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Influence of Biochar and Liquid Smoke Additives from Cacao-Pod Husks on In vitro Ruminal Fermentation Characteristics

| The objective of this study was to investigate the influence of biochar (BC) and liquid smoke (LS) additives from cacao pod husks on in vitro ruminal fermentation products, ruminal microbiota and feed degradation. The in vitro effects of different levels of BC and LS on rumen fermentation characteristics were assessed in a randomized block design experiment with five treatments and four replicates (BC and LS were separately analyzed). The treatments consisted of a basal ration (forage: pollard; 60:40 w/w) with various BC additions: BC0 (basal ration + 0 mg/ml biochar), BC1 (basal ration + 0.1 mg/ml biochar), BC2 (basal ration + 0.2 mg/ml biochar), BC3 (basal ration + 0.3 mg/ ml biochar), and BC4 (basal ration + 0.4 mg/ml biochar) and LS additions: LS0 (basal ration + 0 μl/ml liquid smoke), LS1 (basal ration + 0.25 μl/ml liquid smoke), LS2 (basal ration + 0.5 μl/ml liquid smoke), LS3 (basal ration + 0.75 μl/ ml liquid smoke), and LS4 (basal ration + 1 μl/ml liquid smoke). These treatments were subjected to in vitro incubation with buffered rumen fluid for 48 hours. Results showed that the addition of BC and LS did not affect pH, ammonia, or total gas production. Compared to the BC0 treatment, BC addition significantly increased acetate production (P < 0.01), and significantly decreased butyrate, isobutyrate, valerate, and isovalerate (P < 0.05). Compared to the LS0 treatment, LS addition significantly increased total volatile fatty acids (VFA), acetate, and acetate to propionate ratio (P < 0.05). Biochar addition above 0.1 mg/ml but not LS addition significantly decreased (P < 0.01) in vitro ruminal dry and organic matter degradabilities. Biochar addition above 0.1 mg/ml, but not LS addition significantly decreased total bacteria population (P < 0.001). Addition of BC above 0.2 mg/ml decreased protozoa population (P < 0.001) while LS decreased protozoa population, dependent on dose (P < 0.001). In conclusion, biochar and liquid smoke from cacao-pod husks had different effects on ruminal fermentation characteristics. However, both of them partially defaunated ruminal microbiota and shift VFA composition.

INTRODUCTION
T he use of antibiotics in animal feed is often aimed at improving growth performance and preventing the adverse effects of pathogens and non-pathogens. However, this use has been prohibited in many countries over the last two decades. The prohibition of antibiotic use is attributed to increasing antibiotic resistance in humans and animals, especially in Salmonella spp. and Escherichia coli (Qomariyah et al., 2019). Antibiotics can increase the weight, growth, and performance of livestock, but if used continuously, antibiotics cause microbial resistance to livestock and have residues for livestock and humans (Marshall and Levy, 2011). The use of antibiotics as growth promoters has been banned by the European Union since 2006 (EC Regulation No. 1831/20031) because of their negative effects on animal health and food safety (European Food Safety Authority, 2009). This prohibition has led farmers to switch to natural non-antibiotic alternative ingredients such as prebiotics, probiotics, organic acids, phytogenics, and zeolites which improve the health and growth performance of livestock (Papatsiros et al., 2012). The use of these materials has disadvantages, such as low potency, expensive production costs, and short-term effects.
Given these issues, to minimize or eliminate pathogenic infections and colonization in livestock it is necessary to develop new strategies and commercial products that are effective, cheap, and environmentally friendly. One of the plants sources of alternative bioactive compounds is cacao (Theobroma cacao). At present, Indonesia is the third largest producer of cacao, at 777,750 MT/year (FAOSTAT, 2014). Cacao beans are an industrially important crop because cacao is the key ingredient of chocolate, one of the most common foods in the world, and its processed goods. However, waste in the form of cacao-pod husks is also produced (Mansur et al., 2014). Cacao fruit has four component parts: (a) 73.7% pod husk; (b) 10.1% pulp; (c) 2.0% placenta; and (d) 14.2% seeds (Watson et al., 2012;Young, 2007). The cacao-pod husks contain crude fiber as their main component (55.7%) (Laconi and Jayanegara, 2015) and this fiber contains 35.4% cellulose, 37% hemicellulose, and 14.7% lignin (Daud et al., 2013). The use of this biomass to increase added value can be carried out by physical, chemical, and biological processing (Lehmann and Joseph, 2015).
Pyrolysis is one of the physical processes used to produce low-cost and environmentally friendly compounds. Pyrolysis is a thermal decomposition process in which, in the absence of oxygen, biomass is heated at moderate to high temperatures, resulting in the conversion of biomass into three main products: carbon-rich solid residues (biochar), the liquid condensation portion of the released volatile matter (liquid smoke) and the remaining non-condensable gases, such as CO, CO 2 , CH 4 and H 2 (Kambo and Dutta 2015;Liu and Wang 2018). Biochar compounds are porous and have a large surface area (Hernandez-Mena et al., 2014;Tomczyk et al., 2020) that allows them to absorb gases and carbon, bind to toxins, and provide a biofilm habitat in which microbiota can reproduce (Hansen et al., 2012;Lehmann and Joseph, 2015). Biochar also exhibits electron-mediating properties in biological redox reactions (Yu et al., 2015) and provides benefits in the form of feed efficiency in ruminants and the reduction of greenhouse gas emissions (Leng et al., 2012a). The phenolic compounds contained in liquid smoke are known to be antimicrobial agents (Beker et al., 2016) and to also have antioxidant properties (Crozier et al., 2011). Liquid smoke from cashew-nut shells can reduce the number of E. coli bacteria in an in vitro trial (Sinurat et al., 2018). The supplementation of 1% bamboo vinegar powder in feed ration provides an immune effect similar to antibiotics (Qin et al., 2018). The use of biochar and liquid smoke from cacao-pod husks is expected to support antibiotic-free programs in which farmers are directed to utilize the functional ingredients in their surroundings as natural and environmentally friendly growth-stimulating agents.
This study aimed to investigate the influence of biochar (BC) and liquid smoke (LS) additives from cacao pod husks on in vitro ruminal fermentation products, ruminal microbiota and feed degradation.

ethical aPProval
The experiment was approved by the Animal Welfare Commission of the Indonesian Agency for Agricultural Research and Development (Balitbangtan/BPTP Sulsel/ Rm/02/2020) and was conducted at the Laboratory of Dairy Nutrition, IPB University, Indonesia. This study used the rumen liquor from one ruminally non-lactating Holstein dairy cow located at the field experimental station, Faculty of Animal Science, IPB University, Indonesia. Animal were cared for according to the IPB University standard for animal welfare.

SamPle PreParatioN
Cacao-pod husks were obtained from a cacao plantation in Bogor Regency, West Java Province. The fresh cacao pod husks were chopped and dried and then processed at the Central Laboratory for Forest Products Research and Development, Bogor, and BC and LS were obtained as pyrolysis products. The chemical composition and physical properties of the BC and LS used can be seen in Table 1. Basal rations (based on dry matter) consisted of 60% grass and 40% pollard. The basal ration was composed to resem

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April 2021 | Volume 9 | Issue 4 | Page 535

