Zanieczyszczenia mikrobiologiczne procesu fermentacji etanolowej (ang.).pdf

ACTA Acta Sci. Pol., Technol. Aliment. 8(4) 2009, 25-31

ISSN 1644-0730 (print) ISSN 1889-9594 (online)

THE OCCURRENCE AND IDENTIFICATION

OF MICROBIOLOGICAL CONTAMINATION

IN FUEL ETHANOL PRODUCTION *

Katarzyna Leja, Magdalena Broda

Poznań University of Life Sciences

Background. Bacterial contamination is a major problem for commercial fuel ethanol

production in distilleries all over the world. The contaminating microorganisms produce

acetic and lactic acid that has a detrimental effect on fermentation efficiency. The aim of

this work was to calculate the number of bacterial contaminants in some distilleries.

Moreover, in this study it was signified what kind of bacteria contaminate ethanol produc-

tion process.

Material and methods. Grains were obtained from five distilleries from some regions in

Poland, in this work hereafter referred to as α, β, γ , δ, and ε distilleries. Corn was the raw

material in the α, β, and γ distilleries, triticale in δ distillery, and rye in the ε one. From

these five distilleries, sweet mashes during fermentation and after it, were also analysed.

The total number of microorganisms, the number of lactic acid bacteria, the number of

anaerobic bacteria and the quantity of yeasts and moulds in raw materials were calculated.

Results. The number of total viable bacteria (CFU/g), lactic acid bacteria (CFU/g), anae-

robic bacteria (CFU/g), moulds, and yeasts (CFU/g) occur in the samples were deter-

mined. In all distilleries tested, all groups of microorganism were present.

Conclusions. The results of our study show that all tested distilleries have a lot of diffi-

culties with microbiology pollution which leads to a decrease of ethanol production and

economical problems. From the economical point of view, reduction of microbial conta-

mination makes it possible to increase the production volume.

Key words: contaminants, distillery, fuel ethanol production, lactic acid bacteria

* This work was supported by the Polish Ministry of Science and Higher Education as a POL-

-POSTDOC III (grant number PBZ/MNiSW/07/2006/18).

Corresponding author – Adres do korespondencji: Mgr inż. Katarzyna Leja, Department of Bio-

technology and Food Microbiology of Poznań University of Life Sciences, Wojska Polskiego 48,

60-627 Poznań, Poland, e-mail: katleja@poznan.up.pl

K. Leja, M. Broda

INTRODUCTION

Currently ethanol as transportation fuel is of interest to many factories branches of

industry. However, bacterial contamination is an outgoing problem for commercial fuel

ethanol factories. In distilleries, contaminants can originate from tankage, transfer lines,

heat exchangers, raw materials, active dry yeasts, poorly stored backset, or yeast slurry

used as the inoculums [Narendranath et al. 1997, Reed and Nagodawithana 1999]. They

can significantly limit the scale of ethanol production from agricultural sources [Schell

et al. 2007, Skinner and Leather 2004]. Fuel ethanol is not produced under homogene-

ous culture conditions; moreover, chronic infections are expected and tolerated, al-

though, they are frequently deleterious to the ethanol production process. Infections can

lead to stuck fermentation, and bioreactors must be shut down for cleaning. Thus, they

decrease the efficiency of ethanol production and increase its costs. Contaminants re-

duce carbon available for conversion to ethanol. Simultaneously, they compete for nu-

trient factors needed by yeast cells and can produce toxic byproducts (lactic and acetic

acids). The primary bacterial contaminants of fuel ethanol fermentations are lactic acid

bacteria (LAB) [Skinner-Nemec et al. 2007]. LAB are the most troublesome because

of their tolerance to high temperature and low pH, and their ability to grow rapidly

[Narendranath et al. 1997]. Removal of contaminating bacteria reduces the microbial

competition for nutrients in the growth media thereby increasing the efficiency and

productivity of the culture.

