INTRODUCTION

Solid waste is the biggest problem of environmental impact worldwide; they affect the
soil and air quality by the gases they produce at their decomposition. Additionally, they
affect water when they are deposited into it or dragged by rain. Organic waste resulting
from animal, agricultural and industrial production is the main source of pollution in
several countries. 1.
In 2014, approximately 11,203.24 tons of solid waste were collected daily in Ecuador,
of that, 62% was organic waste, 25% was recyclable inorganic waste, and 13% was
non-reusable hazardous waste (2-3). In 2010, an average of 150 tons of solid waste per
day was generated in the city of Riobamba (Chimborazo, Ecuador) by 225,741
habitants 4. The biggest problem in Riobamba is odor pollution, due to the
accumulation of waste in landfills located in markets, corners and public places.
The decomposition of organic waste is a serious problem due to the large amount
produced, in addition, air pollution due to bad odors has been increasing in recent
years. In Spain for example, 25% of the population feels affected by this problem and
in Ecuador, 26.76% of the population indicates having problems due to bad
environmental odors 5.
Some gases are generated as a result of waste rot: acetic acid, acetaldehyde,
ammonia, amines, mercaptans, phenol, toluene, sulfuric acid and other sulfur
compounds 6.
Additionally, the incorrect way of waste storage can generate the production of
pathogens, which present a high risk to the health of the population, and mainly to
people who handle the waste for final disposal 7.
Effective Microorganisms (EM) are cultures of mixed organisms that degrade organic
matter and allow its use for plants, improve soil characteristics and therefore,
conditions for agriculture 8(Higa, 2010). The EM was formulated as a microbial cocktail
using: photosynthetic bacteria, lactic acid bacteria, yeasts, fungi, and actinomycetes
(Ramírez, 2006).
In the process of rotting organic matter, the EM produce organic acids that are not
usually in the soil such as: lactic acid, acetic acid, amino acids, malic acid and vitamins
that could be absorbed by plants (Higa, 2010).
The aim of this research was formulate a microbial cocktail that reduces odors caused
by the decomposition of organic waste. To obtain an EM consortium, four strains of
microorganisms were chosen: Lactobacillus plantarum, Rhodopseudomona palustris,
Streptomyces albus, and Aspergillus oryzae; this in order to check the ability of the
microbial consortium to reduce the substances that produce bad odors from the waste
by removing pathogenic microorganisms through competitive exclusion.
MATERIALS AND METHODS
This study was carried out in the Molecular Biology-Genetics and Microbiology
laboratory at Science Faculty, Escuela Superior Politécnica de Chimborazo
(ESPOCH), Ecuador. The culture collection belonging to Plantsphere Laboratories,
Quito, Ecuador (Table 1). The purity of the microorganisms was checked by
observation under the microscope: fungal staining using lactophenol blue dye (López,
et al., 2014) and the Gram staining technique were used for bacteria (Madigan, et al.,
2004).
Initial treatment of bacterial strains

Table 1
Summary of the methods for microbial strains activation
Microorganisms Code Treatment Provenance
Lactobacillus PSL 40215 enriched in TSB broth,
plantarum seeded in MRS Probiotics
medium / 24 hours -30
°C
Rhodopseudomonas PSL 40460 Seeded in PDA organic substrates, and
palustris medium / 5 days-28 °C dairy products
Streptomyces albus PSL 40123 seeded in PDA
medium / 7 days-28 °C organic material
Aspergillus oryzae PSL 50127 seeded in PDA
medium / 5 días-28°C rice seed

Antagonism test
A confrontation was made between the four microbial strains in Petri dishes with PDA
medium at 28 °C for 7 days. The microbial suspensions were prepared in
concentrations of 1x104 CFU / mL; in saline solution for L. plantarum and R. palustris,
and in tween 80 0.1% for S. albus. Then 5mm diameter discs of A. oryzae were taken,
using sterile punches (Suárez, 2007). Three assays of the antagonistic effect and three
controls were performed with the fungus.
To determine the inhibition rate the formula was used:
I = [(C-T) /C] x 100.
Where, C is the radius of the mycelium of the control, T is the radius of the mycelium.

Treatment design
Four treatments (T1 to T4) and Control treatment (T5) were performed with three
repetitions each, with concentrations described in Table 2 (Huang, 2011).