In VItro iNcubatioN Procedure
The sample was evaluated for in vitro ruminal fermentation and degradability using the procedure used by Theodorou et al. (1994). The in vitro incubation was performed in four consecutive runs (replicates) at different weeks, in which each sample per run was represented by six fermentation tubes. Approximately 750 mg of sample was placed into a serum bottle (100 ml capacity) and 25 ml ruminal fluid and 50 ml bicarbonate buffer were added. All serum bottles were exposed to CO 2 for 30 s and then closed with a butyl rubber plug and aluminum crimp seal to begin incubation in a water bath at 39°C. Four tubes were removed after 24 h incubation and the remaining tubes were continued for another 48 h incubation. Gas was released and recorded regularly at 2, 4, 6, 8, 10, 12, and 24 hours incubation using a 100 mL syringe. Shaking was performed manually each time gas was taken. After the first stage of incubation (24 h), the tubes were centrifuged at 3500 rpm for 15 min. The supernatant (fermentation fluid) was separated and sampled for pH, ammonia concentration, and partial VFAs. Ammonia concentration was determined by the Conway micro-diffusion method (General Labo-ratory Procedure, 1966). Partial VFA concentration was analyzed by gas chromatography (Bruker Scion 436 GC) with a capillary column BR-Wax fame containing WCOT of fused silica with a column length of 30 m x 0.32 mm. Ionization detection was used to evaluate the VFA products of fermentation. Nitrogen 25 ml/min was used as the carrier gas and hydrogen 30 mL/min as the combustion gas. The temperature of the injector was 250ºC, and in 11 minutes, the column temperature gradient was 70-150ºC. The flame ionization detector (FID) temperature of the detector used was 275°C. Gas-production kinetics was calculated through the exponential equation (Orskov and McDonald, 1979) as follows: where p is the cumulative gas production at t hours, while a, b, and c are constants of the exponential equation. The constants a, b, and c can be interpreted as the production of gas from the soluble fraction (a), the production of gas from the insoluble but fermentable fraction (b), and the rate of reaction of gas formation (c). Thus a + b can be interpreted as the maximum gas production that can be formed during the fermentation process at time t approaches infinity (Orskov and McDonald, 1979). Biochar and liquid smoke were separately analyzed. The calculations were carried out using a nonlinear regression equation in SPSS Version 20 software.

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April 2021 | Volume 9 | Issue 4 | Page 536 to measure methane concentration, using a Shimadzu 14A GC with an Flame Ionization Detector (FID). The total bacterial population was analyzed using Ogimoto and Imai's (1981) methods. The protozoa populations in the rumen fluid were calculated using a hemocytometer with methyl-green formalin saline solution (Ogimoto and Imai, 1981). In the second stage of in vitro fermentation, the supernatants in the remaining tubes were discarded after centrifugation (3500 rpm for 15 min). The residue was analyzed for dry matter (DM) and organic matter (OM) to identify in vitro ruminal dry matter degradability (IVRDMD) and in vitro ruminal organic matter degradability (IVROMD) values (Tilley and Terry, 1963).

exPerimeNtal deSigN aNd data aNalySiS
This study used a randomized block design with five treatments and four replications (BC and LS were analyzed separately). The treatments consisted of a basal ration (forage:pollard, 60:40 w/w) with additions as follows: BC0 (basal ration + 0 mg/ml biochar), BC 1 (basal ration + 0.1 mg/ml biochar), BC2 (basal ration + 0.2 mg/ml biochar), BC3 (basal ration + 0.3 mg/ml biochar), BC4 (basal ration + 0.4 mg/ml biochar), and LS additions: LS0 (basal ration + 0 µl/ml liquid smoke), LS1 (basal ration + 0.25 µl/ml liquid smoke), LS2 (basal ration + 0.5 µl/ml liquid smoke), LS3 (basal ration + 0.75 µl/ml liquid smoke), and LS4 (basal ration + 1 µl/ml liquid smoke). Different in vitro processes were divided into groups due to differences in the population variations and ruminal microbial activity in each sampling time. Detected outlier data were removed from the data set when their Z score was lower than -2 or higher than 2. Data were analyzed using ANOVA at a 5% significance level. The comparison between the different treatments was analyzed using Duncan's multiple range test at a 5% significance level. Data analysis was conducted using IBM SPSS Statistics version 20.

RESULTS
The addition of biochar (BC) and liquid smoke (LS) did not affect pH, ammonia, total gas or methane production ( Table 3). The in vitro gas-production kinetics with BC and LS additions during the 24 h incubation period can be seen in Figures 1 and 2, respectively. In both, it can be seen that there was a quite large increase in gas production in the first 8 h of incubation, then it decreased along with the fermentation period. Compared to the BC0 treatment, BC addition tended to increase total VFA production (P < 0.1) and significantly increased acetate production (P < 0.01). However, increasing the dose of BC did not cause any significant difference in acetate production. Addition of BC significantly reduced butyrate, isobutyrate, valerate and isovalerate production (P < 0.05), however, there was no significant difference in their values with increasing levels of BC.
Compared to the LS0 treatment, LS addition significantly increased total VFAs, acetate, and acetate to propionate Table 3: In vitro total gas production, gas production kinetics and methane concentration of basal ration added with biochar (BC) and liquid smoke (LS).

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April 2021 | Volume 9 | Issue 4 | Page 537  .001 a-e : means in the same column with varying superscript differ significantly (P<0.05); IVRDMD: in vitro ruminal dry matter degradability; IVROMD: in vitro ruminal organic matter degradability ratio (P < 0.05). However, increasing the level of LS addition did not cause any significant difference in total VFA, acetate, and acetate to propionate ratio. Addition of LS significantly decreased isobutyrate and isovalerate production (P < 0.05); however, no significant difference in their values with increasing level of LS was seen. The production

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April 2021 | Volume 9 | Issue 4 | Page 538 of butyrate and valerate tended to decrease (P < 0.1) with addition of LS.
In vitro ruminal dry matter and organic matter degradabilities (IVRDMD and IVROMD) decreased significantly with BC addition above 0.1 mg/ml, while non-significant difference in IVRDMD and IVROMD were seen with LS addition.
Biochar addition above 0.1 mg/ml significantly decreased total bacteria population (P < 0.001) but this was not the case for LS addition. Addition of BC above 0.2 mg/ml decreased protozoa population (P < 0.001) while LS addi-tion produced dose-dependent decreased protozoa population (P < 0.001).

DISCUSSION
Gas production in the rumen is derived from the fermentation of feed by various microbes. Volatile fatty acid (VFA), carbon dioxide (CO 2 ), and hydrogen (H 2 ) molecules are produced during the fermentation of carbohydrates by bacteria, protozoa, and fungi in the rumen. Volatile fatty acids function as the main energy source for ruminants (Vyas et al., 2016). Furthermore, overall production of gas provides a representation of nutrient profiles (Wahyono et al., 2019). The in vitro gas production rate for each treatment decreased with increased incubation time, due to the reduction in the amount of easily fermented substrate. Jayanegara and Sofyan (2008) attributed the decrease in the rate of gas production as being related to VFA production indicating a reduced energy supply for livestock. The positive correlation between incubation time and in vitro gas production is associated with degradation of the incubated feed samples over a longer incubation time (Maccarana et al., 2016). Previous research has shown similar results, in which there were no differences in gas production when BC was used during in vitro ruminal fermentation (Hansen et al., 2012;Leng et al., 2012a;Pereira et al., 2014;Saenab et al., 2018;Teoh et al., 2019). Biochar has been used as a feed additive to build microbial habitats and to alter biofilm activity in the rumen (Leng, 2014). Also, BC can increase the efficiency of microbial growth (Leng et al., 2012b(Leng et al., , 2012c. The degree of acidity (pH) of the ruminal fluid indicates the fermentation process in the rumen. The addition of BC and LS produced an average rumen pH of 6.9. The pH value was in the normal range of ruminal pH of 5.5-7.5 (Franzolin et al., 2010) and so did not interfere with microbial activity in the rumen, supported the growth of ruminal microbes, and produced VFAs and NH 3 . Although the pH of BC and LS were 10 and 3.75, respectively, they did not harm total gas production because the buffer solution added to the ruminal fluid was able to maintain the ruminal fermentation pH. The addition of 1-4% DM of BC had no negative effect on ruminal fermentation in either total gas production or the rate of gas production. Biochar supplementation in this experiment did not reduce CH 4 in the rumen. As a greenhouse gas, CH 4 contributes to global warming (Eisler et al., 2014) and also represents a certain level of energy loss from livestock ( Jayanegara et al., 2018). A previous study by Hansen et al. (2012) which tested four types of BC added by 9% DM showed a reduction in CH 4 emissions of 11-17%. Leng et al. (2012a) added 1% rice husk-based BC (DM) to a mixture of cassava and urea feed and showed a decrease in CH 4 of 12%.

