Application of compost and biochar mixtures to soils to pro-2 duce parsley plants rich in nutrients and antioxidant com-3 pounds

: Composts and biochar individually or in combination have been used for decades for im-14 proving soil quality and health. To date, very few studies have focused on the quality of food pro-15 duced using compost-biochar mixtures. In this study, the use of biochar to improve the fertilization effect of composts and the quality of greenhouse-grown parsley was investigated by adding biochar to composts made from a mixture of broiler chicken wastes and sugar bagasse, sawdust, urban trees, napier grass or cotton residues. On average, highest N and P contents were obtained with the ba-19 gasse- and sawdust-biochar substrates. The tree-biochar substrate led to increased levels of phenolic 20 compounds in parsley compared to all the other organic substrates.


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The term "food quality" is often used to represent all attributes of a food item that 25 are acceptable to the customer; these attributes include the sensory, suitability, and nutri-26 tional/health values [1]. Thus, the perception of quality highly affects purchase decisions 27 and dietary patterns. Numerous factors can directly affect the quality of foods before har-28 vesting. Among these are genotypic, soil, and climatic factors [2]. Other factors such cul-29 tural practices (e.g., maturity at harvest) are also important. Before harvest, the quality of 30 crops is predominantly driven by genetic factors and most quality improvement pro-31 grams in the industry are focused on breeding and/or selecting cultivars of high quality 32 [2][3]. The increase in crop yields observed around the world during the last decades has 33 been achieved in part using inorganic fertilizers and pesticides [4][5], and several studies 34 have investigated the quality of food from crops grown with synthetic chemicals. How-35 ever, synthetic chemicals used in agriculture pose potential hazards to the environment 36 and human health [5]. 37 Globally, there is increasing interest in soil organic amendments for the sustainable 38 production of food. Several organic media for growing plants have been proposed, in-39 cluding composts, biochar, and peat [6][7][8]. Biochar is a black carbonaceous, porous, and 40 low-density material that is produced by pyrolysis of various biological residues in the 41 absence of oxygen [8]. Compost is decayed organic material usually produced by aerobic 42 biological decomposition of plant and food wastes [9] Peat is a spongy material formed 43 by the partial decomposition of vegetation or organic matter in the wet acidic conditions 44 of bogs and fens [6,10].
Systematic analyses of available data show that organic matter from composts for 1 example positively affects physical, chemical and biological properties of soils, as well as 2 the physiological development of plants [8,11]. However, the focus in the compositing 3 field has been more on the quality of the compost [12] than the quality of food-crops pro-4 duced using the composts. Besides providing the physical and chemical properties 5 needed for the proper development of plants, organic amendments should ensure the 6 production of crops that are safe, rich in nutrients and bioactive compounds [13][14]. 7 The effect of organic amendments on the quality of foods has recently been found as 8 an area of great interest [3,[9][10][15][16]. In a previous study, we studied the effect of com-9 posts obtained from chestnut, red grape, white grape, olive and broccoli wastes, on anti-10 oxidant compounds in lettuce [6]. Our results showed a high accumulation of phenolic 11 compounds in lettuce grown in the white grape-based compost, and in particular querce-12 tin 3-O-glucoside, luteolin 7-O-glucoside, and cyanidin 3-O-(6″-malonyl)-β-d-glucoside; 13 however, all composts led to decreased vitamin C levels [6]. Tortosa et al.
[4] evaluated 14 "Alperujo" compost form olive mill wastes as an organic amendment for pepper and 15 found that plants experienced a yield increase and an enhancement of the Vitamin C con- 16 tent, which was in contradiction with our study with lettuces. The amendment of biochar 17 and biochar + compost (from several plant leaf residues) significantly increased chloro-18 phyl contents in Alpinia zerumbet, an ornamental and medicinal plant primarily used in 19 traditional medicine [8]. In the study by Ngakou et al. [5], the nutrition value of leaves 20 and seeds of Moringa oleifera grown using different composts and inorganic chemicals 21 (cow dung compost, goat manure compost, poultry manure compost, chemical fertilizer, 22 mixture of fertilizers and control) was assessed; results obtained indicated that dried 23 leaves from plants grown with poultry manure compost contained a high total protein 24 content. Growing plants with compost resulted in increasing chlorophyll a and b in leaves, 25 but not vitamin A [5]. 26 The general perception is that studies often have given contradictory results regard-27 ing the effects of organic amendments on food quality. The effects appear to be depended 28 on the compost type, the amount applied to soil, and the quality parameter measured. 