Gaime Perraud, Isabelle

Martinez-Carrera, D.

Roussos, Sevastianos

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MICOL. NEOTROP. APL. 6 : 95-103,1993. 
NATURAL MICROORGANISMS OF THE FRESH 
COFFEE PULP 
I. GAIME-PERRATJD~, S.  ROUSSOS~ AND D. MARTINEZ- CARRERA^ 
Institut Français de Recherche Scientifique pour le Développement en Coopera- 
tion (ORSTOM), Centre de Montpellier, U. F. Riotechnologie PMC, 911 Avenue 
Agropolis, BP 5045,34032 Montpellier Cedex 1, France. 
Colegio de Postgraduados, CEICADAR, Laboratorio de Biotecnologia en Hongos 
Comestibles, Apartado Postal 701, Puebla 72001, Puebla, México. 
MIGROORGANISMOS PRESENTES EN LA PULPA DE CAFE 
FRESCA 
Accepted for publication September 28,1993 
Coffee pulp is one of the most abundant byproducts in Latin America. A quantitative 
study was accomplished in order to  know the main groups of microorganisms natu- 
rally occurring in this substrate. Samples of fresh coffee pulp from Mexico and 
Colombia, either lyophilized or sun dried during 2 and 10 days, were studied using 
eight different culture media. Yeast population was considerably higher than that of 
fungi and bacteria in fresh coffee pulp lyophilized as soon as it was produced. On the 
other hand, bacterial population became more and more predominant when coffee 
pulp was environmentally exposed (i. e .  sun dried during 2 and 10 days). Independ- 
ently of bacteria, the cellulose, starch, and pectins of sun dried coffee pulp were 
being utilized mainly by fungi, while in lyophilized coffee pulp they were being 
utilized mainly by yeasts. The main physical and environmental factors affecting the 
presence or dominance of a certain group of microorganisms in the fresh coffee pulp 
are discussed, as well as the need of suitable methodologies for its long term 
conservation. 
Key words: Coffee pulp, fungi, yeasts, bacteria, natural fermentation. 
96 I. GAIME-PERMUD ETU.  
RESUR/IEN 
La pulpa de café es uno de los subproductos agrícolas más abundantes en Lati- 
noamérica. Se llevó a cabo un estudio cuantitativo para conocer los principales 
grupos de microorganismos que se desarrollan en forma natural en la pulpa de café 
de México y Colombia. Se estudiaron muestras de pulpa de cdé  fresca liofilizada y 
secada al sol durante 2 y 10 días, empleando 8 medios de cultivo diferentes. La 
población de levaduras fue considerablemente más alta que aquella de hongos y 
bacterias en la pulpa de café fresca que se liofilizó una vez obtenida. En cambio, la 
población bacteriana fue cada vez más predominante en la pulpa de café que 
permaneció expuesta, es decir, cuando se secó al sol durante 2 y 10 días. Indepen- 
dientemente de las bacterias, la celulosa, el almidón y las pectinas de la pulpa de 
café secada al sol estaban siendo utilizadas principalmente por hongos, mientras que 
en la pulpa liofilizada principalmente por levaduras. Se discuten los principales fac- 
tores que afectan la presencia o dominancia de cierto grupo de microorganismos en 
la pulpa de café fresca, así como la necesidad de métodos adecuados para su conser- 
vación a largo plazo. 
Palabras clave: Pulpa de café, hongos, levaduras, bacterias, fermentación natural. 
INTRODUCTION 
Commercial coffee production is an economically important activity in most 
Latin American countries. As the world trade market of coffee grain is now 
being collapsed and fetching very low prices, there is a pressing necessity for 
using the coffee byproducts profitably. Coffee pulp is the most important 
byproduct of the so called wet coffee processing, as it represents about 40% of 
the fruit on a fresh weight basis (Tauk, 1986), and 29% on a dry weight basis 
(Bressani et al., 1972). It has been calculated that nearly 2,500,000 tons of 
fresh coffee pulp are produced around the world each year (Roussos et al., 
1989), most of which remain unused so far causing serious pollution prob- 
lems. 
Several alternatives have been proposed for the utilization of coffee pulp, 
such as animal feed, organic fertilizer, or as a substrate for enzyme produc- 
tion (Antier et al., 1993; Boccas et al., 1993), for biogas production, for edible 
mushroom production (Martinez-Carrera, 1989), and for vermiculture 
(Bouché, 1991). However, the practical application of these technologies has 
been difficult because a high moisture content (8045%) and the presence of 
many other nutrients, easily available for a wide variety of microorganisms, 
MICROORGANISMS OF COFFEE PULP 97 
make the fresh coffee pulp a substrate of very difficult handling and iong 
term conservation. 