Table 2. Treatment design

R.
Strains L. plantarum A. oryzae S. albus
palustris
CFU/mL spores/mL spores/mL
CFU/mL
Treatment
Treatment 1 (T1) 1x104 1x104 1x104 1x104
Treatment 2 (T2) 1x106 1x106 1x106 1x104
Treatment 3 (T3) 1x106 1x106 1x104 1x106
Treatment 4 (T4) 1x106 1x106 1x106 1x106
Control (T5) - - - -

To determine a concentration of microorganism to work, the spore counts of A. oryzae

and S. albus strains was performed in triplicate using a Neubauer chamber. Once the
required concentration was found, the inoculums were stored at 5 °C until the
preparation of the EM cocktails.
Additionally, the inoculum of L. plantarum and R. palustris was suspended into 10 mL
of saline solution, homogenized in a vortex for 5 minutes, and successive dilutions
were made until 10-5 [CITATION San11 \l 12298 ]. To define a concentration, each dilution
was counted by triplicate in the Neubauer chamber (Arredondo, et al., 2014).
Treatment Formulation

To determinate the treatment formulation, a relationship was made with the volume of
the container used (5 L) as follows:

1 L ME∗5 L container
X=
1000 L
X =0.005 L 5 mL

100 mL of each cocktail was prepared with 5% sterile molasses ME (5 mL), 5% of

microbial inoculums (5 mL) and 90% distilled water (90 mL); the volume that was
placed in the microbial cocktail of each inoculum was 1.25 mL. The cocktails were
incubated at 28 °C for 24 hours measuring the pH.

Substrate preparation

The biomass used for the preparation of the substrate was obtained from the markets
of the city of Riobamba. For this, 1 kg of vegetable waste was chopped until a
homogeneous mixture of density of 200 Kg.m-3 was obtained.

Application of microbial cocktails

For the pilot test, 5 mL of the microbial cocktail was applied in 18 mL of distilled water 3
times per week. Then for a second test, 5 mL of cocktail was placed in 10 mL of
distilled water, with the help of a sprinkler the cocktail was sprayed on the walls of the
container and on the organic waste, allowing a good distribution of the microorganisms
in the waste.

Treatment Evaluation
Assessment of the growth of the strains in the cocktail

Before the first application and at the end of each test, 1 mL of cocktail was seeded in
cell culture dishes with PDA, then were incubated for 5 days and a Gram and
lactophenol blue staining tests were performed to check the presence or absence of
the 4 microbial strains into the cocktails.

Odoriferous evaluation of cocktails.

The sensory procedure of (Vallejo, 2014 pág. 12) was used as reference in this
evaluation. A group of 40 heterogeneous people from 19 to 23 years old was selected.

An 8 days pilot test was performed to validate the procedure and to establish the rating
scale of the evaluation. Cocktails were applied on days 1, 4 and 6 and volunteers
began to evaluate from day 5 to 8.

Then another 8 day test was performed, the cocktails were applied on days 1, 3 and 6,
and the odor evaluation was performed on days 1, 3, 6 and 8.

Before starting the experiment, on day 1 the vegetable wastes were placed into the
experimental devices, the volunteers checked if the smell was homogeneous or if it
was in an advanced decomposition in each of the containers.
The scale used for the analysis of the tests was proposed by the American Society of
Heating Refrigerating and Air Conditioning Engineers (ASHRAE).

Table 3. Relative odor force scale

VALUE CORRESPONDS TO
0 Without smell
1 Light smell
2 Moderate smell
3 Strong smell

In addition, temperature, pH and humidity were measured using the Consort C562
multiparameter; the conductivity was measured in microsiemens and the temperature
in Celsius degrees (Alvarez, 2014 págs. 3-20).

Leachate Analysis

At the end of the experimentation, 200 ml of the leachates from the treatment with the
highest efficiency in the odoriferous evaluation tests and from the control group were
collected and subjected to analysis of BOD5, COD, turbidity, color, temperature,
conductivity and pH.

These analyzes were performed in the Water Quality laboratory of the Faculty of
Science-ESPOCH-Ecuador.

BOD5
This parameter was determined by the gasometric method using a BOD Digester Hach
BODTrak TM 11 for 5 days, subsequently, the results were read in mg.L -1, following the
methodology of (INGELAB, s.f. pág. 2).

COD
The volumetric method was used to determine this parameter, a distillation equipment
was used following the methodology of (Arias, 2003 pág. 93).

Turbidity
The turbidity was determined using The Hach RATIO / XR Turbidimeter in a scale of 1-
2000 NTU units following the methodology of (Carpio, 2007).

Color
The apparent color was determined using dilutions with a factor of 200, these were
evaluated on a DR 2800 photometer at a wavelength of 465 nm in cobalt platinum units
(PtCo) following the technique of (Aguilar , 2001 p. 9).

Temperature, conductivity and pH

For the measure of these parameters a Consort C562 multiparameter equipment was
used. pH was determined at room temperature. Conductivity was measured in
microsiemens and temperature in Celsius degress accordin with (Alvarez, 2014 págs.
3-20).

