Substrates for seedlings with sewage sludge and biochar

Submitted on April 17 , 2018 and accepted on April 20 , 2019. 1 Project funded by F APEG. 2 Universidade Federal de Pelotas, Departamento de Solos, Capão do Leão, Rio Grande do Sul, Brazil. alexbeckermonteiro@gmail.com 3 Empresa Brasileira de Pesquisa Agropecuária, Pelotas, Rio Grande do Sul Brazil. ivanspereira@gmail.com; adilson.bamberg@embrapa.br 4 Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, Capão do Leão, Rio Grande do Sul, Brazil. crisstocker@yahoo.com.br 5 Universidade Federal de Pelotas, Departamento de Engenharia Rural, Capão do Leão, Rio Grande do Sul, Brazil. luisctimm@gmail.com Corresponding author: alexbeckermonteiro@gmail.com Substrates for seedlings with sewage sludge and biochar 1


INTRODUCTION
Sewage sludge (SS) is a by-product of the domestic wastewater treatment, and its safe use is still a major challenge for today's society (Bai et al., 2017). Currently, the agricultural use of SS has been indicated as the main alternative for its recycling role (Tessaro et al., 2016), with positive effects when used as a fertilizer, a soil conditioner and in the recovery of land degraded areas (Caldeira et al., 2012;Faria et al., 2013). However, its use as a substrate component for seedling production is still poorly studied, although recent research indicates a high potential Freitas & Melo, 2013).
The use of SS as a component of substrates can also be a better destination when compared to its final disposal in landfills (Caldeira et al., 2012). However, it is fundamental that SS has undergone a process that guarantees its innocuousness. Batches of SS must be monitored, in order to know that the levels of potentially toxic elements and pathogens for human health are in accordance with the limits established by current regional legislation.
Black wattle (Acacia sp.), timbó (Ateleia glazioveana Baill), teak (Tectona grandis) and eucalyptus (Eucalyptus sp.) are examples of forest species already tested with substrates formulated from SS , Silva et al., 2017. On the other hand, studies with substrates from SS in horticultural species are still scarce, perhaps due to the contamination risks of their consumible parts. Among them, we can mention the use of SS derived substrates in the production of tomato seedlings (Solanum lycopersicum) (Freitas & Melo, 2013). Another reason is the difficulty to formulate such substrates, since these species are more sensitive to chemical and physical characteristics of the growth medium.
Although positive results have been noticed by researchers regarding the agronomic efficiency of SS derived substrates, its practical application has been limited, particularly when inactivation of pathogenic contaminants is not properly carried out.
Heat treatment is currently considered the most effective approach (Liu et al., 2014), with pyrolysis being one of the most promising thermal methods for SS treatment worldwide (Agrafioti et al., 2013;Méndez et al., 2013;Yuan et al., 2016;Agbna et al., 2017).
The resulted product of the pyrolysis process of organic carbon sources is known as biochar, a stable carbon rich material with high ammount of reactive sites. Biochar has been widely tested and used as a soil conditioner, since it benefits soil quality overall, in particular soil structure, porosity, bulk density, water storage, microbial activity and nutritional status (Silva et al., 2017). Jin et al. (2016), evaluating SS pyrolysis temperatures ranging from 400 to 600 ºC, performed during one hour, verified that with the increase in temperature there is a decrease in SS volume, an increase in pH levels and a decrease in the bioavailability of a wide set of potentially harmful elements, therefore resulting in a lower risk of SS-biochar application to the environment.
Production and use of SS based biochar in the formulation of substrates for seedling production in Brazil represent a new opportunity for this material, with potential economic gains, reduction of environmental impact and satisfactory agronomic performance. In this context, the objective of this work was to evaluate the performance of substrates formulated with SS-solarized and SS-biochar in the production of lettuce and black wattle.