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April 2021 | Volume 9 | Issue 4 | Page 539 Saenab et al. (2018) showed a decrease in CH 4 when 0.3 mg/ml BC from cashew nut shells was added. The differing results of this experiment to other published reports may be due to differences in the biomass source of BC affecting BC properties such as pore size, adsorptive potential, added into the rumen fermentation, feed composition that may give different effect on methane production (Teoh et al., 2019). In addition, the level of BC addition affected methane reduction in a quadratic relationship, as reported by Saleem et al. (2018).
Ammonia (N-NH 3 ) is the main source of nitrogen for microbial protein synthesis in the rumen and so concentration of ammonia in the rumen is a point for consideration. Ammonia is also the end product of protein degradation of ration by ruminal microbes (Suharlina et al., 2016). The concentration of ammonia therefore represents the degree of degradation of protein by rumen microbes (Sujarnoko et al., 2020). High concentration of ammonia allows increased microbial protein synthesis in the ruminal system because ammonia is the main precursor in the formation of microbial cells. There is no effect on ammonia concentration from the addition of BC or LS at different levels.
The BC or LS addition may not inhibit bacterial growth or activity in the deamination process in the ruminal fluid, and this is in line with previous research which stated that there was no influence of BC addition on the concentration of NH 3 in ruminal fluid in vitro (Pereira et al., 2014). In addition, NH 3 ruminal concentration is influenced by the crude protein quality of the incubated substrate. Difference in the quality and number of different types of soluble protein, quickly degraded, moderately degraded, slowly degraded, and undegradable protein fractions in the substrate often affect the degree and rate of NH 3 production in the rumen .
Volatile fatty acids (VFA) are the final product of fermentation and are the main source of energy for ruminants and of carbon for the formation of microbial protein. The amount of VFAs indicates the fermentability of rations by ruminal microbes. The production of VFAs in the ruminal fluid can be used as a measure of ration fermentability. In this study, the addition of BC had the tendency to increase total VFAs compared to the BC0 treatment. Various effects of BC addition on total VFAs or individual VFAs have been reported. Some BCs create no response in total VFA production (McFarlane et al. 2017;Cabeza et al., 2018;Teoh et al. 2019) while others cause a significant increase of total VFAs (Pereira et al., 2014;Saenab et al., 2018;Saleem et al. 2018). Production of VFAs is consistent with an increase in the molar proportion of acetate, indicating an increase in fiber digestion. In contrast, there was a decrease in the ratio of butyrate, isobutyrate, valerate, and isovalerate, isobutyrate, and isovalerate being the products of branched amino acid degradation. Biochar addition sig-nificantly decreased the proportion of molar butyrate in the ruminal fluid starting from BC2, BC3, and BC4, however, BC1 did not significantly decrease the molar proportion of butyrate compared to the control (BC0). Likewise, the molar proportions of isobutyrate and isovalerate showed a decrease starting from BC1. Isobutyrate and isovalerate are products of degradation of branched amino acids. Reduction of isoacids by biochar indicates that there may be an inhibition of some protein degradation to branched amino acids. However, the inhibition might occur partly and the degradation of amino acids to ammonia didnot get affected since the ammonia production in the rumen was not significanly different among treatments. The mechanism of biochar that reduces certain proteolytic bacteria is unknown. The reduction of protein degradation in the rumen may result in a higher level of by pass protein entering the duodenum. Further in vivo research on BC addition and bypass protein is warranted. The addition of LS significantly (p < 0.05) increased total VFAs, acetate, and the ratio of acetate to propionate. The addition of LS had the tendency (P < 0.1) to lower butyrate and valerate amounts. Liquid smoke addition significantly decreased the molar proportion of isobutyrate and isovalerate in the ruminal fluid starting from LS1. A similar effect of BC and LS additions in reducing molar proportion of isobutyrate and isovalerate indicated an inhibition of protein degradation to isoacids. The mechanism of isoacid reduction of LS was different from that of BC. Liquid smoke contains phenolic compounds (Table 1) which could bind protein in feed and in microbes  and therefore might increase bypass protein. Acetic acid is minimally used in the liver, and is oxidized to produce ATP in most of the body. Another important use of acetate for lipid synthesis is as the main source of acetyl CoA. Higher ratio of acetate:propionate has some effect on an animal's efficiency, for example, higher acetate:propionate ratio appears to result in higher butterfat than milk yield in dairy animals (i.e. increased milk fat at the cost of milk yield).
Meanwhile, the proportion of propionate was not influenced by BC or LS addition. Pereira et al. (2014) reported that decreasing proportion of propionate had a relationship with total VFA production. Propionate development, on the other hand, implies a net incorporation of dihydrogen. Therefore, propionate competes in rumen fermentation with CH 4 as a hydrogen drain, while acetate and butyrate formation release hydrogen that can be used by methanogens to reduce CO 2 to CH 4 ( Janssen, 2010). Dihydrogen concentration in the rumen is influenced by factors influencing the growth of methanogens and the rate of feed fermentation, which in turn regulate the balance between pathways generating and integrating metabolic hydrogen, deciding the production of methane and the profile of VFAs (Ungerfeld, 2020). Biochar supplementa-

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April 2021 | Volume 9 | Issue 4 | Page 540 tion can increase the efficiency of energy use in ruminants. Acetate, propionate, and butyrate are the main products in ruminal fermentation processes, while VFAs are an important part of the energy for ruminants (Li et al., 2013).
Biochar addition significantly and progressively decreased IVRDMD and IVROMD compared to the control from BC2, BC3 and BC4; however, BC1 did not significantly decrease IVRDMD and IVROMD compared to the control (BC0). This is different from a study by Saenab et al. (2018) which added BC at different levels that did not significantly influence IVDMD and IVOMD compared to the control. In addition, different BC sources can give different ruminal fermentation responses, due to differences in structure and composition of basic ingredients, as well as the effectiveness of BC as a feed additive due to changes in feed digestion in the rumen (McFarlane et al., 2017). The addition of LS did not influence IVRDMD and IVROMD. This is in line with Saenab et al. (2018) who found that the supplementation of LS from cashew-nut shells did not influence feed degradation or gas production. The total phenol content in LS was 3.12%, and the supplementation of LS had no negative effect on the total values of gas production, the rate of gas production, IVRDMD, or IVROMD. Jayanegara (2009) reported that phenol concentrations of more than 5% can suppress livestock performance, so their use in rations should be below this level. Furthermore, phenolic compounds can interact with protein (Kondo et al., 2014) in cell walls or membranes and various pathogenic microbial enzymes and so can destroy microbes.
Increasing the level of BC and LS additions caused a decrease in the number of protozoa (about 6-7%). The decrease in the number of protozoa might contribute to the decrease in total bacterial counts and thus contribute to a decrease in IVRDMD and IVROMD (Newbold et al., 2015). Protozoa play a role in the ruminal fermentation process because they can reduce the main components of feed. One of the ciliated protozoa that has an important role in the rumen is Diploplastron affine, which can digest cellulose and carbohydrates from grains (Wereszka and Michałowski, 2012). Also, the holotrich protozoa, although in small amounts, also produce enzymes that are responsible for the degradation of cellulose and hemicellulose. Mosoni et al. (2011) suggest that a decrease in the protozoan population will harm fiber digestion, which is the main function of the rumen. Also, BC addition significantly decreased the bacterial population in the rumen from BC2, BC3, and BC4, however, BC1 treatment did not significantly decrease the total bacteria compared to the control (BC0). This is differs from a study by Teoh et al. (2019) which added 800 mg/d biochar (7.2% DM) and found that this did not significantly influence the total bacteria in the rumen, but suppressed several bacterial species, such as Methanomethylophilaceae, with a decrease of 19.8 times population, and Lactobacillus spp., with 31.7 times decrease in population. Rumen protozoa have a symbiotic relationship with methanogens involved in the transfer of hydrogen to reduce carbon dioxide to methane (Machmüller et al., 2003). When protozoa are reduced, these symbiotic methanogens would also reduce. In this experiment, however, the reduced methanogens did not decrease methane production in the rumen. The same result was recorded by Hegarty et al. (2008), who found that protozoa did not have a significant impact on the production of rumen methane when investigated in chemically defaunated, birth-defaunated, and faunated lambs. In addition, the release of methane without changes in protozoa numbers suggests that methanogenesis is often regulated by other non-protozoa mechanisms (Guyader et al., 2014).

