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Current Trends on Biotechnology & Microbiology

Short Communication(ISSN: 2641-6875)

Nutritional and Protein Characterization of the Leachate Produced in the São Paulo Zoo Composting Unit Volume 1 - Issue 5

PFM Eugênio1, PL Ramos2, JV Alberice1, ANM de Araújo3, NA Assunção3, L Juliano4, ENVM Carrilho5, E Carrilho1 and LTD Cappelini1,4*

  • 1Instituto de Química de São Carlos, Universidade de São Paulo, São Paulo, Brazil
  • 2Departamento de Pesquisas Aplicadas, Fundação Parque Zoológico de São Paulo, São Paulo, Brazil
  • 3Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Brazil
  • 4Instituto de Farmacologia e Biologia Molecular, Universidade Federal de São Paulo, Brazil
  • 5Centro de Ciências Agrárias, Universidade Federal de São Carlos, Brazil

Received:March 14, 2020;   Published: June 18, 2020

*Corresponding author: LTD Cappelini, Instituto de Química de São Carlos and Instituto de Farmacologia e Biologia Molecular, Universidade Federal de São Paulo, Brazil

DOI: 10.32474/CTBM.2020.01.000124

Abstract PDF

Keywords:Leachate; organic fertilizer; proteins; waste


In recent years the, Foundation Zoological Park of São Paulo (FZPSP) implemented and put into operation the Organic Composting Production Unit (OCPU). The main goal of this action was to give a new destination to the organic waste collected daily in the park since before that sustainable attitude, this waste had the same fate as the usual household waste: landfills. Currently, the composting produced in OCPU serves as fertilizer for the park gardens and food cultivation in the Rural Production Unit (UPR), where much of the food served to the animals in the Zoo (ZOO) and Zoo Safari comes from Bernal et al [1].
Leachate is a liquid residue of high organic contents and strong color produced during the percolation of water through the composted organic waste. During the passage of this water, various chemical, physico- chemical and fermentation processes occur concurrently and, for this reason, various organic and inorganic compounds, besides that microorganism and its metabolites can be transferred to the leachate. Thus, leachate from different sources can be considered as a distinct matrix with specific characteristics.
Currently, what is known of leachate are those generated in landfills that have a high toxic potential. But it can be considered that the one produced during the composting in FZPSP is different, due to the nature of the organic material and also the water used to cool the composters, which runs several times by the composting systems. Therefore, because there is very few information on leachates from different sources other than municipal landfills, by the differentiated characteristics of the process and also of the materials used during composting at OCPU there was interest in studying this particular leachate, the potential of this matrix in bringing nutritional and proteomic information.

Material and Methods

Digestion of the leachate sample

The materials used for composting are shredded tree branches and leaves from the surrounding Atlantic rain forest, manure, waste food and carcasses from small and large animals (previously reduced in the room necropsy).
The leachate sample collected for the experiments circulated by the cooling system of the production of composting for a period of 40 days. After this period, about 5 L of this leachate was collected in an amber bottle and stored at 4˚C.

Determination of macro and micronutrients

The digestion of the leachate for analysis of macro and micronutrients was done according to the literature Carrilho et al. [2]. After digesting the sample, the nitrogen was analyzed according to the specifications of the spectrophotometer protocol HACH, DR 6000 (Loveland, CO, USA).The nutrients As, Cr, Pb, Cd, Ca, K, Mg, Mn, Cu, Zn, P, Fe, Na, and Al were determined inductively coupled plasma optical emission simultaneous spectrometer with radial view ICP OES VISTA RX (Varian - now part of Agilent Company - Mulgrave, Australia) was used for elements determination according to the conditions cited in literature Carrilho et al. [2].

