Elucidating the Chemical Properties and Potential Applications of Wood vinegars Produced by Controlled Thermal Treatments

Wood vinegar (WV) has been used for ages as fertilizer and antimicrobial agent, but its impacts on ecosystems are not well understood. Our objective was to investigate potential uses for WVs made from biomass resources and evaluate conditions for possible agronomic, chemical/pharmaco-chemical purposes and potential impact on the environment. We studied four woods vinegars made by slow pyrolysis using: Cambara (WVCam), Eucaliptus (WVEuc) at 350oC for 72 h, Nim (WVNim) and Nim mixed with fresh Nim leaves and branches, soaked into WVNim (WVMix) at 120oC for 40 h. WVs from WVMix, WVEuc and WVCam demonstrated significant potential chemical products, while WVNim for pharmaceutical purposes. WVs can be used to partially substitute fertilizers and chemicals, most especially WVMix. Soil bacteria tests suggest a concentration of 0.8% or less for WVs application in soils. No WVs tested presented risk for environmental and human health due to absence of carcinogenic poly-aromatic hydrocarbons (PAHs). Keywords— biomass, controlled pyrolysis, pyroligneous acid.


I. INTRODUCTION
Large amounts of agricultural residues, such as sawdust, sugarcane straw and rice husks are produced worldwide annually, mainly in developing countries like Brazil. Current disposal methods for these residues are not usually adequate and have been responsible for a wide environmental impact 1 . Thermal treatments, such as pyrolysis, could be an alternative for treatment of these residues, converting biomass into bio-products with higher added value 2-4 , such as wood vinegar 5 . Traditional vinegar production utilizes sugar crops that compete with food as feedstock. There are newer processing technologies, via thermal means to produce similar wood vinegar products.
Thermal conversion technologies, such as controlled pyrolysis may be optimized to generate specific ranges of organic liquids through the condensation of the gaseous products during the thermal treatment. The ranges of chemicals and their properties vary according to operational temperature and exposure time (Capareda, 2013).
This study investigates the chemical characteristics of WVs made using different biomass resources and using controlled thermal conversion processes and conditions to generate highly valuable chemicals or pharmaco-chemicals including agronomic chemical substitutes. The study also identifies risks to the environment through its agronomic use. The overall goal is to find high value chemicals and products from materials that are onsidered wastes. The specific objectives are as follows: a. Evaluate wood vinegar for its agronomic fertilizer or chemical partially substitutes, b. Identify wood vinegar whose chemical components are considered beneficial and suitable high value chemical products, such as potential for pharmaco-chemicals, and c. Evaluate other properties such as various polyaromatic hydrocarbons (PAHs) that are potential risk for environmental and human health Review of Related Literature

Thermal Conversion Processes
Wood vinegar, also called pyroligneous acid or wood distillate, is the water soluble organic fraction of the liquid that is produced during the pyrolysis of biomass 6 , many times treated as pyrolysis residue. The pyroligneous acid is brown, with strong smoke smell

Beneficial Antimicrobial Properties and Soil Microbiota Improvements
Depending on the dosage, wood vinegar exhibits a high degree of antimicrobial activity against various microorganisms 5,26 , such as Legionella pneumophila 27 , Escherichia coli and Corynebacterium agropyri 9 . Wood vinegar also has antioxidant activity 5,18,26,28 .
In addition, wood vinegar can be developed into useful sterile products for medical, aquaculture and livestock breeding applications 5 . It has shown benefits as animal diet or feed ingredient [34][35][36] . Finally, it has numerous dermatological purposes in humans 37 and used for smoked food products 38 .
Wood vinegar's impact in the environment can be predicted by soil microbiota, which is easily altered by soil changes 39,40 . WVs can also be used as bio-indicators of high sensitivity that reflect environmental conditions 41 . Among these bio-indicators, microorganisms functional groups density, such as bacteria, is one of the most effective indicator 42 . The quantification of colony forming units (CFU) has been used to estimate the variation in bacterial density in ecotoxicology studies 43 .
The WVs were produced in hot-tail kilns on a small scale, originally for agronomic purposes. WVcam and WVeuc were thermally treated into sealed furnace, to prevent air from entering, at around 350 ºC for 72 h. WVnim and WVmix were thermally treated into sealed furnace, at around 120ºC for 40 h. All of these thermal processes are in batch mode. WVs were obtained by condensing the pyrolysis vapors in the chimney. The pyrolysis liquids were left to sit until separated into three layers: at the top is a light oil; on the bottom is the sticky wood tar and in between lies the organic portion with the majority of refined wood vinegar. The unknown chemical composition of WV from different plants can be estimated by a comparison with the mass spectral libraries available on gas chromatography mass spectrometry (GC-MS) database 44,45 . This technique is an easy, cost-effective and sensitive method to identify and quantify the chemical compositions of natural products.