CONCLUSION
The lowest level of biochar (0.1 mg/ml) and the lowest level of liquid smoke (0.25 µl/ml) from cacao pod husk addition to in vitro rumen fermentation tended to increase and significantly increased total VFAs, respectively. At their lowest level, both biochar and liquid smoke shifted VFA composition, reduced part-degradation of protein to branched amino acids and partly defaunated rumen microbes without affecting IVRDMD and IVROMD.

ACKNOWLEDGEMENT
This study was supported by the Indonesian Agency for Agricultural Research and Development, Ministry of Agricultural, Republic of Indonesia.

AUTHORS CONTRIBUTION
NQ conducted the experiment, performed data analysis, and wrote the article draft; EW checked data analysis and revised the article draft; YR and IGP supervised the experiment; AJ designed the experiment, checked data analysis, and revised the article draft.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Yield of Tomato Crop Irrigated with Untreated Industrial Sewage Effluent and Remediated with Potassium Silicate and Compost Applications

An experiment was conducted for the two successive seasons of 2017 and 2018 to investigate the deleterious effect of irrigating tomato plants with untreated industrial waste water and the possible ameliorating effects of compost and potassium silicate applications on the growth and production of the grown plants. Tomato seedlings of hybrid K186 were transplanted at the four-true leaf stage and irrigated with untreated waste effluent. Compost was applied during the soil preparation at rates of 0 (control), 10, 20, and 30 m/feddan. Potassium silicate was sprayed on the plants three times; at 20, 40 and 60 days after transplanting in the concentrations of 0 (control), 3, 4 and 5 cm/l. Results revealed that vegetative growth and fruit yield of treated plants were increased by increasing compost and potassium silicate rates compared by the control (without compost and potassium silicate).The interaction effect of the treatment showed an added effect of both treatments on all measured parameters. Plant length, number of leaves and number of branches increased as the application rate of compost and potassium silicate increased. Similarly, SPAD readings showed similar positive and significant trend. In the contrary, contents of Zn, Pb, Ni, Cd and Mnresponded negatively and significantly to the interaction effect of the treatments showing the highest effect with the treatment 5 cm potassium silicate associated with 30 m compost application compared to the control. Similar to the positive effect on vegetative growth, the positive additive effect of both treatments was clear on total fruit yield where the highest effect was recorded with the treatment combining the highest rate of application of both potassium silicate and compost. It could be concluded that potassium silicate and compost applications can ameliorate the harmful effects of heavy metals in the soil.

Introduction
Egypt was classified as an arid land with a very little amount of rain fall mainly in the north coast and having the Nile Rivera's the main source of irrigation water. Officials revealed that with the ongoing agricultural expansion and climate changes, Egypt is facing a serious shortage of fresh water supply forcing the country to recycle all available sources of water. Some growers in remote areas are using raw industrial sewage effluent to fulfill their crop water needs ignoring the possible dangerous of contamination of heavy metals of such water.
The negative effects of heavy metal on many aspects of agriculture have been reported. For example, heavy metal can be leached to the underground water (He et al 2004, Rattan et al 2005 causing the spread of pollution to other layers of the aquifers. Heavy metal polluted soil can negatively affect plant growth and production causing serious economic losses (Nagajyoti et al 2010). Moreover, Arao et al (2010) and Khan et al (2008) reported high health risks for people exposed to polluted agricultural soil. The problem of heavy metal pollution to the soil is

Rana, Eman, Hashim, El-Shinawy, Abd El-Gawad
more difficult compared to other types of pollution such organic contamination because they cannot be degraded by microorganisms and last in the soil for longer time.
The application of compost decreased heavy metal concentration in plants (Ramachandran and D'souza 1998). In this respect, Sharma and Dhaliwal (2019) found that decreased concentrations of toxic metals in soils with regular application of wastewater sewage sludge with compost treatments.
Some researchers used soil amendments to change the mobility and bioavailability of such heavy metals (Bolan et al 2014, Udeigwe et al 2011. One of such amendments was compost (Paradelo et al 2011). Compost is rich with mineral ions, humic substances, and microbes which influence the immobilization of heavy metals resulting in reduction of the ecological and environmental risk of heavy metals in agricultural soils (De la Fuente et al 2011, Udovic andMcBride 2012). Adsorption, complexation, precipitation, and redox reactions may all be one or more process involving heavy metal immobilization (Huang et al 2010, Lagomarsino et al 2011, Park et al 2011, Vaca-Paulin et al 2006. Thus, in addition to the benefit of compost as an alternative for waste management, its application can reduce the harmful effects on the crop, lower economic losses, and decrease human health risks from heavy metals existing in the root zone. Beneficial effects of compost on the growth and production of some vegetable crops such as green beans were reported under different levels of irrigation (Abdel-Mawgoud 2005) as well as salinity (Abdel-Mawgoud et al 2010).
Another alternative to reduce heavy metals deteriorating effects on plants is the application of some nutritional and/or beneficial elements such as silicon (Emamverdian et al 2018). Epstein (1999) described silicon (Si) as a beneficial and possibly essential element for plants, which plays important roles in plant growth and development Yamaji 2006, Gu 2012). Many researchers reported various evidence that the application of Si to soils can alleviate Cd or Zn toxicity in many plant species, including rice , maize (Liang et al 2005), wheat (Hussain et al 2015) and cotton (Farooq et al 2013).
Therefore, this work aims to investigate the effect of compost and potassium silicate applications, on the growth and production of tomato crop grown in soils irrigated with untreated industrial sewage effluent.

Materials and Methods
Seeds of tomato plant (Solanum lycopersicum L.) hybrid K186 were sown on 24 th and 28 th of April 2017 and 2018, respectively. After one month when the seedlings reached the fourth true leaf, they were transplanted in the open field in a sandy soil at a private farm in the area of Borg Al-Arab, Alexandria Governorate, Egypt. The soil physical and chemical analyses are shown in Table 1. Individual transplants were grown at the bottom of ridges 100 cm width and at 50 cm apart. Planting distances between plants are 50 cm . The furrow irrigation was used and plants were irrigated using water from an industrial sewage channel. The chemical analysis of the irrigation water is shown in Table 1. Also, heavy metals analyses in soil and irrigation water during the two seasons of study are shown in Table 2.  All standard agricultural practices other than experimental treatments were applied according to the recommendations of the ministry of Agriculture, Egypt.

Experimental treatments
During the preparation of the soil and before transplanting, four levels of compost were applied namely, 0 (control), 10, 20, and 30 m 3 /feddan and mixed well with the upper 50 cm of soil. A complete analysis of the applied compost is shown in Table  3.

Measurements
Plant destructed samples were taken in the end of the seasons to determine plant height, and number of leaves and branches. Total yield/plant was measured by the end of the season when all ripe fruits were harvested. Zn, Mn, Pb, Ni and Cd contents were determined with a Model SOLAR 969 Atomic Absorption Spectrometer (FAO/WHO, 2001). SPAD readings at 90 days after transplanting was measured in fully expanded leaves using Minolta SPAD 501 chlorophyll meter.