Protein extraction and analysis

The proteins from leachate were extracted using the method outlined by Wang and colleagues Wang et al. [3].Digested peptides were subjected to analysis by nano-LC/MS-MS using a nano-LC system (EASY-NLC II, Thermo Scientific) coupled online to a hybrid ion trap linear-Orbitrap (LTQ Orbitrap Velos, Thermo Scientific) mass spectrometer, through a nanospray source Nano-Flex II nanospray ion (Thermo Scientific). The mobile phases used were: A) 0.1% formic acid in water and B) 0.1% formic acid in ACN. The pre-column used was (C18, 100 μm ID x 2 cm, Thermo Scientific) and C18 column (10 cm × 75 μm ID, 3 μm, 120 Å, Thermo Scientific). The gradient used was: 5% B isocratic, 0-5 min; 5% - 35% B, 5-65 min; 35 - 90% B, 65-80 min, 5% B isocratic, 80-90 min. The total analysis time, from column equilibration to the analysis, was approximately 105 min. All LC/MS-MS data were acquired using X Calibur software, version 2.0.7 (Thermo Fisher Scientific). LC-MS data files (MS2 centroided) were used for database searching with MASCOT (Matrix Science, version


The results of N present in the FZPSP leachate can be seen in (Table 1), together with published data from other leachates. Currently, there aren’t studies in the literature reporting amounts of nutrients in leachates with the same characteristics of those produced by FZPSP. There are only studies on leachates from landfills, piggeries, poultry farms, among others.

Table 1: Macro and micronutrients analysis of the FZPSP leachate.


*n = 4; ** Limit of Detection. The standard deviation of all means of the samples analyzed was below 7%.

The nutrients found in FZPSP leachate can be observed in (Table 1). The use of leachate as an adjunct source of nutrients to the crop occurs mainly to reduce costs in agriculture. However, a preliminary analysis of this material is necessary as there may be excess of certain nutrients that cause damage to the soil and crops.

The results obtained from the shotgun of the leachate proteins returned results with a low score, which was expected since the leachate is a waste from composting and has much interference that make it difficult to extract and identify the proteins. Therefore, few proteins could be identified. Only those with a score equal to or greater than 30% (acceptable value for the shotgun technique reference[1] were considered. Thus, 16 proteins were identified, all belonging to bacterial genera as described below: MEMO1 family protein APE_1771 (Aeropyrumpernix); Uncharacterized protein AF_1654 (Archaeoglobusfulgidus); Protein translation factor SUI1 homolog (Cenarchaeumsymbiosum); tRNA-guanine(15) transglycosylase(Methanobrevibactersmithii); Phosphoenolpyruvate guanylyltransferase and L-fuculose phosphate aldolase (Methanococcusaeolicus); L- lysine 2,3-aminomutase (Methanococcusmaripaludis); Probable L-aspartate dehydrogenase (MethanospirillumhungateiJF-1); 50S ribosomal protein L1 (Pyrobaculumislandicum); Adenosylhomocysteinase(Saccharolobussolfataricus); DNA double-strand break repair Rad50 ATPase (Saccharolobussolfataricus); S- adenosylmethionine decarboxylase proenzyme (Sulfolobusacidocaldarius); Phosphoribosylaminoimidazole- succinocarboxamide synthase (Sulfurisphaeratokodaii); NAD kinase (Sulfurisphaeratokodaii); Maltodextrin phosphorylase (Thermococcus litoralis); Probable tRNA pseudouridine synthase B (Thermococcus onnurineus).It is interesting to note that the bacteria related to the proteins found live in extreme environments (such as extremophiles, hyperxtrmophiles, acidophiles,etc) . and/or those capable of generating energy from sulfur. That is, they are able to survive in the leachate which is an extreme condition. In general, the proteins identified have the function of obtaining energy to maintain the bacteria.


Observing the results presented, it can be concluded that the leachate produced from the FPZSP composting process can be used in fertilizing plants as an organic fertilizer in addition to being a potential source of molecules to be explored for different applications.


The financial support provided by Fundação de Amparo a Pesquisa do Estado de São Paulo (2014/07037-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (163888/2013-2).


  1. Bernal MP, Alburquerque JA, Moral R (2009)Composting of animal manures and chemical criteria for compost maturity assessment. A review.Bio resource technology 100(22): 5444-5453.
  2. Carrilho ENV, Gonzalez MH, Nogueira ARA, Cruz GM, Nóbrega JA et al. (2002) Microwave-assisted acid decomposition of animal and plant derived samples for elements. Journal ofagricultural and food chemistry 50(15): 4164-4168.
  3. Wang W, Scali M, Vignani R, Spadafora A, Sensi E, et al. (2003) Protein extraction for two-dimensionalelectrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 24(14): 2369-2375.

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