29 This highlights the importance of formulating different organic amendments for specific 30 plants under specific environmental conditions and cultural management practices. The 31 purpose of this study was to evaluate the impact of five composts made from poultry 32 industry wastes and added or not with biochar on the yield and nutritive/health value of 33 greenhouse-grown parsley (Petroselinum crispum Mill.). The following parameters were 34 assessed: mineral composition, antioxidant activity, total phenolic compounds, and indi-35 vidual flavonoids. The following materials were used for the preparation of organic amendments: plant 39 wastes, chicken wastes, and biochar. Plant wastes were collected form a farm and com-40 prised of sugarcane bagasse, sawdust, urban tree residues, napier grass, and remnants 41 from the defibrillation of cotton. Different wastes (broiler litter, hatchery wastes, floating 42 sludge, cellulose gut, and charcoal) were collected from a poultry processing company 43 and mixed. Biochar was produced by the slow pyrolysis of eucalyptus woods. Five com-44 posts were produced by (i) mixing each of the plant waste with the same quantity of 45 chicken waste to create piles with a C:N ratio of approximately 30; and (ii) processing the 46 piles in a windrow turner as described in detail in Santos  Thirty-day old parsley seedlings were purchased from a local market and planted in 2 pots (1 L) filled with the organic amendments as growing substrates. Parsley plants were 3 grown for 50 days in a non-controlled greenhouse with day/night temperatures of 32/18 4 °C. At harvest, shoots were weighted, and fresh matter recorded in g pot -1 . Dry matter (g 5 pot -1 ) and moisture content (%) were recorded after freeze-drying the shoots for 18 h. 6 2.3. Biochemical measurements 7 Dried shoots were ground and used for the determination of nutritive and health 8 parameters as described by Santos et al. [13]: total nitrogen (N) was determined using a 9 Kjeldahl analyzer, phosphorus (P) was quantified by UV/vis spectrophotometry, potas-10 sium (K) by flame photometry. Mineral contents were expressed in g kg -1 . The phenolic 11 extract was prepared by adding 2.0 mL of 70% methanol to 40 mg of ground shoot. Fol-12 lowing incubation at 75 °C for 30 min, the mixture was centrifuged (3500 rpm, 15 min, 27 13 °C ) and the supernatant collected through a 0.45 μM polyvinylidene difluoride filter. The 14 total phenolic content (TPC) of the shoot extract was determined by a modified Folin- 15 Ciocalteu method and quantified at 750 nm using a UV-vis spectrophotometer; TPC was 16 expressed as mg gallic acid equivalent per g (mg GAE g -1 ). The total flavonoid content 17 (TFC) of the shoot extract was estimated using the aluminum chloride colorimetric 18 method, using catechin (CE) as a standard. Absorbance was measured using a UV-vis 19 spectrophotometer at 520 nm, and TFC expressed as mg CAE g -1 . The antioxidant activity 20 of the shoot extract was estimated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical 21 scavenging activity, and results expressed as % sequestration and mg trolox equivalent 22 per g (mg TE g -1 ). Six parsley flavonoids were separated and identified using High Per-23 formance Liquid Chromatography coupled to a diode array detector; levels were ex-24 pressed as mg apigenin equivalent per g (mg g -1 ).
[13]. In this experiment, we set up four pots per treatment (n = 4) using a completely ran-27 domized block design consisting of five composts and five biochar amounts. For each 28 compost data, replicate values for all biochar treatments were pooled and used in statisti-29 cal analyses. The objective of data pooling was to have a global picture of the compost 30 effect, regardless of the amount of biochar added to the organic amendment mixture. A 31 descriptive analysis of the data was carried out to produce means and standard devia-32 tions, using SPSS 16.0. An analysis of variance (ANOVA) was also carried out to compare 33 mean values. A p < 0.001 was considered to indicate differences among means (Duncan 34 test).  In this study, we tested the effect of chicken-based composts added with biochar on 38 the yield and mineral composition of parsley. The results are shown in Figure 1. The water 39 content of parsley shoots was not affected by the treatments. Highest yields were obtained 40 with the bagasse and sawdust composts, irrespective of the amount of biochar. The napier 41 compost presented the worse results in terms of fresh matter yield. However, no statistical 42 differences in dry matter yield was found among the napier, tree and cotton composts. 43 Although we did not include a control treatment (non-amended pots) in this study, our 44 previous data showed higher yields in composted growing media than in non-amended 45 media using some composts [6, 11,13]. The application of biochar and composts has also 46 been reported in several studies to increase yields relative to unamended controls 47 [8,10,13]. Highest N and P contents were also measured in plants grown on the bagasse 48 and sawdust composts, and lowest in the napier compost; this pattern agrees with the 49 yield data, although plants grown using the cotton compost also exhibited high N con-1 tents. K levels varied in their response to the organic amendments. In the study by Tortosa 2 et al. [4], macro-and micronutrient levels in pepper leaves from non-amended soils were 3 lower than those obtained from compost-amended soils. However, different results have 4 been reported by different authors [5,[9][10]14,17]. 