Experimental design and statistical analysis
The treatments were arranged in a split plot design with four replicates where potassium silicate treatments were in the main plot and compost treatments in the sub main plots. All data collected were subjected to the statistical analysis according to Snedecor and Cochram (1968). The data of treatments were compared, using least significant difference (LSD) method at 0.05 as mentioned by Gomez and Gomez (1984). Table 4 show that potassium silicate spraying increased significantly plant length and number of leaves and branches per plant as the concentration of the application increased in both growing seasons.

Data in
As for the effect of compost adding, compost application showed a gradual positive effect on plant length, and number of leaves and branches per plant with the highest effect recorded with the highest rate of application (30 m 3 /fed.) Table 4.
The interaction effect of potassium silicate and compost showed an added effect of both treatments on all measured parameters. The highest concentration of potassium silicate with the highest rate of compost gave the tallest plants and highest number of leaves and branches. Data in Table 5 indicate that potassium silicate spraying increased significantly SPAD reading and total yield per plant as the concentration of the application increased in both growing seasons.
As for the effect of compost adding, compost application showed a gradual positive effect on SPAD reading and total yield per plant with the highest effect recorded with the highest rate of application (30 m 3 /fed.) Table 5.
The interaction effect of potassium silicate and compost showed an added effect of both treatments on all measured parameters. The highest concentration of potassium silicate with the highest rate of compost gave the highest SPAD reading and total yield per plant. Table 6 indicated that Zn and Mn contents responded negatively and significantly to the increment in potassium silicate application rates Table 6.

Data in
Also, increasing compost application rate significantly decreased the contents of Zn and Mn in the tissue of the plants with the highest negative effect recorded with the highest rate of application (30 m 3 /fed.) in two seasons of study Table 6.
Data in Table 6 showed Zn and Mn contents responded negatively and significantly to the interaction effect of the treatments showing the highest effect with the treatment 5 cm 3 potassium silicate associated with 30 m 3 compost application compared to control. Data in Table 7 indicated that Pb, Ni and Cd contents responded negatively and significantly to the increment in potassium silicate application rates Table 7.

Yield of tomato crop irrigated with untreated industrial sewage effluent and remediated with potassium silicate and compost applications
Also, increasing compost application rate significantly decreased the contents of Pb, Ni and Cd in the tissue of the plants in two seasons of study Table 7. Table 7 showed Pb, Ni and Cd contents responded negatively and significantly to the interaction effect of the treatments showing the highest effect with the treatment 5 cm 3 potassium silicate associated with 30 m 3 compost application compared to control.

Discussion
Heavy metals have been reported to cause deteriorating effects on different plant growth aspects resulting in reduction in plant production and quality. Cd, Ni and Pb are such example of those heavy metals exist in agricultural soils because of misuse of agrochemicals and/or pollution from other sources such as irrigation of raw untreated sewage effluent. In this study, the deteriorating effect of heavy metal polluted irrigation water has been observed leading to the lowest growth and production of tomato plants as well as the highest contents of heavy metals in plant tissue. Such accumulation has been observed earlier in many crops (Nagajyoti et al 2010). In this study, compost application reduced the harmful effect of heavy metals existed in irrigation water. This can be due to changing the physicochemical property of soils and reacting with heavy metals (Bolan et al 2014, Liu et al 2009. The beneficial effect of compost application in reducing the harmful effects of heavy metals has been reported earlier by Huang et al (2016). On the other hand, potassium silicate showed also an ameliorating effect on plant growth and production as revealed from our data. These positive effects are Rana, Eman, Hashim, El-Shinawy, Abd El-Gawad mainly due to the presence of Si. These beneficial effects have been explained on the basis that Si increase plant resistance to some heavy metals such as Cd by inhibiting Cd uptake in roots and the enhancement of light-use-efficiency in leaves (Nwugo and Huerta 2008 a&b). Silicon has been reported to have a beneficial effect on growth and yield for various horticultural plant species such as bean, cucumber (Zhu et al 2004), tomato (Romero-Aranda et al 2006) and Zucchini squash (Savvas et al 2015).
Because of the different mode of action for compost and potassium silicate against heavy metals, the interaction of the two treatments was additive and its combination resulted in enhancing the effect of each other which reflected on higher plant growth and production.

Conclusion
It can be concluded that the treatment with compost and potassium silicate, as well as the interaction between them, reduced the harmful effect of irrigation with untreated industrial wastewater, as well as increased the vegetative growth and yield of the tomato plants under study.


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Effect of Crop Residue and Nitrogen Level in Yield and Yield Attributing Traits of Rice under Rice-Wheat Cropping System

A 3 years (2015, 2016 and 2017) >ield study was carried out at National Wheat Research Program, Bhirahawa, Rupandehi, Nepal to evaluate the influence of crop residues and nitrogen levels on rice. The experiment was conducted in split plot design with three replications. Main plots were two crop residue levels (with crop residue and without crop residue) and sub plots consisted of seven nitrogen levels (0, 25, 50, 75, 100, 125 and 150 kg N ha-1). Combined analysis of three years data revealed that crop residue levels did not differ significantly in terms of grain yield; however, crop residues incorporation increased the rice grain yield slightly. Significant difference was observed with application of different nitrogen levels in grain yield and yield attributing characters viz; tillers m-2, panicle length, number of filled grains per panicle and thousands grain weight. Rice grain yield was found at increasing rate with the increased level of nitrogen @ 150 kg ha-1. Application of nitrogen @ 150 kg ha-1 gave highest grain yield of 4831 kg ha-1 which was found at par with nitrogen @ 125 kg ha-1 with grain yield of 4722 kg ha-1. Crop residues with nitrogen @ 125 kg ha-1 resulted in 216 kg ha-1 higher rice grain yield than residues removed treatment. The overall conclusion drawn from the three years field experiment was an improved crop residue management with 125 kg N ha-1 increased the grain yield of rice.

Introduction
In recent years, use of combined harvester for harvesting and threshing of rice and wheat crop has been increasing in Western Terai region of Nepal. The crop residue left over after harvesting of previous corp by using combined harvester is major concern to manage for planting succeeding crops under rice-wheat cropping system. So, farmers are compelled to burn such huge amount of crop residues. Burning of wheat residues is cost effective and the predominant method of disposal in areas under combined harvesting in the Indo Gangetic Plains (Samra et al., 2003). However, disposal of crop residues by burning is no longer environmentally acceptable and often criticized for accelerating losses of soil organic matter and nutrients, increasing C emissions and reducing soil microbial activity (Kumar and Goh, 2000). Crop residues incorporation can improve soil quality and reduce air pollution on a longterm basis. However, where residues have been soil incorporated, farmers often have concerns for reduced soil fertility from nutrient immobilization and problems for cultivation associated with slow rates of residues decomposition (Cookson et al., 1998). Effective mitigation of these effects depends on developing crop residue management strategies that enhance residues decomposition. Realizing the potential benefits of cereal residues incorporation depends on synchronizing the release of N with the crop demands, while minimizing the risks to nutrient losses (Powlson et al., 1985;Hooker et al., 1982). Where residue has been incorporated before planting the next crop, grain yield was lower than where residues were removed or burned, resulting in N immobilization (Bahrani et al., 2002;Singh et al., 2004). The most influential factor on wheat yield is N fertilization, although the degree of influence is governed principally by weather conditions and residual soil N (Garrido-lestache et al., 2005). Hence, this study was conducted to determine the effect of crop residue and nitrogen level on growth and yield of rice under rice-wheat cropping system.