Wood Vinegars Chemical Characterization
The WVs were treated with phosphoric acid 1% to measured total carbon (TC), total inorganic carbon (TIC), total organic carbon (TOC), using TOC-Analyzer (Elementar Analysersystem GmbH). Additional WVs inorganic elemental analysis was performed 46 to determine the total Ca, Mg, Cu, Mn, K and Zn, by extraction with nitric-percloric acid solution and determination by atomic absorption spectrometry. The total P was analyzed by colorimetry. WVs pH titration curves were measured by incrementally adding 0.5 ml of 0.1 N NaOH to a 50 ml of WV. After each increment, the solution was stirred until the pH stabilized.

Organic Chemical Analysis
The organic compounds were analyzed by GC-MS, using Agilent 7890 GC apparatus (Wilmington, DE. USA) equipped with a 5975C MS detector and 7683B automatic liquid sampler with a capillary column: HP-5MS 5%-phenyl-95%-dimethylpolysiloxane (30 m, 0.25 mm i.d., film thickness 0.25 μm) (J&W Scientific, Folsom, CA. USA). The oven temperature was programmed to start at 150°C (1 min), increase to 210°C at 15°C min -1 (10 min), and then increase by 5°C min -1 to 310°C for 1 min. The injector temperature was set at 270°C in splitless mode, when 1 μl of sample was injected. The mass selective detector temperatures of the ionization chamber and MS Quad were set at 230 and 150°C, respectively. The electron ionization energy was 70 eV, and the mass range was m/z 30-500 (amu). Results (chromatographic peaks) from WV samples were used to identify the organic compounds by comparison with the experimental m/z values of the compounds from NIST data base. Only those compounds with match above 70% were considered. The relative percentage was calculated by the relation between the peak area of the compound identified and the sum of the peak areas of all majority compounds in the chromatogram.

Poly-aromatic Hydrocarbon (PAH) Analysis
The total PAH analysis was measured in triplicate. About 4 g of s ample with 20 ml of toluene were agitated for 30 min at 50% on an Kline agitator NT151 (Novatecnica, SP. BRA). Then, the sample was sonicated for 30 min in an ultrasonic bath (Quimis, SP. BRA). After the extraction, the sample was centrifuged at 4500 rpm for 5 minutes on a Sigma 3-16KL centrifuge (Sigma Laborzentrifugen GmbH, DE). The supernatant was collected in a glass tube. The solid residue was extracted twice. All the supernatant were cleaned up on a silica gel column (3 cm) and then eluted with heptane. The solvent was evaporated under vacuum at 60°C until a volume of 1 ml is reached. The samples were analyzed using the same apparatus used for the organic compounds analysis. However, the oven temperature was programmed to start at 50 °C (2 min), increase to 150°C at 10°C min -1 , then increased to 280°C at 5°C min -1 and held for 9 min. It is then ramped up at a rate of 40°C min -1 to 310°C. It is held at this temperature for 8 minutes.
The majority and main chemical and/or pharmacy usefulness of the organic compounds found by GC-MS were investigated by the SciFinder ® (scifindercas.ez103.periodicos.capes.gov.br) and ChemSpider (http://www.chemspider.com) databases.

Wood Vinegar Antibacterial Tests
We assessed the antibacterial effects of wood vinegar in four types of soils: (1) crop: integrated system with a sequence of soybean and maize, this last one intercropped with pasture; (2) pasture: Urochloa brizantha cv. Marandu (L); (3) planted forest: Eucalyptus urophylla x Eucalyptus grandis hybrid and (4) native forest: classified as "Evergreen Seasonal Forest" soil.
Soil samples were collected in 2016 during the dry season from 0 to 10 cm using a sterile Dutch Auger. The sample was composed of twenty samples per plot.
The antibacterial study was adapted from 47. Dilution was performed starting from 10 g of soil with natural moisture in 90 ml sterile phosphate buffered saline (PBS: 1.44 g ml -1 Na2HPO4, 0.24 g ml -1 KH2PO4; 0.20 g ml -1 KCl, 8.00 g ml -1 of NaCl and pH 7.4).
The dilution of about 10 -3 was plated onto 10% Trypticase Soy Agar plates (TSA-Merk) with WVCam, WVEuc, WVMix and WVNim at 0.8 and 100%. The antibacterial tests were performed with five replicates and incubated for 7 days at 28°C and related with the biological oxygen demand (BOD). The quantification of total bacteria was carried out by Colony Forming Units (CFU).