SS-solarized and SS-biochar
The SS was collected at the Sewage Treatment Station of Rio Grande-RS, during the spring of 2015. After collect, SS-solarized was spreaded in 10 cm thick layer in fiberglass boxes, placed inside an agricultural greenhouse made of transparent plastic with automated control of aeration and heating. The heating system was activated every time the temperature reached 15 ºC and turned off when it exceeded 19 ºC, remaining in this condition for approximately 45 days until the moisture content of SS reached less than 20%. After the solarization, part of the material was identified and stored and the other part was submitted to the pyrolysis process, with partial air supply and variable temperature (300-600 ºC) for three hours. Finally, SS-solarized and SS-biochar used in this study was crushed and sieved until granulometry 100% < 2.0 mm.
Samples of SS-solarized were submitted to the characterization of the presence of pathogenic contaminants (thermotolerant coliforms, viable eggs of helminths, salmonella and enteric virus contents), inorganic contaminants (As, Ba, Cd, Pb, Cu, Cr, Cr 6 , Hg, Mo, Ni, Se and Zn) and chemical characteristics (pH, N, P, K, Ca, Mg, S, Cu, Zn, Fe, Mn, Na, Al, B and CTC) at the Eurofins ALAC Laboratory. This characterization was performed before the formulation of the substrates and the implantation of the experiments.

Experiment 1: Lettuce (Lactuca sativa L.)
The formulated substrates were homogenized and distributed in 110 cm 3 cells (styrofoam trays containing 78 cells). Commercial seeds of lettuce, cv. Simpson, with germination power of 74%, were sown on 20 June 2016. From this moment, the trays were kept in a floating system during the experiment time. The plants were evaluated at 29 days after their emergence, when the crop stage ideal for transplantation was reached.
The response variables evaluated were: plant height, dry mass of aboveground parts (PDM) and dry mass of roots (RDM), total chlorophyll index and total leaf area. Plant height (cm) was measured in six plants of each plot with a graduated ruler. The PDM (g) and RDM (g) were determined in the same six plants of each plot, which were packed in paper bags and dried out at 65 ºC until reaching a constant mass. The total chlorophyll index was evaluated in three leaves per plant, using a portable chlorophyllometer (ClorofiLOG -Falker Automação Agrícola Ltda., Porto Alegre-RS). The total leaf area of the plants (cm 2 ) was obtained by scanning all leaves from the same six previously used plants using computer and scanner. Subsequently, the digital images were processed with the AFSoft program (Silva & Jorge, 2009). The experimental design was a randomized block, with four replications, where each plot had six cells with one seedling per cell.

Experiment 2: Black wattle (Acácia decurrens Willd)
The substrates were disposed in polypropylene tubes with 53 cm³, thenblack wattle seeds (germination power of 72%) were sowed at 20 August 2016, with two seeds per tube, and then thinning when necessary. On 24 November 2016, when the plants reached the transplanting stage, the following variables were evaluated: plant height, dry mass of aboveground parts (PDM) and dry mass of roots (RDM). The plant height (cm) was measured in four plants of each plot with graded ruler, while the PDM (g) and RDM (g) were determined in the three plants of each plot, being conditioned in paper bags and dried at 65 ºC until reaching a constant mass. The experimental design was randomized blocks, with three replications and three plants per plot.
The wet and dry densities of the substrates were determined by the self-compacting method described by Brasil (2007). The granulometric fractionation was performed with samples of 100 g of air dried substrate, with a set of sieves (4.76, 2.00, 1.00 and 0.50 mm) coupled to a mechanical stirrer, working for three minutes. The variables TP, AS, WA, WR 100 and WRC were determined according to De Boodt & Verdonck (1972). The pH and CE values of the substrates were determined in 1:5 (substrate:water ratio) according to Brasil (2007).

Statistical analysis
The data sets obtained in experiments 1 and 2, and in the characterization of the substrates, were evaluated with relation to the normality of their distributions and about the presence of discrepant values. Each response variable was then submitted to analysis of variance and for those variables with significant treatment effect, the Scott-Knott test was applied at a 5% error probability level.