In this work, a quantitative study was performed in order to know the 
main groups of microorganisms naturally occurring in the fresh coffee pulp 
with different treatments from Mexico and Colombia. 
MATERIALS AND METHODS 
Substrates 
Three samples of fresh coffee pulp were obtained by the wet coffee processing 
from Mexico and Colombia. Sample A was coffee pulp sun dried during 10 
days and collected in Xalapa, Veracruz, Mexico, with a moisture content of 
about 10%. Sample B was coffee pulp sun dried during 2 days and cdllected in 
Chinchina, Caldas, Colombia, with a moisture content of about 10%. Sample 
C was lyophilized cpffee pulp also collected in Chinchina, with a moisture 
content of about 5%. All samples were stored in plastic bags under humidity 
controlled conditions. 
Microbiological analysis 
Culture media. Sterilization was accomplished by autoclaving at 121°C for 15 
min. Three different culture media were used for determining the main 
groups of microorganisms. Potato dextrose agar (Difco) was used for fungi 
and prepared by adding 0.250 g L-l chloramphenicol as antibiotic (PDA + A), 
the final pH was adjusted to 5.6. Sabouraud dextrose agar (Difco) was used 
for yeasts and also prepared by adding 0.250 g L-l chloramphenicol (SAB + 
A), with a final pH of 5.6. Plate count agar (PCA, Difco) was used for bacteria 
and had a final pH of 7.0. 
Four selective culture media were used for studying microorganisms 
(bacteria were not studied), according to their nutritional requirements. 1) 
Cellulose group. Medium of Mandels and Weber (1969) whose composition 
was (g L-I): (NH,),SO, 1.4, KH,PO, 2.0, urea 0.3, CaC1,.2H,O 0.3, 
MgS04.7H,0 0.3, Carboxy-methyl cellulose 2.0, Tween 80 (1 ml L-l), yeast 
extract 0.25, peptone 0.25, FeS04.7H,0 0.005, MnS04.7H,0 0.00156, 
ZnS0,.7H,O 0.00140, CoCl, 0.002, agar 15.2) Starch group. The composition 
of the agar medium was (g L-l): starch 2, urea 0.05, (NH,),SO, 0.15, agar 15. 
3) Pectin group. The composition of the agar medium was (g L-9: citric pectin 
98 I. GAIME-PERRAKD ET AL. 
(SIGMA) 2, urea 0.05, (NH,),SO, 0.15, agar 20 (Boccas et al., 1993). 4) Lactic 
group. The culture medium was prepared by adding'l5 g L-l agar to the 
Lactobacilli MRS broth (Difco), and adjusting the final pH to 6.5. 
4 
The culture medium based on coffee pulp extract was prephred as follows: 
40 g of coffee pulp are ground and boiled in 1 L distilled water during 10 min. 
This solution is filtered (filter paper Whatman No. l), adjusted to 1 L, and 
mixed with (g L-l): KH,PO, 1.3, Na,HPO, 0.12, MgSO, 0.3, CaC1, 0.3, 
and agar 15. Bacteria were not studied in this medium. 
Inoculation. 10 g of coffee pulp from each sample studied were ground and 
mixed with 90 ml sterile distilled water and 0.1 ml of Tween 80. This solution 
was homogenized in Ultra-Turrax at  maximum speed during 4 min..Eight di- 
lutions were prepared from the homogenized solution. The optimal dilutions 
were selected by inoculating a microdroplet of each dilution on 2 petri dishes 
of every culture medium (eight microdroplets per petri dish); as can be seen 
in figure 1, which were incubated at 28°C. Subsequently, for the final 
counting of microorganisms, 0.1 ml of the selected dilutions were again 
inoculated and homogeneously distributed on petri dishes of all culture 
media (3 replicates, 28OC). Microorganisms were identified under the 
microscope. 
10-1 
10-7 
10-5 
Fig. I. Method of inoculation of a microdroplet in petri dishes used in this study for 
selecting optimal dilutions in different culture media. 