Statistical Analysis
The WVs chemical properties and antibacterial tests were compared among various treatments by performing Tukey´s test. The p-value of less than 0.05 was considered significant. Statistica Soft Version 10 was used in the statistical analysis of all the data generated from these studies.

International Journal of Advanced Engineering Research and Science (IJAERS)
[  Table 1 shows the pH, complete elemental analysis important for fertilizer and nutrients as well as total organic and inorganic carbon. These properties will represent the wood vinegar's capability to partially substitute the use of chemical fertilizers. Of fertilizer importance in the concentrations of macro-nutrients (phosphorus and potassium), two of the basic inorganic chemical fertilizer component.

III. RESULTS AND DISCUSSION a. Wood Vinegar Chemical Characteristics 3.1 Chemical Fertilizer Potential
The other elemental components are also vital nutrient needs of plants. Among the WVs samples tested, WVEuc and WVMix showed potential fertilizer P and K substitute having the highest concentrations. WVEuc had reported P and K concentrations of 20.3 and 69.8 mg/ℓ, respectively. WVMix had 23.4 mg/ ℓ of P and similar 69.8 mg/ ℓ of elemental K. WVMix, contained the highest concentrations of Mg and Ca with concentrations of 7.84 and 122.71 mg/ ℓ, respectively. Hence, the elemental components of WVMix is an excellent potential supplement to other high-quality fertilizers inputs.
For example, it has been reported that WV has traditionally been applied in the growing of tea. Report showed that it increases the yield by three times the level of useable phosphoric acid. Plants roots secrete organic acids which dissolve and absorb phosphoric acid in the soil and it is thought that organic acids in WVs have the same effect 48 . Table 1 also showed the total organic carbon (TOC) data showing high values for WVCam and WVEuc (258.6 -275.6 mg/ℓ). High TOC levels indicate great material accessibility with microorganisms. This property may play the role of stimulators (or sometimes inhibitors) of plant growth and plant development because of its high carbonaceous components. This is especially true for lowmolecular weight organic compounds 49 .
The use of WV as fertilizer is gaining importance in many crops. It cannot act as substitute for soil fertilization, but it can supplement a sound soil fertilization program 8 . However, its utilization is generally based on local knowledge and not scientific research 50 , which poses certain environmental risk for soil and water contamination, toxicity and impact on soil microbiota.
It is important to note that, because of low pH values and the high TOC values, the WVs described above will have considerable organic load. Hence, none of WVs should be disposed to the environment indiscriminately. Proper treatment or identification of recommended application rate as pesticide or chemical fertilizer in soil agriculture must be known. The beneficial effect of WVs in soil or plant are directly related to the dosage used 9,32,51 .
The pH of WVs can be correlated inversely with titratable acidity 38 . Unlike strong acids that are fully dissociated, WV acids are only partially ionized. It could be attributed to differences in its buffering capacity, Fig  1. WVNim and WVMix contents have narrower working ranges for buffering than WVcam and WVeuc do. It can positively be correlated with carbonyl and carboxyl groups content on WVs, Fig 2. In addition to potential chemical fertilizer and substitutes, WVs could be recommended as co-solvents for agrochemicals. The majority of agrochemicals are dissolved most easily and their effectiveness most enhanced if they are mixed with organic acid solutions 48 . The pH is a critical factor in the effectiveness of many pesticides and growth regulators, some pesticides particularly carbamate and organophosphate insecticides are broken down when combined with high pH water.
The rate and severity of the reaction is determined by how susceptible the pesticide is to hydrolysis 52 . A pH of 5.5 to 6.5 is the ideal one for mixing most pesticides. This is the primary reason that most commercial pesticides indicate in their directions the addition of a buffering or acidifying agent into the spray tank 53 .
WV can act as a pH buffer and an acidifying agent for mixing pesticides that require low pH. This buffering and acidifying behavior will allow the plant to absorb nutrients more readily 54 . Such mixture between WV and pesticides no doubt improve cost and can even increase the effectiveness of the pesticide 55,56 .