Characteristics of SS-solatized
The concentrations of inorganic and pathogenic contaminants evaluated in SS-solarized were lower than the maximum limits posed by the Resolution Nº 375/2006 (Brasil, 2006) for SS disposal in agricultural areas, and lower that the limits allowed in substrates for plants, posed by the Normative Instruction Nº 07/2016 (Table 1). Therefore, the concentrations of these contaminants were not impeditive to the use of SS-solarized in the composition of plant substrates.
The concentrations of pathogenic contaminants found in SS after solarization demonstrate that this method is efficient in SS sanitation, being similar to observed by other studies (Pereira Lima et al., 2009;Ozdemir et al., 2013;Santos et al., 2015;Alves Filho et al., 2016). Pereira Lima et al. (2009) evaluating the chemical and microbiological characteristics of SS when subjected to hygienization in an agricultural greenhouse, observed that after 28 days of solarization, SS reached the limits of the current legislation for pathogenic contaminants. Thus, considering that SS-solarized meets the limits posed by the current legislation (Table 1), and SS-solarized is the raw material for biochar production, these contaminants were not evaluated in the SS-biochar.
For the SS-solarized, the chemical results are shown in Table 1. Based on agronomic aspects, SS-solarized resembles animal manures such as from cattles and equines both used in large scale in agriculture (Kiehl, 1985).