RESULTS AND DISCUSSIONS

Antagonistic activity

The inhibition percentage associated with: L. plantarum, R. palustris, S. albus, and A.

oryzae, ranged from 4% to 7.8%. The low antagonistic degree of A. oryzae, equal to 1,
suggests that the fungus can invade ¼ of the surface of other microorganism without
damaging it. Additionally, the results show no formation of inhibition halos among the
four microbial cultures, so its use as a single consortium of microorganism is
recommended due to the symbiotic effect presented. Therefore fungi, actinomycetes,
and bacteria can co-exist in a mixed culture (Higa et al., 1994); and they can be
included in a biological treatment system for odour abatement (Nanda, Sarangi, &
Abraham, 2012).

Evaluation of the cultures growth in the microbial consortium

The four microbial cultures, which formed the initial mixed consortium, were remained
after the treatments, as illustrated in Fig.1. Despite the notorious presence of A.
oryzae, the growth of R. palustris, L. plantarum, and S. albus, was not inhibited.

c
d

Fig. 1. Consortium of microorganism: a) S. albus; b) L. plantarum; c) R. palustris; and d) A. oryzae.

The efficiency of a biological treatment system for odour reduction depends on its
heterotrophic microbial consortium (Nanda et al., 2012). Lactobacillus plantarum,
Streptomyces albus, and Aspergillus oryzae are heterotrophic microorganism while
Rhodopseudomonas palustris has a versatile metabolism; for that reason, when they
were placed into organic waste and molasses as substrate, they had nutrients
necessary for gain energy (Wysocka, Gębicki & Namieśnik, 2019) and growing after
the treatments.

Odor analysis
The application of an EM consortium had a significant effect (p<0.05) on the difference
of odor levels from organic waste, in fact, the panel perceived odor variations during
the period of treatment until day eight. The use of pure cultures in biological treatment
system for odor reduction in air, as the used in the mixed consortium for T1 to T4,
ensures the early action against the potentially pathogens that cause possible
emissions of odour (Rybarczyk, Szulczyński, Gębicki, & Hupka, 2019); likewise Fan et
al. (2018) determined the reduction of time for elimination of pungent odours coming
from the decomposition of organic matter on home scale organic waste composting, so
the unpleasant smells of compost with EM varied to earthy smell on week five
compared to control treatment (without EM) which generated earthy smell on week
seven.

According to the sensory procedure performed (Vallejo, 2014), T4 can be considered

as an effective way to odor control of organic waste. The parameter “Strong odor” was:
25%, T1; 17.5%, T2; 2.5%, T3; and 0%, T4; as shown in Fig. 2. The threshold level of
olfactory identification for some malodorous compounds are: 42 ppm, acetone; 17 ppm,
ammonia, and 0.00041 ppm, hydrogen sulphide (Wysocka et al., 2019). In this way, T4
could have eliminated the perception for unpleasant smell compared to T5 (control)
which kept the parameter “Strong odor” on 80% of the panel.

120

100

80
Olor fuerte
60 Olor moderado
Olor ligero
Sin olor
40

20

0
T1 T2 T3 T4 T5
Fig. 2. Odor levels by treatments with effective microorganism (T1 to T4) and control without EM (T5).

By comparison, the T4 contained the highest concentration of effective

microorganisms: ~106 CFU/mL of L. plantarum and R. palustris, and ~106 spores/mL of
S. albus and A. oryzae. Namasivayam and Kirithiga (2010) verified that native EM
increased when EM consortium also increased, they used high concentrations of
compost with EM (12.1x106 CFU/g of bacteria, 21.3x105 CFU/g of actinomycetes, and
15.1x104 CFU/g of yeast and mold) for improving the soil nitrogen, phosphorus and
potassium levels.

Effect of temperature and pH

La temperatura tuvo un incremento paulatino en todos los tratamientos, como se

muestra en la Figura 3. Song et al. (2018) señalan que los cambios de temperatura en
el compostaje de desechos orgánicos se encuentran estrechamente relacionados con
la actividad microbiana, de esta forma se ha establecido que la velocidad metabólica
microbiana aumenta en el tiempo por cada incremento de 10°C.
 35
33
31
29
27
25
23
21
19
17
15
0 1 2 3 4 5 6 7 8 9

T1 T2 T3 T4 T5
Fig. 3. Temperature variations for treatments with EM (T1 to T4) and control without EM (T5).

Los tratamientos con ME presentaron mayores temperaturas a comparación del

tratamiento control; de hecho, la mayor temperatura alcanzada (33°C) se registra para
T4 en el día 6. Este comportamiento se asemeja al presentado en la investigación de
Song et al. (2018) para la descomposición de desechos orgánicos, quienes observaron
mayor temperatura en el tratamiento con un consorcio microbiano comparado con el
tratamiento que no tuvo ninguna inoculación.