Materials and methods
The >ield experiment was conducted in three cropping seasons of 2015, 2016 and 2017 at National Wheat Research Program (NWRP), Bhairahawa, Nepal and the field was laid out in split plot design: two crop residue levels (with and without residue) as whole plot and seven nitrogen levels (0, 25, 50, 75, 100, 125 and 150 kg ha -1 ) as sub-plot which were replicated three times. The rice variety was sabitri and transplanted at the spacing of 20 cm × 20 cm. The plot size was 5 m × 3 m. Urea, single super phosphate and muriate of potash were the source of fertilizers used for supplying nitrogen, phosphorus and potash respectively. Full dose of phosphorus (30 kg P2O5 ha -1 ), potassium fertilizers (30 kg K2O ha -1 ), and half dose of nitrogen was applied at the time of land preparation. Whole Crop residues were retained in the field as natural residues after wheat harvesting. The remaining half dose of Nitrogen was top-dressed in two equal splits viz; first at active tillering and second at panicle initiation stage. Before harvest, plant height, panicle length and tillers m -2 of rice were recorded. After harvest, >illed grains per panicle, 1000 grain weight and grain yield were recorded. Average plant height and panicle length were recorded from five sample plants. Average number of filed grains per panicle was counted from five panicles for each treatment. Grain yield was measured from 8 m 2 area. Grain yield was adjusted to 14% moisture content determined according to Yoshida (1981). Ten panicles were randomly collected from each plot to determine 1000 grain weight and number of >illed grains per panicle.

Statistical analysis
Data were analyzed through GENSTAT statistical package and treatment means were compared using least significant difference (LSD) test at P< 0.05.

Effect of crop residue
Results revealed that yield and yield components were significantly affected by nitrogen levels but crop residue had no significant effects on yield and yield components of rice during three cropping seasons (Table 1). Three years combined data analysis showed that incorporation of crop residue slightly increased plant height, panicle length, tillers m -2 , filled grains panicle -1 , 1000 grain weight and grain yield. This suggests that there was positive role of wheat residue in increasing rice yield as compared to without residue condition. The higher yield under residue incorporation might be due to the faster rate of decomposition of wheat straw due to high temperature and longer window between wheat residue incorporation and rice transplanting. Results obtained showed that both rice and wheat residues can be safely incorporated without any detrimental effects on the crops of rice or wheat grown immediately after incorporation. Application of rice residue to wheat typically has a small effect on wheat yields during the short term of 1-3 years (Singh et al., 2008;Singh et al., 2005) but the effect appears in the fourth year with the incorporation of straw (Gupta et al., 2007). Similarly, Khatri et al., (2017) reported that after two year completion of experiment, biological yield of wheat increased significantly in rice crop residue incorporation treatment.

Effect of nitrogen
Plant height, panicle length, tillers m -2 , filled grains panicle -1 , 1000 grain weight and grain yield of rice significantly increased with increased N rates. Grain yield is a function of interplay of various yield components such as number of productive tillers, panicle length, percentage of grain >illing and 1000 grain weight (Hassan et al., 2003). Application of nitrogen @ 150 kg ha -1 resulted in significantly highest grain yield and yield components (Table 1), which was found statistically at par with the application of nitrogen @ 125 kg ha -1 . The increased grain yield with 125 kg N ha -1 might be due to the cumulative effect of the highest number of effective tillers m -2 , 1000 grain weight and >illed grains panicle -1 obtained from the supply of nitrogen for the plants. Similar results were found previously (Singh et al., 2000;Salahuddin et al., 2009). Whereas lowest yield and yield components were obtained from 0 kg N ha -1 treatments.

Year wise comparison of grain yield
In the first year, rice yield was found lowest under no crop residues incorporation and grain yield and yield components were found at increasing rate with cropping season increment (Figure 1), which is similar to (Rieger et al., 2008) for no crop residue treatment. During third cropping season, yield was recorded highest of 4633 kg ha -1 under crop residue incorporation. Short-term effects of cereal residues (wheat straw) incorporation into paddy field include stimulation of CH4 emissions, immobilization of available N, suppression of rice growth, and accumulation of toxic materials (Singh et al., 2005;Singh et al., 2008).

Interaction effect of crop residue and nitrogen level
The interaction between crop residue and nitrogen level treatment were investigated in Figure 2. The grain yield of rice increased by increasing N levels in both without crop residue and with crop residue treatments. However, higher grain yield was obtained in crop residue incorporation treatment. Incorporation of crop residue into the soil with 150 kg N ha -1 gave the highest grain yield in rice of 4884 kg ha -1 which was found at par with nitrogen rate of 125 kg ha -1 (4830 kg ha -1 ). Crop residues with nitrogen @ 125 kg ha -1 resulted in 216 kg ha -1 higher rice grain yield than residues removed treatment. Incorporation of crop residue into soil without N application gave the lowest grain yield of rice. This may be attributed to the soil N imbalance due to the slower decomposition ( Figure 2).

Conclusion
From the results of the present study, it can be concluded that 125 kg N ha -1 along with crop residue incorporation may be used to obtain the highest grain yields of rice under rice-wheat cropping system. Thus, farmers are suggested to incorporate crop residues with nitrogen dose of 125 kg ha -1 into the soil instead of burning; which is environmentally hazardous to human and soil health.


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Acidity Control After Surface Application of Micronized Liming Material in a Typic Distrudept Under No-Tillage

The objected to evaluate the pH and soil base saturation (V) values, during 45 months after, surface application and without incorporation of the liming materials in deeper layers of soil. The experimental design was completely randomized blocks in split-plot with four replications. In the plots were studied the dolomitic limestone (DL) and micronized liming materials (MLM) – granulated micronized calcite (GMC) and dolomite (GMD), and carbonated suspension (CS). In the subplots were studied doses of all the liming materials aiming to increase V to 50, 70 and 90% besides of control treatment. We measured the pH and V values in 0-5, 5-10, 10-20, 20-40 and 40-60 cm soil layers over time (5, 15, 28, 37 and 45 months). The MLM were more efficient to increase and maintain the pH and V, over time, than DL. The doses of MLM aiming V to 50-70% increasing the pH and V of Typic Distrudept. Despite the MLM are more fineness than DL, the effectiveness for soil acidity control in subsoil were inefficient, because theses pH and V increases/maintains occurred just in 0-5 cm layer. However, the MLM present lower gap between the V estimated and obtained, in the 0-5 cm layer, than DL.

INTRODUCTION
Around 40% of the land used for agriculture in the world have their productivity limited by soil acidity [25]. The acidification occurs due to following factors: rainfall higher; basic cations displaced by H + ; basic cations leached; use of nitrogen (N) fertilizer and/or N fixation [12]; oxides-Al and oxides-Fe accumulation; higher aluminum (Al) and manganese (Mn) concentration; release of H + from soil organic; Al and Fe hydrolysis; uptake of cations favoring extrusion of H + [13,14]; and acidity deposition by fossil fuel combustion [16].