Characteristics of substrates
The proportion of SS-solarized or SS-biochar utilized in the mix influenced significantly the physico-hydric and chemical characteristics of the substrate. In general, there is a tendency to increase the values of wet density (WD), dry density (DD), total porosity (TP) and pH, and to decrease the electrical conductivity (EC) with the increase of the proportions of SS-solarized (T1 -T5) and SS-biochar (T6 -T10) in the substrates (Table 2). On the other hand, this trend was not detected for the aeration space (EA).
The tendency of increasing in the values of WD and DD as SS-solarized and SS-biochar increase in the substrates are in agreement with the results obtained in the characterization of these materials (SS-solarized: WD = 888.50 kg m -3 and DD = 718.80 kg m -3 ; SS-biochar: WD = 897.52 kg m -3 and DD = 728.24 kg m -3 ), which indicated higher densities compared to rice husk ash and vermiculite (rice husk ash: WD = 385.87 kg m -3 and DD = 249.12 kg m -3 ; vermiculite: WD = 489.81 kg m -3 and DD = 265.60 kg m -3 ). The high WD and DD values presented by SS-solarized and SS-biochar resulted in compacted substrates where these materials are predominant. This is a negative aspect Rev. Ceres, Viçosa, v. 67, n.6, p. 491-500, nov/dec, 2020 in comparison with the commercial substrates (T11 and T12), since the commercial substrates have 22% and 26% lower DD than substrates with SS-solarized and SSbiochar, respectively.
The TP was higher in the substrates with SS-solarized (T1 to T5), value 29% higher than the SS-biochar (T6 to T10) and 22% higher than commercials T11 and T12 ( Table  2). The TP of substrates with SS-biochar did not differ statistically from commercial substrates T11 and T12. However, substrates with SS-solarized and SS-biochar showed, respectively, a TP of 17.84 and 32.81% below the reference value of 85%, established by De Boodt & Verdonck (1972) and Verdonck & Gabriels (1988).
The highest pH values were found on substrates with 50% and 60% SS-solarized (T4 and T5, respectively). Among the SS-biochar substrates, the highest pH value (7.69 to 8.09) was obtained in T2 to T5 (20% to 50% SSsolarized) and T9 (substrate with 50% SS-biochar) ( Table   Table 1 2). The lowest pH values were found in commercial substrates 1 and 2 (pH = 5.24 in T11 and pH = 5.26 in T12). Thus, it is noted that both substrates with SS have higher pH than commercial ones. In addition, a positive correlation was found between the increase in the proportion of SSsolarized (r = 0.88, p = 0.001) and SS-biochar (r = 0.92, p = 0.001) and increase in pH of the substrates. Substrates having pH below 5.0 may cause deficiencies of nutrients such as nitrogen, potassium, calcium, magnesium and boron; on the other hand, deficiencies in phosphorus, iron, manganese, zinc and copper may happen in those with pH values above 6.5 (Valeri & Corradini, 2000). In this study, the substrates based on SS-solarized and SSbiochar presented an average pH of 7.64, being classified in the second case. The highest EC values were found on the substrates T1 to T7 (1,230.83 µS cm -1 to 1,629.67 µS cm -1 ), followed by substrates SS-biochar (T8 to T10) and commercial T11 (Table 2), respectively. The lowest EC value was found on the substrate T12 (309.10 µS cm -1 ). The highest percentage of available water (WA = 20%) was presented by T9 (50% SS-biochar) ( Figure 1A), remaining at the lower limit of the range of 20% to 30% adopted as a reference by De Boodt & Verdonck (1972).
The substrates with 50% and 60% of SS-solarized (T4 and T5) had the lowest WA values (6.93% and 7.13%, respectively) ( Figure 1A), a result that may be related to the higher water retention capacity (WRC) from SSsolarized when compared to SS-biochar. This fact is evident when analyzing the WR 100 values of T4 and T5 substrates (50% and 60% of SS-solarized), since in these substrates the highest values of WR 100 were obtained (Figure 1A). This relationship can be corroborated by the positive correlation (r = 0.94, p = 0.001) between the increase of SS-solarized and WR 100 in the substrate. The substrates containing SS-solarized presented, in general, a content of WR 100 on average 39% higher than the substrates with SS-biochar and 54% higher than the commercial ones.
In general, the formulated substrates presented low percentages of particles larger than 4.76 mm ( Figure  1B). On the other hand, expressive differences were verified in relation to particles lower than 0.50 mm, so that the substrates composed by the lower concentrations of SS-solarized (T1, T2 and T3) and SSbiochar (T6, T7, T8 and T9) formed the two highest percentage groups ( Figure 1B).
Only the substrates T4, T5, T9 and T10 showed granulometry similar to the commercial substrates. The main granulometric differences of the substrates are verified in the granulometric bands between 4.76 and 2.00 mm and less than 0.50 mm. There was an increase in particles between 4.76 and 2.00 mm as the proportions of SS were increased in substrates, while lower particles than 0.50 mm in substrates formed by lower amounts of SS (solarized and biochar) were due to the greater proportion of rice husk ash, which has 86% of particles within this grain size range.