MICROORGAN~MS OF COFFEE PULP 99 
RESULTS AND DISCUSSION 
Bacteria, fungi and yeasts were the main groups of microorganisms found 
growing in all studied samples of coffee pulp. The number of microorganisms 
varied according to each sample (Table 1). The population of bacteria in 
samples A (98.8%) and B (94.7%) was considerably higher than that fQund 
in sample C (37.0%). Conversely, the population of yeasts was considerably 
higher in sample C (60.6%) than in samples A (0.1%) and B (1.2%). There was 
not a significant variation among the three samples with regard to  the fungal 
population. Accordingly, it can be said that the fresh coffee pulp is particu- 
larly rich in yeasts and bacteria, as sample C was directly lyophilized as soon 
Table 1. Main groups of microorganisms which occur naturally on fresh coffee pulp 
with different treatments, in comparison with other tropical substrates. 
Substrate Sample Microorganisms (%)* 
(Country) 
1 - '  
Fungi Yeasts Bacteria Total 
Sun dried 
coffee pulp 
Sun dried 
coffee pulp 
Lyophilized 
coffee pulp 
Sugar cane 
bagasse** .\ 
Cassava 
meal*** 
A 1.24x106(1.1) 1.O7x1O5(0.1) l . l l~ lO~(98.8)  1 . 1 2 ~ 1 0 ~  
(Mexico) 
B 2 .85x104(4. 1). 8. 52x103( 1.2) 6 .64x105( 94.7) 7. O 1 ~ 1 0 ~  
(Colombia) 
C 8.56x104(2.4) 2.18x1O6(6O.6) 1.33x1O6(37.O) 3 . 6 0 ~ 1 0 ~  
(Colombia) 
1.08x104 2 . 6 3 ~ 1 0 ~  2 . 6 4 ~ 1 0 ~  
2 . 5 0 ~ 1 0 ~  
* Number of microorganisms per gram of dry weight substrate. 
** Data from Roussos (1985). 
*** Data from Raimbault (1980). 
P . 
100 I. GAIME-PER” ET AL. 
as the pulp was produced. However, the bacterial population becomes pre- 
dominant when the coffee pulp is environmentally exposed; this was the case 
of samples A and B, which remained exposed to be sun dried during 10 and 2 
days, respectively. In comparison with other tropical substrates, such as 
sugar cane bagasse and cassava meal, the colffee pulp showed a relatively 
high number of total microorganisms (Table 1). 
The number of fungi and yeasts from the samples studied grown on differ- 
ent selective media is shown in Table 2. Independently of bacteria, it can be 
seen that cellulose, starch and pectins are being utilized mainly by fungi in 
samples A and B, while mainly by yeasts in sample C. In the case of lactics, 
there were no fungi or yeasts d’egrading them in samples A and B, which 
Table 2. Fungi and yeasts from fresh coffee pulp grown on selective culture media 
and grouped according to their nutritional requirements. 
Group Sample Microorganisms* 
(Country) 
Fungi . Yeasts 
Starch 
Pectin 
~ ~~ ~ ~ ~ ~~ ~ 
Cellulose A (Mexico) 8 .33~10~  4 . 4 1 ~ 1 0 ~  
B (Colombia) 2 .59~10~ 9 . 6 3 ~ 1 0 ~  
C (Colombia) - 6 .25~10~  2.18x106 
A (Mexico) 5 .67~10~ 6 . 6 7 ~ 1 0 ~  
B (Colombia) 1 . 5 7 ~ 1 0 ~  3 . 7 0 ~ 1 0 ~  
C (Colombia) 5 .26~10~ 2 . 6 0 ~ 1 0 ~  
A (Mexico) 8 .43~10~  2 . 0 4 ~ 1 0 ~  
B (Colombia) 2 .17~10~ 3 . 8 9 ~ 1 0 ~  
C (Colombia) 5. 16x104 1 . 8 9 ~ 1 0 ~  
Lactic A (Mexico) 0.0 0.0 
B (Colombia) 0.0 0.0 
C (Colombia) 0.0 6 . 0 2 ~ 1 0 ~  
* Number of microorganisms pes gram of dry weight substrate. 
MICROORGANISMS OF COFFEE PULP 101 
could mean that bacteria are mainly involved in this process; on the other 
hand, a good number of yeasts were counted from sample C in this medium. 
The number of fungi and yeasts found in the culture medium based on 
coffee pulp extract can be seen in Table 3. Fungi were more abundant than 
yeasts in samples A and B, while a higher population of yeasts was found in 
sample C. 