HIGHLIGHTS
 The fineness particles of micronized liming do not favor the soil reaction in the 5-60 cm layer.
 The micronized liming increase pH values in the 0-5 cm layer, in an Inceptisol under no-tillage.
 The gap between the soil base saturation estimated and obtained is smaller with micronized liming.
 The liming materials not changed chemical attributes below the point of placement, in an Inceptisol.
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Brazilian Archives of Biology and Technology. Vol.63 no.spe: e20190411, 2020 www.scielo.br/babt Therefore, the soil acidity control is important for keeping of crop yield and reduce of soil fertility loss, mainly in variable charge soils (as Typic Distrudept).
In no-tillage (NT), the soil acidity control occurs with surface application of liming materials, without incorporation. Then, the reaction of the liming material in NT is lower and gradual [5], but can resulting, along the time in amelioration of the soil fertility below the point of placement [18]. The liming materials provide ideals conditions of change in the pH and exchange complex, favoring the crops yield and amelioration of soils attributes [4,6]. Besides, in soils with controlled acidity there is increase of crops yield due to carbon input [20] and this influence the soil reaction [13,14].
Therefore, the understanding of the MLM effects application on soils with variable charge, as Inceptisol, will allow the development of rational strategies for acidity control. We hypotheses that, in soils with high buffering power and variable charge, the MLM applied in the surface and without incorporation can be more efficient than DL concern to increase and keep pH and base saturation (V) values below the point of placement. The objective of this study was to evaluate the pH and V values along of the 45 months as a result from surface application (without incorporation) of liming materials in the soil profile under NT.
The experimental design employed was the randomized complete block in split-plot with four replications. In the plots (384 m 2 ) were studied the dolomitic limestone (DL) and three micronized liming material (MLM) granulated micronized calcite (GMC), granulated micronized dolomite (GMD) and carbonated suspension (CS). In the subplots (96 m 2 ) the treatments were the control (V = 0%) plus three doses of liming material aiming to increase the V to 50, 70 and 90%. The liming requirement was performed by the method of elevation of soil base saturation. The doses are V estimated. The liming materials were applied in total area, on the soil surface, without incorporation, once in jun/2012.
The effective calcium carbonate equivalent (ECC) of DL, GMC, GMD and CS was of 952.0, 962.7, 1006.5 and 770.0 g kg -1 , respectively. The fineness factor (FF) value adopted for the MLM was of 1000 g kg -1 and for the DL was of 942.8 g kg -1 [10]. The GMC and GMD were pelletized lime, the CS was fluid lime (density is 1.89 kg dm -3 ) and DL is powder lime (Figure 1). The doses aiming to increase of V to 50, 70 and 90 % was obtained through of the equations was suggested by current methodology [7]. The doses applied of DL were 3.22, 6.28 and 9.34 Mg ha -1 to increase of V to 50, 70 and 90%, respectively; of GMC were 3.18, 6.21 and 9.23 Mg ha -1 to increase of V to 50, 70 and 90%, respectively; of GMD were 3.05, 5.94 and 8.83 Mg ha -1 to increase of V to 50, 70 and 90%, respectively; of CS were 3.98, 7.76 and 11.54 Mg ha -1 to increase of V to 50, 70 and 90%, respectively.
After The nutrients inputs via fertilizers during the experimental period were 686, 700 and 538 kg ha -1 of N (as urea or as monoammonium phosphate), P2O5 (as single or triple superphosphate, or monoammonium Brazilian Archives of Biology and Technology. Vol.63 no.spe:e20190411, 2020 www.scielo.br/babt phosphate) and K2O (as potassium chloride), respectively. The common agronomic practices used regionally for adequate crops development were employed.
The aleatory soil samples from 0-5, 5-10, 10-20, 20-40 and 40-60 cm were sampled by auger and dutch auger. Twelve simple samples were collected for performed a composite sample, in each layer of the subplot. The soil was sampled in 0 (at the beginning of the experiment), 5 (after the harvest of wheat 2012), 15 (after the harvest of soybean 2012/13), 28 (after the harvest of maize 2013/14), 37 (after the harvest of soybean 2014/15) and 45 (after the harvest of maize 2015/16) months after surface application of liming materials.
These samples were taken to the laboratory, dried in an oven at 40ºC with forced air circulation over a period of 48 hours, ground and sieved in a 2.0 mm mesh sieve. Afterwards, pH, exchangeable-Ca, Mg and K values was performed according to regional methods [21]. The pH value was determined in a 0.01 mol L -1 CaCl2 solution using a 1:2.5 (v:v) soil-to-solution ratio suspension and after was determined H+Al with buffer solution SMP. The exchangeable-Ca and Mg values were determined in a 1.0 mol L -1 potassium chloride (KCl) solution and titration using a standard 0.025 mol L -1 EDTA (Ethylenediaminetetraacetic acid). The exchangeable-K value was determined in Mehlich-1 (0.05 mol L -1 chloride acid (HCl) and 0.0125 mol L -1 sulfuric acid (H2SO4)) solution. The V was calculated by the relationship between the Ca, Mg and Kexchangeable values sum and cation exchange capacity (in pH = 7.0) [12].
Statistical analysis was performed used the computer program SAS Version 9.1.2 [23]. The variance analysis was carried out using the PROC GLM command and Tukey test (p < 0.05) to averages the liming materials (DL, GMC, GMD and CS). The analysis of regression was carried out using the PROC REG command to adjust the dose (to increase V to 50, 70 and 90% besides to the control treatment) of each liming material and least significant difference (LSD) to compare the liming materials. The analysis of profile was carried out using the PROC GLM and REAPEATED command procedures to measure the effects of doses and liming materials over time (0, 5, 15, 28, 37 and 45 months).

RESULTS
( Table 1) There were interactions between liming materials, doses and periods after application for pH and V values. (Figure 2) In the control treatment there was increase of the pH, in the all layers, along the time. However, the application of liming materials resulted in the pH changes just in the 0-5 cm layer than in others layers. In the 0-5 cm layer, all the doses of liming materials increased and remained the pH along the time, when compared with the control treatment.
( Figure 2) In the 0-5 cm layer, the pH increased and remained as from 28 months after application of DL in the dose V to 50 %. Being that, after 45 months, the pH measured was of 5.7 (the biggest pH for DL). Already for MLM, the pH increased and remained as from 5 months of application in the dose V to 50 %. Whereas the highest pH was observed, in the 0-5 cm layer, 28 months after the application of MLM in the dose V to 50 %. These pH values were of 6.2, 5.6 and 6.1 to GMC, GMD and CS, respectively. Already, the pH, after 45 months, were of 5.0, 5.4 and 5.5 to GMC, GMD and CS, respectively.
( Figure 2) The pH increased and remained during 5 up to 34 months of liming materials application in the dose V to 70 % and in the 0-5 cm layer. However, the major increased in the pH were observed for GMC, GMD and CS than for DL. Already, the highest pH observed was 23 and 11 months after application of DL (pH = 6.0) and GMC (pH = 6.4), respectively. And, the pH, after 45 months, were of 5.0 and 4.8 to DL and GMC, respectively. The highest pH was observed after 5 months of GMD and CS application and were of 6.1 and 6.7, respectively, both in the dose V to 70 %. After 45 months, the pH was of 5.4 and 5.5 to DL and GMC, respectively.
( Figure 2) Yet, in the 0-5 cm layer, the pH was increased and remained as from 5 months of MLM application in the dose V to 90%. And, the increased in the pH were observed in the following order GMC < GMD < CS < DL. Yet after 5 months, the highest pH values were of 5.7, 6.9, 6.5 and 6.9 to DL, GMC, GMD and CS, respectively. Already, after 45 months of DL, GMC, GMD and CS application in the dose to 90 %, the pH was of 5.5, 5.9, 5.1 and 5.5, respectively.
( Figure 3) The V in control treatment and in the all layers was increased along the time. However, after the application of liming material doses, the more variations in the V occurred in the 0-5 cm layer than 5-60 cm layer. In the 0-5 cm layer, the V increased and remained of 23 up to 45 months of DL application in the dose to 50 %. Besides, the V obtained after 45 months after application of DL was of 44 % more than V estimated. Yet, in the 0-5 cm layer, the V increased and remained as from 5 months after application of MLM in the dose to 50 %. Being that, the highest V in the dose to 50 % was observed 28 months after application of GMC (V obtained was 58 % more than V estimated) and after 34 months of GMD and CS application (V Brazilian Archives of Biology and Technology. Vol.63 no.spe: e20190411, 2020 www.scielo.br/babt obtained was of 38 and 52 % more than V estimated, respectively). Already, the V obtained, after 45 months, by GMC was 42 % and by GMD and CS was 30 % more than the V estimated 50 %.
( Figure 3) The V, in the 0-5 cm layer, increased and remained of 5 up to 34 months after application of DL and GMC in the dose V to 70%. Already, the highest V obtained was observed 28 months after the application of DL and GMC in the order of 2 and 14 % more than V estimated, respectively. Besides, the V obtained, after 45 months and in the 0-5 cm layer, by DL and GMC, for both, it was reached 82 % of the V estimated. And, the only treatment that the V obtained equal the estimated of 70 %, was the GMC after 34 months. Still in the 0-5 cm layer, the V increased and remained as from 5 months of GMD and CS application in the dose V to 70 %. The highest V obtained was observed 23 and 34 months after application of GMD and CS, in the order of 14 % more than V estimated, for both. Already, the V obtained, after 45 months, by GMD and CS, was reached 97 % of the V estimated of 70 %. (Figure 3) The V, in the 0-5 cm layer, increased and remained as from 5 months after application of all liming materials in the dose V to 90 %. The highest V obtained, was observed 34 months of DL and GMD application, both reached 88 % of V estimated. Already, the V obtained, after 45 months, by DL and GMD reached 73 and 65 % of V estimated, respectively. Besides, the highest V was observed 28 months after application of GMC and CS, both reached 93 % of V estimated. Already, the V obtained, after 45 months and in the 0-5 cm layer, by GMC and CS, both reached 82 % of the V estimated of 90 %.
( Figure 4) The pH was more influenced by doses of liming material in the 0-5 cm layer. The linear increases the pH was observed of 5 up to 34 months of application of liming materials, except for 28 months. Thus, the higher pH was observed in the dose V to 90 % after 5 months of DL and GMC application; after 11 months of all liming materials application, after 28 months of GMC, GMD and CS application; and after 34 months of DL and GMC application. The increase pH occurred 28 months after application of DL in the doses V to 50-70 %.
( Figure 5) The V was influenced by doses of liming material up to 20 cm depth than 20-60 cm. In the 0-5 cm layer, higher V was observed in the dose V to 90 % after 5, 23, 34 and 45 months of DL, GMD, GMC and CS application, respectively. In the 0-5 cm layer and in the dose V to 50-70 %, higher V was observed after 5 months of GMC, GMD and CS application; after 11 months of all liming material application; after 28 months of DL, GMC and CS application; after 34 months of CS application; and after 45 months of GMD application. Already, in the 5-10 cm layer, higher V was observed in the dose V to 50-70 % after 5 and 28 months of DL application. And, in the 10-20 cm layer, higher V was observed in the dose V to 50-70 % after 11 months of CS application. Besides, after 5 months of GMC application, in the 10-20 cm layer, higher V value was observed in the dose V to 90%.