Treatments¹ pH
Rev. Ceres, Viçosa, v. 67, n.6, p. 491-500, nov/dec, 2020 There were significant differences between the studied substrates in relation to the water retention at the tensions of 0, 1, 5 and 10 kPa ( Figure 1C). At the tensions of 0 and 1 kPa, the substrates composed with SS-solarized presented greater water retention. However, in the higher tensions (5 and 10 kPa), only the substrates with higher proportions of SS-solarized (T3, T4 and T5) formed the group of highest water retention ( Figure 1C). Thus, introducing SS in substrates clearly increase their WRC.
In relation to water retention capacity at 1 kPa (WRC1), it was observed that SS-solarized at proportions of 30, 40, 50 and 60% (T2, T3, T4 and T5, respectively) formed the group of substrates with the highest WRC1, differing significantly from the other treatments ( Figure 1D). The T1, with the lowest proportion of SS-solarized (20%), did not differ from SS-biochar and commercial substrates (Figure 1D), indicating that the SS-solarized increased the water retention of the substrates, even in comparison to commercial substrates. Data from literature suggest that WRC1 between 20 and 80% is ideal for substrates to vegetables (De Boodt & Verdonck, 1972;Verdonck & Gabriels, 1988), confirming that the studied substrates are within the ideal values for this variable.
Rev. Ceres, Viçosa, v. 67, n.6, p. 491-500, nov/dec, 2020 respectively, as well as commercial substrates (T11 and T12). On the other hand, the plants submitted to the substrates with the highest proportions of SS-solarized (T3, T4 and T5) had the lowest heights. In relation to the dry mass of aboveground parts (PDM) and dry mass of roots (RDM), there was an identical effect of the substrates on these variables (Table 3), occurring four distinct groups for the two variables. The highest mass resulted from T6, the lower concentration of SSbiochar. The substrates T7 and T10, containing 30 and 60% SS-biochar, respectively, together with commercial substrates 1 and 2 (T11 and T12) also resulted in high PDM and RDM, being lower only to T6 treatment. On the other hand, treatments with SS-solarized, especially T3, T4 and T5, resulted in lower values of PDM and RDM among the evaluated substrates.
The total leaf area was significantly influenced by the substrates, with the formation of four response groups ( Table 3). The largest leaf area was presented by the smallest amount of SS-biochar (T6). After T6, the better performance resulted from the commercial substrates and T7, T8 and T10, with 30, 40 and 60% of SS-biochar, respectively. On the other hand, the substrates that resulted in the smallest leaf areas were those with the highest proportions of SS-solarized (T3, T4 and T5). For total chlorophyll, there was no significant differences among all the substrates.
In general, SS-solarized resulted in plants with lower performance than SS-biochar for lettuce seedlings production (Table 3). This fact is possibly related to the higher content of WR 100 presented by SS-solarized substrates ( Figure 1A), being on average 39% higher than SS-biochar substrates, which caused water excess and therefore deficiency of root aeration.
This water excess was possibly aggravated by the production system of seedlings (floating). Considering that in the floating system the trays are suspended in a water table, water is largely availablefor plants. In floating systems it is strongely important to have substrates with high macroporosity and water free pores, to ensure gas exchange in the plant root system zone.

Experiment 2 -Black wattle (Acacia decurrens)
In the black wattle cropping, there was no significant effect of the studied substrates on plant height (Table 3). On the other hand, for PDM and RDM, the substrates T1, T8, T9 and T12 presented the largest mean values for both PDM and RDM variables. The low biomass production of black wattle crop on T2, T3, T4 and T5 substrates, based on SS-solarized, is probably related to the high WRC of the SS-solarized. This high WRC provided by SS-solarized is related to the high WR 100 content ( Figure 1A), causing an oxygen deficiency and less gas exchange in the root system, as observed in the lettuce crop. This decrease in gas exchange is aggravated by the fact that black wattle seedlings are produced in plastic tubes, which are impermeable and therefore avoid the connection of substrates with the free air environment.