Coffee pulp is a byproduct very rich in nutrients, which can be immediatly 
used in nature by a number of microorganisms. The presence or dominance of 
a certain group will depend on many physical and environmental factors, 
which can be summarized in figure 2. Natural fermentation develops rapidly 
and can take different pathways (e.g. acetic, lactic, anaerobic, aerobic). These 
microorganisms can be beneficial or even harmful if the coffee pulp is used as 
a substrate for other purposes, e.g. edible mushroom cultivation (Martínez- 
Carrera, 1987). If any of the available technologies for using the coffee pulp is 
to be applied on a local or regional basis, the following aspects shoud be 
considered: 1) Plant variety and quality of the fruit, 2) Method of coffee 
processing, either “wet” or “dry”, considering the water quality used in 
the process, 3) Management of the coffee pulp after discarding, 4) Environ- 
mental conditions (temperature, humidity) in which the coffee pulp is being 
Table 3. Microorganisms counted from the culture medium based on coffee pulp 
extract. 
Sample 
(Country) 
Microorganisms“ 
Fungi Yeasts 
A (Mexico) 
B (Colombia) 
C (Colombia) 
* Number of microorganisms per gram of dry weight substrate. 
102 I. GAIME-PERRAUD ET AL.
ENVIRONMENTAL 
CONDITIONS 
OF THE FRUIT 
METHOD OF COFFEE 
PROCESSING 
MANAGEMENT AFTER I DISCARDING 
Fig. 2. Different factors affecting the natural microorganisms growing on coffee 
PulP. 
produced, and 5) Quantity and quality of coffee pulp currently produced. 
Each one of these factors, independently or as a whole, are responsible for the 
heterogeneity of coffee pulp and directly affect its quality as a raw material. 
Accordingly, suitable methods of long term conservation of this substrate are 
needed. 
ACKNOWLEDGEMENTS 
The authors thank Mme. Laure Hannibal for her excellent technical assistance. Dr. 
Jaime Zuluaga, CENICAFE, kindly provided coffee pulp samples from Colombia. 
LITERATCTRE CITED 
ANTIER, P., A. MINJARES, S. ROUSSOS, M. RAIMBAULT and G. VINIEGRA- 
GONZALEZ, 1993. Pectinase hyperproducing mutants of Aspergill us niger 
C28B25 for solid state fermentation of coffee pulp. Enzyme Microb. Technol. 
15: 1-17. 
MICROORGANISMS OF COFFEE PULP 103 
BOCCAS, F., S .  ROUSSOS, M. GUTIERREZ, L. SERRANO and G. VINIEGRA- 
GONZALEZ, 1993. Fungal strain selection for pectinases production from 
coffee pulp in solid state fermentation system. J. Fd. Sci. Technol. 30: in 
press. 
BOUCHE, M., 1991. La lombricultura en el tratamiento de residuos orgánicos. 2nd. 
Inter. Symp. on Biotechnology in the Coffee Industry, Manizales, Colombia. 
BRESSANI, R., E. ESTRADA and R. JARQUIN, 1972. Pulpa y pergamino de café, I. 
Composición química y contenido de aminoácidos de la proteína de la pulpa. 
Turrialba 22: 299-304. 
MANDELS, M. and J. WEBER, 1969. The production of cellulases. Adv. Chem. Ser. 
MARTINEZ-CARRERA, D., 1987. Design of a mushroom farm for growing PZeurotus 
on coffee pulp. Mushroom Journal for the Tropics 7: 13-23. 
MARTINEZ-CARRERA, D., 1989. Simple technology to  cultivate Pleurotus on coffee 
pulp in the tropics. Muslaroom Science 12(II): 169-178. 
RAIMBAULT, M., 1980. Fermentation en milieu solide. Croissance de champignons 
filamenteux sur substrat amylacé. Thèse de Doctorat d'Etat. Sciences 
Naturelles (Université Paul Sabatier), Toulouse, France. 
ROUSSOS, S., 1985. Croissance de Trichoderma harzianum par fermentation en 
milieu solide: physiologie, sporulation et production de cellulases. Etudes et 
Thèses (Université de Provence), ORSTOM, Paris. 
ROUSSOS, S.,A. AQUIAHUATL, J. CASSAIGNE, E. FAVELA, M. GUTIERREZ, L. 
A. SALAS, R. SANCHEZ, M. TREJO and G. VINIEGRA-GONZALEZ, 1989. 
Detoxificación de la pulpa de café por fermentación sólida. 1st. Inter. Symp. 
on Biotechnology in Coffee Industry, Xalapa, Mexico. 
TAUK, S. M., 1986. Estudo decomposiçao da polpa de café a 45°C atraves do uso de 
microorganismos isolados da polpa. Turrialba 136: 271-280. 
95: 391-414. 
HANNIBAL, S .  HUERTA, G. NAVA, M. RAIMBAULT, W. RODRIGUEZ, cT. 


Natural microorganisms of the fresh coffee pulp

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