DISCUSSION
The MLM (GMC, GMD and CS) increased the pH and V as from 5 months of your application while the DL increased as from 28 months of your application, in all doses applied (Figure 2 to 5). This fact shows that, the MLM presented faster reaction and higher responsiveness than DL, in soil with high buffering power and variable charge under NT, under field conditions. Similar results also were observed in soils under controlled conditions, where the MLM are able to increase the pH and the V in the first month of application [10,11]. Therefore, in short-term, the MLM can be strategic more efficient to soil acidity control and then, reduce loss of crops yield. These results, also point to differences of Typic Distrudept with others soils, as the Oxisol under NT, whom the DL (with 840 g kg -1 of ECC) reacted after 48 months of application [15]. And also, as the Typic Hapludox under NT, whom the DL (with 710 g kg -1 of ECC) reacted after 12 months of application [9]. However, the highest pH and V observed along the time, was only 28 months of MLM application and 45 months of DL application (Figure 2 to 5). Begging that, after 28 months of MLM application there was slight decrease in the pH and V, until 45 months (Figure 2 to 5). Hence, we concluded that, the MLM presented lower residual effect than DL.
All the major increased in the pH and V occurred more in the 0-5 cm layer than 5-60 cm layer, for all the liming materials and doses applied (Figure 2 to 5). This fact suggests that, the reaction of liming material applied in a Typic Distrudept (high buffering power and variable charge) under NT, occur only in the surface layer and not below the point of placement. Even though the MLM present fineness particles much lower than DL (MLM of 1.91 to 6.58 μm and DL of 269 μm) this fact not favored your reaction in deep layers (5-60 cm). In others soils, as the Haplic Umbisol, the reaction of DL (878 g kg -1 of ECC) with fineness particle (< 0.25 μm) was much faster than with DL of coarsest particle (2-4 mm), during 36 months and 0-20 cm layer [1]. Already, the absence of liming material effect in 5-60 cm layer can be attributed the following factors that (i) the soil presented higher potential acidity [22], (ii) the dissolution/dissociation as far from acidity zone and reaction time [26]; (ii) subsoil pH is adequate [12], (iii) the neutralization of aluminum (Al 3+ ) and of hydrogen (H + ) on soil functional groups is limited [26], (iv) the soil buffering power is strongly by Al and iron (Fe) oxides [8], (v) lower water infiltration via macroporos [2] and (vi) lower adsorption/desorption of cations by organic anions soluble [17].
Also, in Typic Distrudept, can occur the reprecipitation of carbonate due to decrease of partial pressure of CO2 (by microbial activity) and increase of solute concentration, limiting the acidity control only in 0-5 cm layer [3]. This layer may have occurred increase of microbial activity by liming material, favoring the organic matter mineralization and the decrease of the carbon stocks (less CO2) [20].
The majority of Brazil, especially in the Paraná State, the liming requirement by V is calculated for 0-20 cm layer of soil in conventional tillage, but this also is used for NT [24]. However, in NT, with surface application and without incorporation of liming materials, the V estimated is not reached, across the 0-20 cm layer. But, only, in some part of 0-20 cm layers, as the 0-5 cm layer. All the major increased in the V occurred more in the 0-5 cm layer than 5-60 cm layer, for all the liming materials and doses applied (Figures 3 and 5). For the MLM in the dose of V to 50 %, the highest V observed along the time as much as the V after 45 months of application, it was of V obtained > V estimated, approximately 40 % greater. Already, in the dose of V to 70 %, the highest V observed, along the time, it was of V obtained > V estimated, approximately 10 % greater. Yet, in the dose of V to 70 %, the V after 45 months of application was of V obtained < V estimated, approximately 20 % smaller. For the MLM in the dose of V to 90 %, the highest V observed along the time as much as the V after 45 months of application, was of V obtained < V estimated, approximately 80 % smaller. This fact was stronger in MLM than DL. Begging that, among the MLM, the GMC was the only where V obtained equal the V estimated of 70 % (Figures 3 and 5). Therefore, the gap between the V estimated and measured is smaller in soil treatment with MLM than DL. This way, the MLM present greater assurance that what was applied really reacted. But, when the V obtained is higher than V estimated, can be occur a problem with the overliming in the pH in surface layer. This fact was common with surface application of CS in all the doses (Figures 3 and 5). The overliming in the pH may cause, in short-time, micronutrient unavailability [12]. Therefore, in short-term, the MLM can be strategic more efficient to increase and remain the V and then, reduce loss of crops yield.
As from results obtained, the doses of all liming materials more recommended is aiming V between 50 and 70 % for a Typic Distrudept under NT (Figure 2 to 5). Therefore, the dose aiming V over 70 % and below 50 % will not occur interesting results. In these doses and among the MLM, the GMC and CS were more efficient than others liming materials studied with regard to increase of pH and V. However, for Typic Distrudept the dose aiming V to 70 %, commonly used in Oxisol [6], may not be appropriate.

CONCLUSION
The micronized liming material increase and keeps pH and V values, only in the 0-5 cm layer, during 5 up to 45 months after surface application, without incorporation, in a Typic Distrudept under no-tillage. However, the fineness particles of micronized liming material do not favor the soil reaction in the 5-60 cm layer. The doses of all liming materials more recommended is aiming base saturation between 50 and 70 % for a Typic Distrudept. The gap between the soil base saturation estimated and obtained is smaller in soil treatment with micronized liming material than dolomitic lime, manly in the 0-5 cm layer. Therefore, the liming materials, applied superficially and without incorporation, were not efficient to increase the pH and V values below the point of placement, in soils high buffering power and variable charge, as the Typic Distrudept.