Physico-hydric characteristics versus morphophysiological characteristics of lettuce and black wattle
In general, the substrates formulated with SS-biochar have shown similar results to commercial substrates in the production of lettuce and black wattle seedlings (Table  3). On the other hand, substrates containing SS-solarized have shown a poorer performance than others, particularly with the greater proportions of this material.
Correlation analysis indicates that the physico-hydric parameters that best explain the growth of lettuce seedlings are TP, WA, WR 100 and WRC. This means that, for lettuce, physical aspects of substrates are of major importance during the early crop development days. It was observed that higher average values of TP, WRC and WR 100 , verified in the substrates composed with SS-solarized, are related to the worse performance of these substrates.
The pore diameter distribution and the macro and micropores affect the physico-hydric characteristics of a substrate, so that it can retain more or less water. In the case of SS-solarized, it probably promotes the micropores and these are occupied in greater proportion by water, but in unavailable form for plants, being the main reason that the material showed a high WRC ( Figure 1C and Figure 1D).
In addition to the higher WRC presented by substrates with SS-solarized, results from AR 100 ( Figure 1A) indicate that this water portion is harder to be accessed by plants. Therefore, the values of WA were low and those of WR 100 were high in these substrates. This condition result in plant stress due to lack of aeration, and certainly contributed to the fact that the substrates composed of SS-solarized, especially in the higher proportions, have provided the worst conditions for lettuce plants development.
In the production of eucalyptus seedlings, Caldeira et al. (2013) observed in substrates with different doses of SS-solarized and vermiculite (100:0, 80:20, 60:40, 40:60 and 20:80) that higher microporosity were presented by substrates with higher doses of SSsolarized and, consequently, also increased WRC. These results are similar to those obtained in the present study. According to De Boodt & Verdonck (1972), it is essential that the substrate retain water in an energy state as low as possible to the plants, in addition to sufficient air in the root zone. However, some stress symptoms due to water excess and lack of oxygen in pores of substrates with SS-solarized were identified in the two evaluated species.
The significant correlation observed between total chlorophyll from lettuce plants and physico-hydric parameters (TP, WA, WR 100 and WRC), indicates that the balance of macro and micropores from substrates plays an important role in water availability and root aeration. Lettuce seedlings are more sensitive during the early stages of life, and a substantial part of water was not available to plants because of its stronger state of retention and, at the same time, this water occupied most of the pores, causing lack of aeration. Lenhard et al. (2010) reported a reduction in chlorophyll concentration in leaves of several species as a consequence of oxygen restricions caused by inadequate substrate pore volume distribution or soil flooding. The two evaluated species had different responses to the substrates (Table 3). The lettuce crop showed to be more sensitive to the different formulations, with the formation of several groups of response of the growth variables to the tested substrates, being able to differentiate substrates with better, similar or inferior performance to the commercial ones. Black wattle presented at most two response groups, one with similar performance and other poorer than the commercial substrates. These results can be related to several aspects, such as the great difference of rusticity between species. For example, tolerance to water stress, which is high in black wattle and small in lettuce (Attias et al., 2013) may explain most of the effects on the morphophysiological variables of the evaluated plants.
The different responses obtained in the two species show the importance of performing multispecies studies while developing and evaluating alternative substrates. Although Abad (1991) points out that there is no substrate that is universally valid for all species, the evaluation of the same substrate for different species can broaden the market for this product. This study goes further, because the combination of raw materials for different crops can be the same, but the proportion of each material that returns the best substrate for each crop may variy substantially.
In relation to the materials used in the alternative substrates (T1-T10), vermiculite and rice rusk ash are already widely tested materials (Kratz & Wendling, 2016). However, SS is a residue whose studies to this employment is still incipient, even though there is a great potential due to its chemical and physical characteristics (Nascimento et al., 2014). It is clear from this study that the main reason for its low utilization is the lack of knowledge about safe and low-cost ways of eliminating the risk of contamination by pathogenic microorganisms (Nascimento et al., 2014) and the preconcept regarding the use of SS in agriculture.
However, biochar produced from SS, when mixed with other materials that complement its chemical and especially physical characteristics, has proved to become a viable alternative. From efficiency aspects, SS-biochar was superior to SS-solarized and similar to commercial substrates. In addition, it can be considered a safe material because after the pyrolysis process, the pathogenic contaminants are eliminated (Agrafioti et al., 2013;Liu et al., 2014) as well as a waste recycling process.
Although the results indicate that several proposed substrates have achieved similar performance and even higher than the commercials tested, further studies should be conducted. There is a need to test new combinations with other materials and/or proportions of the same materials, since some chemical and physical parameters (pH, WD, DD, TP, AS and WA) did not reach the values considered ideal by the literature, indicating that the results obtained in the present study could be maximized or extended to a greater number of species.

CONCLUSIONS
The biochar made from sewage sludge is more effective in comparison to the solarized sewage sludge as component of substrates for the studied species; The formulated substrate composed by 20% sewage sludge biochar, 40% vermiculite and 40% rice husk ash presented better performance among the studied substrates; Solarized sewage sludge increases the water retention capacity of the substrates, but negatively affects the development of lettuce and black wattle seedlings.