Microbial numbers can be significantly reduced by cleaning and sanitizing the

equipment, by maintaining backset at a temperature over 70°C, by pasteurizing or

chemically sterilizing the substrates [Narendranath et al. 1997]. Furthermore, various

agents for control of bacterial contaminants under laboratory conditions have so far

been tested: potassium metabisulfite, hydrogen peroxide, 3,4,4’-trichlorocarbanilide,

and antibiotics such as tetracycline, penicillin, monensin, and virginiamycin. All of the

above-mentioned agents inhibited bacteria over yeasts. Nowadays, penicillin and virgi-

niamycin commercially to inhibit bacterial infections of fuel ethanol production are sold

[Skinner and Leather 2004, Gibbons and Westby 1996, Narendranath et al. 2000, Oliva-

-Neto and Yokoya 1998, Stroppa et al. 2000].

In this paper, the microbiological situation of five distilleries in Poland was dis-

cussed. The number of total bacterial contaminants was calculated, as well as the lactic

acid bacteria, anaerobic bacteria, moulds and yeasts. The aim of this study was also to

specify what kinds of bacteria contaminate ethanol production process.

MATERIAL AND METHODS

The microbiological purity of grains used in fuel ethanol production was tested.

Grains were obtained from five distilleries in Poland, in this work hereafter referred to

as α, β, γ, δ, and ε distilleries. Corn was the raw material in the α, β, and γ distilleries,

while triticale in the δ distillery, and rye in the ε one. Probes of sweet mashes during

fermentation and after it were also analysed. Fermentation went on for three days. Sam-

ples were taken in the 2008/2009 campaign. Bacteria were cultured in MRS Medium,

Lab-Agar, Thioglycollate Fluid Medium, and in Chloramphenicol Lab-Agar (BIOCORP

company).

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The occurrence and identification of microbiological contamination ...

Grains obtained from the distilleries were immersed in physiological salt and shaken

out for two hours. The total number of microorganisms, the number of lactic acid bacte-

ria, the number of anaerobic bacteria and the quantity of yeasts and moulds in raw mate-

rials were calculated. The inoculations of microorganisms into a medium were done.

In case of mash, cykloheximide 100 mg/ml (Sigma) was added to medium to kill yeasts

and make it possible to count live bacteria cells.

Selected bacterial isolates were identified by comparing ribosomal RNA gene se-

quences to known sequences. A 1.6 kb segment of the 16S rDNA was amplified from

genomic DNA of a single colony per reaction using bacterial primers. A cycle sequenc-

ing kit was used to sequence the amplified product. The nucleotide sequence was ob-

tained from both ends of the PCR product. On the basis of 16s RNA the affinity of the

microorganism was determined.

RESULTS AND DISCUSSION

Samples of raw materials (corn, triticale, and rye) were obtained from tested distille-

ries in February and in March 2009. Grains in all the distilleries were stored in the con-

tainers at the room temperature. Purity of these grains was controlled. The number of

total viable bacteria (CFU/g), lactic acid bacteria (CFU/g), anaerobic bacteria (CFU/g),

moulds, and yeasts (CFU/g) occur in the samples were determined. In all distilleries

tested, all the above-mentioned groups of microorganism were present. The level of

total viable, lactic acid, and anaerobic bacteria was similar in the β and γ distilleries

(about 7·10 7 CFU/g). It was higher than the estimated value (e.g., the number of cereal

grains total viable bacteria is assessed at 5·10 4 -1.6·10 6 CFU/g) [Maciorowski et al.

2007]. Both distilleries use corn as the raw material. Corn was also used in the α distill-

ery but in spite of this the level of bacterial contaminants in grains here was lower than

in the β and γ distilleries. It may be a result of different storage conditions of corn in

these three distilleries. The level of contamination with total viable bacteria (about 10 6

CFU/ml) and lactic acid bacteria (about 10 5 CFU/ml) in the α distillery was similar to

the δ distillery. The lowest number of the total viable, lactic acid, and anaerobic bacteria

was observed in the material from the ε distillery and was comparable with the esti-

mated value for this kind of grain (bacterial number for wheat reaches 10 5 -10 6 CFU/g)

[Obuchowski and Strybe 2001]. In this distillery rye is the raw material to fuel ethanol

production. And the lowest level of moulds and yeasts was in the β distillery. The con-

tamination by moulds and yeasts was generally on the lowest level in all five distilleries

as compared to the other contaminants. It was about 10 4 CFU/ml in the α, γ , δ, and ε

distillery and about 10 4 CFU/ml in the β distillery. The detailed data about bacterial

contamination are presented in Table 1.