Por otra parte, los ensayos con ME presentan un pH igual a 6 en el día 8, mientras que
el pH máximo alcanzado por el tratamiento control es igual a 5, como se muestra en la
Figura 4. El rango de pH sugerido para llevar a cabo una apropiada degradación de la
materia orgánica con consorcios de ME se encuentra entre 6 a 8.5 y además, en la
fase inicial de descomposición, el pH decrece durante los primeros días y luego
experimenta un incremento paulatino, pudiendo llegar a valores de 8.16 a los 15 días y
7.90 a los 30 días (Jusoh, Manaf, & Latiff, 2013).

6.5

5.5

4.5
0 1 2 3 4 5 6 7 8

T1 T2 T3 T4 T5

Fig. 4. pH variations for treatments with EM (T1 to T4) and control without EM (T5).
El pH puede relacionarse con la producción de olores puesto a que la acidificación,
neutralización y alcalinización del pH en los procesos de compostaje se encuentran
estrechamente relacionados con la actividad microbiana mediante la liberación de
amoniaco y la conversión de ácidos orgánicos en CO 2 (Fan et al., 2018). Igualmente,
Miller, Macauley, and Harper (1991), identificaron que un pH entre 8 a 9 lleva a la
pérdida de nitrógeno por medio de la volatilización de amoniaco, el cual es un
compuesto identificado como causante del mal olor en compost.

Leachate analysis

Los valores de DOB5, COD, turbidez y conductividad obtenidos para T4, fueron
menores a los obtenidos en el tratamiento control (sin ME), tal como se presentan en
la Tabla 1. En lixiviados generados a partir de desechos vegetales en procesos de
compostaje a nivel laboratorio, los rangos de concentración de COD varían de 18 a 68
g/L, y para DOB entre 10 y 46 g/L (Roy, Azaïs, Benkaraache, Drogui, & Tyagi, 2018);
por lo que, los valores de COD obtenidos para los lixiviados de T4 y T5 están dentro
del rango típico, mientras que la DOB está por debajo del límite inferior.

Table 4
Analysis of organic waste leached from T4 (with EM) and T5 (control).
Parameters T4 T5
BOD5
7.63 8.34
(g/L)
COD
30.67 53.33
(g/L)
Turbidity
4547 8300
(NTU)
Conductivity
1.82 3.75
(mS/cm)

De la misma forma, para poder evaluar el nivel de contaminación causada por la

materia orgánica, es necesario calcular el radio de BOD/COD para dilucidar la
biodegradabilidad del lixiviado (Roy et al., 2018), en este sentido, el lixiviado generado
por el tratamiento con ME es moderadamente biodegradable (radio T4 BOD/COD =
0.25) a comparación del tratamiento sin consorcio microbiano inoculado, el cual
presenta un lixiviado con baja biodegradabilidad (radio T5 BOD/COD = 0.16).

Por otro lado, los valores de T4 y T5 se ajustan al rango de conductividad típico para
lixiviados de procesos de degradación de desechos vegetales obtenidos en laboratorio
provenientes de desechos vegetales, mismo que varía entre 1.42 y 82,6 mS/cm
(Roy et al., 2018). 

CONCLUSION

La aplicación del consorcio microbiano conformado por Lactobacillus

plantarum, Rhodopseudomonas palustris, Streptomyces albus y Aspergillus
oryzae tuvo un efecto significativo en la reducción de olores producidos por la
descomposición de residuos del mercado de Riobamba-Ecuador (cáscaras de
legumbres, hojas de verduras y frutas). El presente estudio se realizó únicamente
sobre residuos vegetales, razón por la cual, debe ser considerado como un estudio
preliminar para el control de olores provenientes de otro tipo de residuos orgánicos.
Las concentraciones de ME utilizadas en el mejor tratamiento (~106 CFU/mL y
~106 esporas/mL) lograron una reducción en la percepción del olor fuerte de acuerdo
al panel de estudio. Las especies de ME utilizadas en el consorcio microbiano se
mantuvieron al inicio y al final de los tratamientos, por lo que se comprueba su acción
simbiótica dentro del sistema de tratamiento biológico de desodorización.
Durante los tratamientos, la medición de pH y temperatura es necesaria para su
utilización como parámetros de operación que garanticen la actividad biológica de los
microorganismos.
Finalmente, en los lixiviados del tratamiento más efectivo para la reducción de olor
(T4), los parámetros DOB, COD y conductividad resultaron inferiores en comparación
con los valores del tratamiento sin ME inoculados.

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