Phenolic Compounds
WVs contains around 68 kinds of organic compounds belonging to 4 main groups , Fig 2. These groups include carbonyl, carboxyl, phenolics, silicates and others. The chemical composition of WVs are quite variable as shown in Table 2. The primary reason is that the chemical characteristics of products are so much dependent on the wood source and the thermal treatment processes. These include temperature, retention time, and even particle sizes or granulometry 57 .
Phenols are the primary chemical grouping found in the WV liquid product. The relative phenolic composition ranking and of increasing order is as follows: WVEuc<WVCam<WVNim<WVMix.
Some phenolic compounds shown in Table 2 are as follows: 1. Guaiacol is used as an antimicrobial agent 58,59 , the compound reduces gastric erosions induced by classic anti-inflammatory drugs (ibuprofen) 60 . It is also considered an antidiarrheal agent 61 and an antioxidant 5 ; and antiinflammatory substance 67 ; 4. Kaempferol is considered an antioxidant and an anti-inflammatory agent 68 . It has been used as agent for the treatment of Alzheime's disease 69 , and an intervertebral disc degeneration treatment compound 70 . Further, it has been used in Leishmanial chemotherapy 71 and as cancer therapeutic agent 72 and, 5. Syringol is used as an antioxidant 5,73 . As previously cited, phenolic compounds have antibacterial properties 74,75 , including membranedisrupting agents 76,77 . This chemical characteristic justifies the higher numbers of pharmaco-chemical compounds in this group.
Note that while those compounds are found in the liquid product, some separation techniques must be developed to recover them in pure form. This should be the subject of future research.

Carbonyl Groups
The carbonyl group appears in WVCam, WVEuc, WVNim, but was not found in WVMix. This results made it clear that the chemical output product of a certain biomass resource changes and can be affected by the various pyrolysis processes used, including how the material was processed (e.g. a case for Nim leaves and fine grinded branches effusion on WVNim).
The main composts derived from the carbonyl group, widely used in the chemical industry are as follows: 1. The compound 5-methylfurfural is used as a chemical for the synthesis of fine chemicals manufacture 78,79 . It is also used as a potential candidate for therapy of sickle cell disease 80 ; 2. Acetol is an important intermediate product used to produce polyols and acrolein 81 . It is being widely used as a reduced dye in the textile industry 82 . Moreover, it is used as a skin tanning agent in the cosmetic industry and to give the unique aroma and flavor to foods 83 ; 3. Furfural is a chemical used for the synthesis of fine chemicals manufacture 84 . It is also commonly used as sustainable intermediate for the preparation of a great variety of chemicals, pharmaceuticals and furan-based polymers 85 , and, 4. Cyclotene only appeared in WVEuc and is reportedly used as anti-inflammatory agent 86,87 . Likewise, techniques must be developed in the future for effective separation, isolation and purification of these valuable chemicals.

Carboxyl Groups
The main chemical compound in the carboxyl group is acetic acid. The percentage composition ranged from 8.1 to 20.74%. This is the major chemical compound in all WVs studied. This is perhaps the primary for the WVs low pH values and buffering capacity as demonstrated in Table 1 and Fig 1. Acetic acid has been widely used in chemical industries, with wide range of uses, such as the production of polymers derived from vinyl acetate production of purified terephthalic acid. This compound is used to produce polyethylene terephthalate (PET). Acetic acid may be precursor or as raw material for acetic anhydride and acetate esters production and are widely used as solvents 88,89 . These upgraded acids from acetic acid are commonly used in the food industry as acidity regulator 90 .
The primary industrial method for production of acetic acid is the carbonylation of methanol. The current method for commercial production of this chemical consumes fossil fuels 91 . The production of these organic acid chemicals by pyrolysis of wood can be a promising approach to obtain renewable chemicals or precursors.
Likewise, there is an additional challenge to efficiently separate the organic acids from a mix of multiple diluted components. This necessary step will improve the purity of other valuable organic acids with similar properties with acetic acids 92 . The chemical industry widely uses the distillation process to separate and purify chemical compounds based on the differences in boiling temperature of the components 93,94 . Other novel and cost-effective methods of separation must be studied. This should be the subject of future research.

