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Revista mexicana de ciencias agrícolas

versão impressa ISSN 2007-0934

Rev. Mex. Cienc. Agríc vol.7 no.8 Texcoco Nov./Dez. 2016


Investigation note

Evaluation of compost with presence of heavy metals on the growth of Azospirillum brasilense and Glomus intraradices

Augusto Rojas Aparicio1 

Jenny Marbella Vázquez Jacinto1 

Nelly Romero Gomezcaña2 

Miguel Ángel Rodríguez Barrera1 

Jeiry Toribio Jimenez1 

Yanet Romero Ramírez1  § 

1Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Universidad Autónoma de Guerrero. Chilpancingo, Guerrero, México. Av. Lázaro Cárdenas s/n. Ciudad Universitaria. Apdo Postal. 39070. (;;;;

2Secretaría de Desarrollo Rural del Estado de Guerrero. Carretera México-Acapulco No. 274, Col. Burócratas, CP. 39090. Chilpancingo, Guerrero. (


Compost is an organic material obtained as a result of the controlled microbial action on organic waste. Nowadays, farming is responsible of the integrated use of biofertilizers and compost, which represent an important alternative to reduce the use of chemical fertilizers. In this study the compost with presence of heavy metals on the growth of A. brasilense and G. intraradices was evaluated. Microbiological analysis of compost only revealed significant results for the growth of S. aureus and P. aeruginosa. The conclusion is that the use of compost with presence of heavy metals does not cause any adverse effects on the growth of A. brasilense and G. intraradices, since the two microorganisms were able to be isolated and identify in the rhizosphere of plants. Furthermore, observed metals did not exceed the maximum permissible limits by the international standards as NTC-5167 and standard 503 of EPA.

Key words: A. brasilense; G. intraradices; compost; heavy metals


Compost es materia orgánica que resulta de la acción microbiana controlada en residuos orgánicos. Hoy en día, la agricultura es responsable del uso integrado de biofertilizantes y compost, que representan una alternativa importante para reducir el uso de fertilizantes químicos. En este estudio se evaluó el compost con presencia de metales pesados sobre el crecimiento de A. brasilense y G. intraradices. El análisis microbiológico del compost solo mostro resultados significativos para el crecimiento de S. aureus y P. aeruginosa. La conclusión es que el uso de compost con presencia de metales pesados no causa ningún efecto adverso sobre el crecimiento de A. brasilense y G. intraradices, ya que los dos microorganismos pudieron ser aislados e identificados en la rizosfera de la planta. Por otra parte, los metales no excedieron los límites máximos permitidos por las normas internacionales como la NTC5167 y la norma 503 de EPA.

Palabras clave: A. brasilense; G. intraradices; composta; metales pesados


The compost is the organic material obtained by the controlled action of microbes on biodegradable organic residues as leaves, fruit and vegetable skins, home organic waste, manure, solid urban waste, sewage waste and agroindustrial wastes. The factors affecting the composting process are: the nature of the organic waste and the conditions of development of the microbial population. Since when environmental conditions are not optimal, the microbial population decreases and thus its action affecting the process. In general the main biodegradable waste included in the composting process are of agricultural origin, animal and vegetable nature, liquid and agro-industrial waste, municipal solid waste, waste from wastewater treatment and wood waste are also included (Muñoz, 2005).

However, when agro-industrial wastes are included in the composting process, the chances to find heavy metals increase considerably, being cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg) and selenium (Se), the most concerning elements for human health. In general, a high quality compost and suitable to use it in agriculture presents the next maximum limits: Cd 10 mg kg-1, Cu 450 mg kg-1, Ni 120 mg kg-1, Pb 300 mg kg-1, Zn 1 100 mg kg-1, Hg 7 mg kg-1 and Cr 400 mg kg-1 (EPA, 2006). The most important factors in the composting process are water content, humidity (50 to 60%) and pH (6.5 to 8) (Muñoz, 2005; Pérez et al., 2010); the ratio carbon-nitrogen (C/N) is a factor that must be controlled to ensure a correct fermentation, the optimal values vary from 25 to 30% of these elements. On the other hand, the microorganisms play an important role during the composting process, where the action of facultative and obligate aerobes, mesophiles and thermophiles is predominant (Pérez et al., 2010). These microorganism are responsible for the degradation of organic matter.

Bio-fertilizers are responsible for stimulating growth and crop productivity (Rueda-Puente et al., 2009). The most commonly used bacteria in agriculture are those corresponding to the genus Rhizobium and Azospirillum (Caballero-Mellado et al., 2007). Some authors have reported that when there is a fungal co-inoculant as the arbuscular mycorrhizal fungi (AMF), the stimulus on the vegetal growth increases significantly, affecting in a positive way the mineral absorption and the availability of N, P and K on the soil (Mendoza and Cruz-Hernández, 2010). AMF associations are formed only by fungi belonging to the Phylum Glomeromycota, from these the most used in agriculture is Glomus intraradices. Guerrero has being considered one of the main maize producers in Mexico during many years, being the seventh position nationally, not only for family consumption but to commercialize it.