5 6 Figure 1. Growth (dry and fresh matter in g pot -1 , moisture in %; y-axis) and mineral content (N, P, 7 K in g kg -1 ; y-axis) in shoots of parsley plants grown in medium composed of bagasse-chicken com-8 post + biochar, sawdust-chicken compost + biochar, tree-chicken compost + biochar, napier-chicken 9 compost + biochar, and cotton-chicken compost + biochar. Biochar was added to the composts at 10 five inclusion rates (0, 15, 30, 45, and 60%, weight basis) and the average values used in statistical 11 analyses. Error bars represent standard deviations. For each parameter, bar values followed by the 12 same letter are not statistically different (p < 0.05; ANOVA Duncan post-hoc test).  14 In agreement with yield and N, P contents, TPC was highest in plants grown using 15 the bagasse, sawdust, and tree composts (Figure 2). Generally, the treatments did not af- 16 fect the TFC content, except for a low TFC for plants grown using the napier compost. 17 However, there seems to be no relationship between TPC/TFC and DPPH. The antioxidant 18 activity of shoot extracts revealed that parsley plants grown using the napier and cotton 19 composts exhibited increased DPPH activities, despite low yields and low TPC; these lat-20 ter plants also tended to exhibit high TAC. It can be concluded that the napier and cotton 21 composts induced a nutritional stress in plants. The plants responded to the oxidative 22 stress by activating their antioxidative system. Therefore, increased DPPH antioxidant ac-23 tivities might not be related to phenolic compounds only, but also to other antioxidant 24 compounds such as glutathione and ascorbate [4, 8,16]. Overall, levels of bioactive com-25 pounds in foods depend on the type and amount of composts used as growing substrates 26 [7,10,13]. For example, Zawadzińska et al. [10] found that tomatoes grown in a medium 27 consisting of 25% compost, 30% high peat, 15% low peat, 20% pine bark and 10% wood 28 fiber reached the highest TPC and vitamin C levels. 1 Figure 2. Antioxidant activity (DPPH in % and mg TE g -1 ; y-axis), total flavonoids (mg CE g -1 ; y-2 axis), total phenolics (mg GAE g -1 ; y-axis), and total anthocyanins (mg CGE g -1 ; y-axis) in shoots of 3 parsley plants grown in medium composed of bagasse-chicken compost + biochar, sawdust-4 chicken compost + biochar, tree-chicken compost + biochar, napier-chicken compost + biochar, and 5 cotton-chicken compost + biochar. Biochar was added to the composts at five inclusion rates (0, 15, 6 30, 45, and 60%, weight basis) and the average values used in statistical analyses. Error bars repre-7 sent standard deviations. For each parameter, bar values followed by the same letter are not statis-8 tically different (p < 0.05; ANOVA Duncan post-hoc test).  Figure 3. Flavonoid composition (apigenin-7-apiosylglucoside, diosmetin-apiosylglucoside, dios-12 metin-apiosylglucoside isomer, apigenin-malonyl-apiosylglucoside, diosmetin-malonyl-apiosyl-13 glucoside, and apigenin-malonylglucoside in mg g -1 ; y-axis) in shoots of parsley plants grown in 14 medium composed of bagasse-chicken compost + biochar, sawdust-chicken compost + biochar, 15 tree-chicken compost + biochar, napier-chicken compost + biochar, and cotton-chicken compost + 16 biochar. Biochar was added to the composts at five inclusion rates (0, 15, 30, 45, and 60%, weight 17 basis) and the average values used in statistical analyses. Error bars represent standard deviations 18 For each parameter, bar values followed by the same letter are not statistically different (p < 0.05; 19 ANOVA Duncan post-hoc test).

Antioxidant compounds
Flavonoids are a vital group of bioactive compounds in plants [18]. At the plant level, 1 these compounds play a crucial role in regulating reactive oxygen species [8]. At the hu-2 man level, flavonoids have various biological activities including anti-inflammatory, anti-3 cancer, and anti-depression properties [19]. The results from our study indicate that com-4 posts can enhance flavonoid levels in parsley (Figure 3). The synthesis of flavonoids was 5 highest in tree compost-grown plants, followed by sawdust-, cotton, and bagasse-grown 6 plants. The napier compost represented an exception with very low levels of flavonoids. 7 This behavior was particularly notable for apigenin-7-apiosylglucoside, apigenin-malo-8 nyl-apiosylglucoside, and diosmetin-malonyl-apiosylglucoside. 9 4. Conclusions 10 Our study shows that organic amendments can greatly affect the nutritive and health 11 value of foods. Composts were manufactured using piles of broiler wastes and sugarcane 12 bagasse, sawdust, urban tree debris, napier grass, or cotton residues. All the compost-13 biochar mixture tested in this study had various effects on mineral and phenolic com-14 pounds in parsley. The bagasse and sawdust composts led to the highest N and P levels 15 and yields. Highest phenolic levels were measured in plants grown with the tree compost. 16 The napier compost generally did not cover the nutritional requirements of the plants.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the 31 design of the study; in the collection, analyses, or interpretation of data; in the writing of the manu-32 script, or in the decision to publish the results. 33