Poly-aromatic Hydrocarbon (PAH) Group
Shown in Table 3 are various concentrations of PAHs found in WVs. The total parent PAH concentrations ranged from 44.77 to 357.12 ng g -1 . One agronomic purpose of WV is to partially substitute fertilizers and pesticides. Contamination rates must be considered however as it may potentially cause cancer to humans once such chemical is spread throughout the surface of the soil. Some PAHs have lipophilic nature, that is, they are easily dissolved and transported by human cell membranes 95 .
According to one study 96 , the Danish PAH Soil Quality Criteria (SQC) limit in soil is 1000 ng g −1 . This

International Journal of Advanced Engineering Research and Science (IJAERS)
[ Vol -6, Issue-5, May-2019]   https://dx.doi.org/10.22161/ijaers.6.5.71  ISSN: 2349-6495(P) | 2456-1908(O) was set for the protection of the environmental and human health. This concentration is much higher than the concentrations found in WVs studied. While some PAHs compounds were found, those analyzed in WVs had no potential carcinogenic risk. The major seven carcinogenic compounds reported but were not found in this study are as follows: Benzo(a)anthracene, Chrysene, Benzo(b) fluoranthene, Benzo(k) fluoranthene, Benzo(a) pyrene, Indeno(1.2.3-cd) anthracene, Dibenz(a,h) anthracene 97 . For example, naphthalene is a common compound found in most household mothballs. The other, acenaphthylene, is a compound found in coal tar and used as ligand in some organometallic processes. Several factors, such as biomass and temperature, affect yields and distribution of PAHs formed. Thermal PAH formation can occur over a wide range of temperatures. At low temperatures, the compound distribution is governed by thermal stability and the most stable isomers are formed, while at high temperatures, PAHs of higher formation enthalpy can be generated 98 .

b. Wood Vinegar Antibacterial Effects
Plating of soil onto treated plates showed that total cultured bacterial densities were significantly different among types and concentrations of WVs, Table 4.
The soil's bacteria were tolerant to WVs assessed at 0.8% for soils cultivated with crop, pasture and native forest. The same was not observed to the soil cultivated with Eucalyptus. This suggested a WV concentration for agronomic purposes of 0.8% or less.
Soil bacterial growth was mightily affected for WVs at the 100% rate, except for WVmix in soils cultivated with crop and pasture. WVs have anti-microbial compounds that affect directly the bacterial growth as well as some organic acids and phenolic substances.
WVEuc and WVCam had the highest toxicity index for soil bacteria. This behavior is due to the higher concentration of anti-microbial compounds in these two WVs. Hence, one must act with due diligence on the use of these compounds as crop soil enhancement for improved and better beneficial microbial growth. Of course, the opposite is also true, that is, WVs may be used to minimize harmful microbiota proliferation in some soils.

IV. CONCLUSION
This study has shown that the chemical composition of wood vinegars (WVs) varied with feedstock, pyrolysis temperature and exposure time. Numerous valuable chemicals were found in the liquid condensate after immediate cooling of the gaseous products produced.
This study has shown that some wood vinegars have high amounts of macronutrients P K, Mg (WVEuc and WVMix) and Ca (WVMix), that are partial substitute of chemical fertilizers. The high amounts of TOC in some WVs (WVCam and WVEuc) showed potential as good material substrate for microbial growth that also act stimulant for plant growth.
The main constituents of the organic portion of WVs were found to include four major compounds groups in order of increasing amounts as follows: (a) phenols, (b) carboxyl groups, (c) carbonyl groups and (d) silicates. Some phenol compounds found were shown to have antimicrobial properties, as antioxidants and antiinflammatory compounds and as food additives. The carbonyl groups showed importance in the chemical industries as pre-cursors to high value chemicals in the pharmaceutical industry. On the carboxyl group, the primary and most common organic acid compound found was acetic acid, another important chemical industry ingredient that may be used to replacer fossil fuel based chemicals.
This study has further shown that WVs have potential uses as chemicals (on syntheses, adhesives and food flavoring), pharmacological (cancer treatment, antioxidant, anti-inflammatory agents) and agronomic purposes (as a partial substitute of chemical fertilizers and pesticides), with highlight to WVNim for both pharmacy and agronomic purposes.