Chemical fertilizers are used in agriculture to help the crops reach an optimal growth and production, reinstating the nutrients removed from the soil; however, the abuse and wrong use of these have caused the soil deterioration (erosion, acidification and salinization), and damage to the environment. In order to reduce the impact caused by fertilizers, the Government of the State of Guerrero, through the Secretaría de Desarrollo Rural (SEDER), has promoted the use of bio-fertilizers (A. brasilense y G. intraradices), as well as the compost production by the farmers. The aim of this strategy is to be used all along the State. In order to reach this goal, the SEDER has suggested the introduction of compost from other sites, however it is necessary to evaluate the quality of it before its distribution since it has been detected the presence of heavy metals, even when they are in the permissive levels. In this project we evaluated the effect of heavy metals present in the compost that it is distributed to farmers in the state de Guerrero on the growth of A. brasilense and G. intraradices.

Materials and methods

Substrate preparation and biological material

Compost was provided by the Government of Mexico DF (GDF) and the bio-fertilizers (A. brasilense and G. intraradices) by the trading house Biosustenta. The compost is made in Texcoco with trash collection from DF and product of the pruning of gardens, no contain carries animal manure and reported a neutral pH (7.31), an organic matter content of 27.36% and humidity very low (4.08%). The soil used presented a slightly alkaline pH (7.65), an organic matter content of 2.88% and very low humidity percentage (1.84%). The compost and soil were autoclaved at 121 °C for 1 h. To sterilize the seeds from Zea mays was determined as described Wenny and Dumroese 1987. After this treatment, they were impregnated with a non-toxic adherent based on carboxymethyl cellulose. To finish, we mixed G. intraradices (333 g) and A. brasilense (117 g) and apply this mixture on the seeds.


The treatments used in this study are CN (soil and seed) T1 (soil, compost and seed), T2 (Soil, compost sterile and seed), T3 (soil, composte, seed and Biofertilizer), T4 (soil, compost sterile, seed and biofertilizer), T5 (soil, compost, seed sterile and biofertilizer) and T6 (soil sterile, compost sterile, seed sterile and biofertilizer.

Determining the compost physicochemical properties

The organic matter content of C, P and N was determined as described González-Chávez et al. (2009). The pH was determined in water and CaCl2 in a ratio 1:2.5 of extractsolution a potentiometer 420ª was used for the measurements (Orion Research, Beverly, MA).

Determining human pathogens in the compost and heavy metals in the compost

To characterize the microbiology of the compost, we did serial dilutions which were inoculated in specific media for bacteria considered potentially pathogenic for the human (Mac Conkey, Salt and mannitol and Salmonella-Shigella). To determine the heavy metals in the compost, the procedure was done in the Laboratory of Geochemistry, belonging to the Academic Unit of Earth Sciencies, located in Taxco el Viejo, Guerrero. Metals were analyzed by ICP-AES (Talavera et al., 2006).

Isolation of A. brasilense and observation of G. Intraradices from maize roots with compost

We did some tests as the glucose fermentation and Gram´s method to identify the microorganism of the roots (Baca et al., 2010). To observe mycorrhizal roots by G. intraradices, we used the technique by Phillips and Haymann (1970). Additionally, we counted spores in the rhizosphere as described by Gerdemann and Nicholson (1963).

Results and discussion

Physicochemical analysis of the compost

One of the goals of this work was to determine the physicochemical properties of the compost. The analysis reported that the compost has a neutral pH (7.31), an average content of organic matter (27.36%) and low humidity (4.08%). The amount of N, K, Ca, Mg, Fe, P, and Mn is average (Álvarez-Solís et al., 2010, Pérez et al., 2010). The electrical conductivity registered a high value and a sandy texture (NTC-5167 2004). Regarding the compost, the three main elements considered for the growth of the microorganism (N, P y K), are present below the levels reported by Álvarez-Solís et al. (2010), N= 0.095%, P= 0.65% and K= 0.83%. These results show that in those composts where trash collection from DF and vegetable waste are included, there is a decrease in the content of those nutrients (Castillo et al., 2000). The organic matter content (OMC), considered an important source of nutrients (Pérez et al., 2010), was 27.36%, which is a good amount for compost. The OMC in the compost has a positive effect on the soil fertility, besides improving its physical, chemical and biological characteristics. However, the percent of OM in the used compost showed a lower value compared to that reported by Zapata et al. (2005), in a range from 46 to 56%, who used compost based on local muds and pine bark residues.

