrate resulted in an improved growth of O. basilicum in pot cultivation, similar to 100% standard substrate in combination only with intensive effluents from C. gariepinus production.It also showed a better chlorophyll content (SPAD) than basil samples from local markets.Therefore, the application of Humicacid Fiber-Substrate in aquaponics with African catfish is a new option to reduce peat without compromising basil quality.
The combination of aquaculture process water from the intensive stocking of African catfish and 15% Humicacid Fiber-Substrate was effective and increased levels of several nutrients, including zinc, NH 4 -N, Fe-EDTA, phosphorus, and magnesium.However, to ensure higher plant quality and growth, zinc should be increased 10 mg/kg, and iron could be added in the form of chelate with 2.5 mg/L every three weeks.Further adaptations www.nature.com/scientificreports/are possible by using resources from the agricultural circular economy for the nutrient composition of the soil conditioner Humicacid Fiber-Substrate, or by increasing the substitution rates of the peat-based standard substrate with Humicacid Fiber-Substrate above 15%.This might further improve basil growth and quality while reducing the amount of peat with its negative impacts through peat mining and increased CO 2 emissions.

Methods
All methods were carried out in accordance with relevant guidelines and regulations.

Experimental facility
The experiment was carried out at the aquaponics experimental facility "The FishGlassHouse" in spring 2018 (April to June) for in total 49 days, using the extensive (EAU, 100 m 2 ) and intensive recirculating aquaculture units (IAU, 100 m 2 ; PAL Anlagenbau GmbH, Germany) and one hydroponics unit (100 m 2 ) in a VENLO greenhouse (GTW Gewächshaustechnik Werder GmbH, Germany) at the University of Rostock (UoR), Faculty of Agricultural and Environmental Sciences, Northern Germany in Mecklenburg-Western Pomerania (GPS: latitude: 54.075714, longitude: 12.096591, Fig. 4).The aquaculture units consisted of nine fish tanks (F 1-9, water volume 1 m 3 ; Fig. 4) arranged in triplicates (3 × 3) for staggered fish production of three different fish weight classes, one solids separation unit / sedimenter (Se-E in EAU: 1.1 × 1.2 × 0.9 m and 1.2 m 3 ; Se-I in IAU: 1.5 × 1.3 × 0.9 m and 1.7 m 3 ), nitrifying trickling filters (TF-E in EAU: 2.9 m 3 ; TF-I in IAU: 11.8 m 3 ), and communicating sumps (S-E in EAU: 1.6 m 3 ; S-I in IAU: 4.0 m 3 ) 7 .Aquaculture process water was transferred semi-continuously via a water management system and tanks (WT-E-1/2 in EAU and WT-I-1/2 in IAU approx.1,500 L) into the hydroponic cabin with corresponding aquaculture tanks for the EAU (AET-E), IAU (AET-I), and an additional hydroponics control group tank (Control) with a fertiliser solution (each approx.1,000 L).For semi-coupled aquaponics conditions, nutrient solutions were pumped back from the hydroponics unit via water management system to the extensive and intensive aquaculture units twice a week.

Fish production
All fish were delivered in January 2018 (30 g/fish initial weight) from a local fish farm (Fischzucht Abtshagen GmbH & Co. KG, Germany) and stocked in the extensive (33.8 fish/m 3 and tank) and intensive (132.4 fish/m 3 and
The Biologically pure three-phase natural-fertiliser Humicacid Fiber-Substrate (HFS, old name "BIO-HUMIN ® ") consisted of a mineral-humus substrate mix made from natural substances without pollutants and

Figure 1 .
Figure 1.(a-c) Comparison of O. basilicum growth parameters between (a) control with commercial standard growing media substrate in each pot (S1 of Trial I and S2 of Trial II: 100% Einheitserde + dried commercial starter fertiliser) of plants irrigated with hydroponic fertiliser solution (Universol ® Orange); (b) basil cultivated with standard growth media substrate "Einheitserde" (E, Trial I) and 15% HFS-substrate (HFS, Trial II) irrigated with process water from the extensive aquaculture unit (EAU); and (c) comparison of basil grown in standard growth media substrate "Einheitserde" (E, Trial I) and 15% HFS (HFS, Trial II) irrigated with process water from the intensive aquaculture unit (IAU); different letters showing different groups (p < 0.05).

Figure 4 .
Figure 4. Schematic illustration of the experimental design in the FishGlassHouse with aquaculture section: intensive aquaculture unit (IAU) with nine fish tanks (F1-9), sedimenter (Se-I), sump (S-I) and trickling filter (TF-I); extensive aquaculture unit (EAU) with nine fish tanks (F1-9), sedimenter (Se-E), sump (S-E), and trickling filter (TF-E); water management system with tanks supplying waste water to hydroponics (WT-I-1 of IAU; WT-E-1 of EAU), and discharging waste water tanks (WT-I-2 of IAU; WT-E-2 of EAU); hydroponics unit with aquaculture effluent tanks for the IAU (AET-I) and EAU (AET-E), dissolved fertiliser liquid tank for control (Control) and nine ebb-and-flood planting tables corresponding to aquaculture effluent and control fertiliser tanks (T-EAU-1/3, T-C-1/3, T-IAU-1/3), substrate variants were symbolised from Trial I: with control as S1 and "Einheitserde" as E, and Trial II: control (S2) and "Humicacid Fiber-Substrate" as HFS with used pot numbers (11) for analytics in brackets.Fluid supply pipes are shown solid and return pipes are drawn dashed.

Table 1 .
The

Table 2 .
The

Table 4 .
Comparison of chemo-physical water parameters (mean ± SD) of the extensive (EAU) and intensive (IAU) aquaculture units with C. gariepinus production; different letters show different groups (p < 0.05).

Table 5 .
Hydroponic physico-chemical water parameters (means ± SD) in the fertiliser control tank (Control) and the experimental aquaculture process water tanks (extensive: AET-E; intensive: AET-I) with light parameters between planting tables of Humicacid Fiber-Substrate (HFS) and standard growth media substrate (Einheitserde, E) trial (light intensity, PPFD); different letters showing different groups (p < 0.05; p-I: significance value (p) between Control & EAU; p-II: between Control & IAU; p-III: between EAU and IAU)..001) by staggered production, and a total feed use of 58.2 kg for EAU, and 182.1 kg for IAU, this corresponds to a daily feed quantity for EAU of 1.2 kg feed/day, and for IAU of 3.7 kg feed/day.