The level of bacteria contaminants in sweet mash at the beginning of the fermenta-

tion, in the sweet mash after 24 hours and after complete fermentation in all tested dis-

tilleries is presented in Figure 1.

In the α distillery the highest level of total viable and lactic acid bacteria was ob-

served in sweet mash after 24 hours of the fermentation. Also the level of anaerobic

bacteria increased after 24 hours of fermentation and was the same up to the end of this

process. The number of mould and yeast cells was the same in sweet mash at the begin-

ning of the fermentation and after 24 hours. Decrease to zero was observed only just

Acta Scientiarum Polonorum, Technologia Alimentaria 8(4) 2009

K. Leja, M. Broda

Table 1. Microbiological purity of raw materials from five Wielkopolska distilleries

Materials

α β γ δ Ɛ

Total viable bacteria

6.39

7.60

7.39

6.08

4.75

Lactic acid bacteria

5.69

7.87

7.17

5.50

4.00

Anaerobic bacteria

6.53

7.81

7.17

5.00

4.20

Moulds and yeasts

4.08

3.74

4.53

4.79

4.68

The raw material from the α, β and γ distilleries was corn, triticale from the δ distillery and rye from the Ɛ one.

Fig. 1. The number of bacteria contaminants in five distilleries from Poland. Ab-

breviations: SM – sweet mash, SM_24_hrs – sweet mash after 24 hours of

the fermentation, SM_CF – sweet mash after complete fermentation

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The occurrence and identification of microbiological contamination ...

on the end of the fermentation. It is connected with run-down of oxygen – moulds and

yeasts need oxygen to grow and live. In the β distillery all groups of microorganisms,

in spite of moulds and yeasts, increased through the first 24 hours of the fermentation.

The level of this increase was lower than in the α distillery. After 24 hours of fermenta-

tion there was no moulds and yeasts. In the γ distillery situation was similar to the α one.

However, the number of bacteria contaminants in this distillery was lower. There were

no mould and yeast cells after 24 hours of fermentation yet (like in the β distillery).

All three distilleries used the same raw material – a corn.

In the δ distillery, the number of total viable, lactic acid, and anaerobic bacteria in-

creased during the first 24 hours of fermentation process. The number of bacteria con-

taminants in the sweet mash was twice as big as lower than in the α distillery, but after

24 hours it was almost the same in both distilleries. After the complete fermentation no

changes were observed. The number of moulds and yeasts increased after first 24 hours

of fermentation, and decreased at the end of the process. As similar situation was ob-

served in the Ɛ distillery – the number of moulds and yeasts increased at first and de-

creased to zero at the end of fermentation. The number of total viable and anaerobic

bacteria cells increased after first 24 hours of fermentation and at the beginning of the

fermentation. The level of anaerobic bacteria was the highest here because the fermenta-

tion is an anaerobic process. Lactic acid bacteria numbers during first 24 hours of fer-

mentation increased and decreased later – as in the α and γ distilleries.

Table 2. Bacterial contaminants in Polish distilleries

Bacterial species

Occurrence of the genus within the samples

percentage of total isolates

α β γ δ Ɛ

Bifidobacterium adolescientis

Bifidobacterium angulatum

Clostridium aerotolerans

Lactobacillus acidophilus

Lactobacillus brevis

Lactobacillus buchneri

Lactobacillus casei

Lactobacillus delbrueckii

Lactobacillus fermentum

Lactobacillus lactis

Lactobacillus paracasei

Lactobacillus reuteri

Leuconostoc carnosum

Pediococcus parvulus

Unidentified

Acta Scientiarum Polonorum, Technologia Alimentaria 8(4) 2009

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