Determining human pathogens in the compost

The microbiological analysis of the compost reported the presence of Staphylococcus aureus and Pseudomonas aeruginosa. We confirmed this result with the coagulase test for S. aureus and a biochemical set for P. aeruginosa. The results obtained in the microbiological analysis of the compost (Table 1) showed the existence of S. aureus, although the growth was not significant to affect the health of those manipulating the compost (NMX-F-310-1978). Several authors have reported the growth of this bacterium in samples of vermicompost, suggesting that its components have a fecal animal origin (Ogefere et al., 2010) nonetheless, the compost used in this study does not have these origins, no carries animal manure.

Table 1 Microbiological analysis to find human pathogenic bacteria in compost samples. 

UFC g-1 composta
Medio de cultivo Núm. de colonias (UFC g-1) Resultado GRAM colorante
Sal y manitol 5 500 S. aureus ns Cocci +
Mac Conkey 16 116 000 P. aeruginosa ns Bacillus -
0 0 E. coli -
SS 0 0 Salmonella sp. -
0 0 Shigella sp. -

ns= no significativo; CFU= unidades formadoras de colonias; SS= medio de cultivo de Salmonella and Shigella.

The growth of P. Aeruginosa was observed due to the capacity of this bacterium to grow at high temperatures which makes it resistant to the auto-sterilization process undergone by the compost. The auto-sterilization consists in a temperature rise because of the metabolic activity of the microorganisms present in the compost (Muñoz, 2005). Pérez et al. (2010) showed that the manure compost has higher densities of the genus Pseudomonas (420 x 103 CFU g-1), which has a high adaptation capacity.

Determining heavy metals in the compost

The analysis of heavy metals in the compost is shown in Table 2. We compared three international norms, considering the amounts of heavy metals found in the compost falling inside the maximum permissive limits. Despite the presence of heavy metals in the analyzed compost, the concentrations of As, Cd, Cr, Cu, Ni and Pb are below the maximum permissive levels in the NTC-5167 2004, Norm 503 of EPA and the EPA in 2006. These concentrations suggest that the compost was made using a big variety of waste, which normally is not subject to an appropriate selection and residue separation. Even more, some authors report the use of waste water to maintain the humidity during the compost process, being one of the ways to incorporate heavy metals to the compost. Likewise, heavy metals as Pb, As, Cd, Cu, Zn, Ni and Hg, could have been added in a continuous manner to the soils through agricultural and/or industrial activities, and incineration (Khan, 2006).

Table 2 Heavy metal concentrations in the compost. 

Metales pesados Composta (mg kg-1) Niveles máximos permisibles
Norma 503 EPA* (mg kg-1) NTC-5167, 2004** (mg kg-1) EPA 2006*** (mg kg-1)
As 0.4375 54 41 --
Cd 0.5 18 39 10
Cr 23.4375 1 200 1 200 400
Cu 91.1875 1 200 450 450
Ni 24.6875 180 420 120
Pb 62.375 300 300 300

*= valores en la norma 503 de la agencia de protección ambiental (EPA); **= valores en el NTC-5167, 2004, que establece los requisitos que deben cumplir los productos orgánicos utilizados como fertilizantes y abonos; ***= Agencia de Protección Ambiental de los Estados Unidos.

Isolation of A. brasilense in the roots and observation of G. intraradices in the maize rhizosphere

We used the method reported by Baca et al. (2010) to obtain pure cultures A. brasilense, here we observed that the concentration of heavy metals from compost used in this work did not affect the native or external microbiology that could exist in the compost and the soil since the analysis showed positive results to microbial growth and to the isolation of A. brasilense. The observation of how the fungus colonized the roots was performed at the end of the experiment. We used two techniques, the first one was to isolate the fungus from the roots, being able to observe a high amount of vesicles, spores and hyphae (Figure 1a), mainly belonging to the genus Glomus. The second technique was the one described by Gerdemann and Nicholson, (1963), where we counted and analyzed the rhizosphere (Figure 1b). In this case, we were able to identify more than one sample of the genus Glomus and particularly G. intraradices in the treatments where we added bio-fertilizers (T3 and T6 mainly).

Treatment 6. Image A) spores (sp); B) hyphae (hf) and spores (sp); C) spores (sp); D) vesicles (vs) and spores (sp); E) spores (sp) and F) spores (sp) and vesicle (vs). Usual taxonomy shown by the spores belonging to G. intraradices.

Figure 1 Morphology (a) and spores of G. intraradices (b) observed in the maize roots and the rhizosphere by the technique of Phillips and Haymann, 1970


It was shown that the heavy metals present in this compost introduced by the SEDER in the Guerrero state, did not affect totally the growth of A. brasilense and G. intraradices. This fact was corroborated when we found the fungus and the bacterium in the roots of all the samples analyzed, even in those where the bio-fertilizer was not added. No significant values were found for the growth of human pathogenic bacteria (S. aureus y P. aeruginosa), showing that they are not a risk to manipulate the compost. Finally, we found that the bacterium is tolerant to high concentrations of As, as well as lead and copper but in lower concentrations. This was not the case of cadmium, where the bacterium did not grow.

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Received: June 2016; Accepted: July 2016

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