1

PREFACE

When I started studying at the KULeuven, it was interest for nature and fascination for water
that made me choose this study. I am glad that up to now I had the chance to focus on this
subject and that the crowning work of these studies was so much in the field of my interests.
It was a bit of a struggle, the research and all the consequences that a stay abroad carries with
it. But that was what made it exciting, and whenever I am asked if I would do it again, the
answer is a whole-hearted Yes.

However, I am most aware of it that it were other people who gave me the chance to go to this
lovely country that Greece is and do this work. First of all there is my promoter, Professor
Verachtert, who also made the contacts with his colleagues in Greece. Then there is professor
Balis, the soul of the project I was working for. The people in the Nagref Institute in
Kalamata, who patiently underwent my stress, assured my comfort and helped me with their
experience. Sofie, my companion in arms who assured that there was always a bit of Belgium
around. The people back home who supported and helped me implicitly: in the first place my
mother, who gave me linguistic help. Professor Van Impe who gave me technical advice and
juggled his computer to a solution for my mathematical problems. And of course Kris, my
predecessor and tutor, and now also the severe but wise judge of my work. To all these
people I would like to say: thank you very much.

It would be nice if I had contributed my mite with this work for the improvement of the
environment in the south. But the primary objective of this work was learning. And one
thing it indisputable: I have learned a lot. And not just science.

Kontich, April 1998

SHORT SUMMERY
In the Mediterranean countries the production of olive oil has grown spectacularly the last
decades. One of the side effects of this growth is that the production of by-products that arise
during the olive pressing has rocketed. Among those by-products is the so-called alpechin or
olive mill waste water (OMW).

This black fluid contains a vast spectrum of organic

substances, among which polyphenols, is poor in nitrogen and has a very high BOD.

Presently several remediation techniques for this waste are under investigation. One of these
is the land application preceded by an aerobic treatment with a culture of Azotobacter
vinelandii. Not only does this bacterium degrade many phytotoxic components of the waste
water, but also it enriches it with nitrogen that it fixes enzymatically from the atmosphere.

The topic of this dissertation is the study of growth characteristics of Azotobacter vinelandii.
The growth of this micro-organism is first studied in batch cultures and in a next stage in a
continuous bioreactor.
The findings of this research have lead to the conclusion that A. vinelandii's growth requires
high quantities of substrate. The highest rate of nitrogen fixation in batch cultures is obtained
in the very beginning of the logarithmic growth phase, and in a continuous reactor at
residence times close to the wash-out flow rate. Isolated cultures of A. vinelandii grow well
in solutions of OMW, but when other micro-organisms are present these seem to be more
competitive.

CONTENTS
I.
II.

III.

IV.
V.
VI.
VII.

INTRODUCTION4
STUDY OF LITERATURE.5
A. The olive culture.5
1. The olive tree..5
2. History5
3. Geographic distribution..6
4. Importance of the olive culture..7
5. olive culture by-products9
B. Characteristics of olive mill wastewater...12
1. Physico-chemical properties.12
2. Toxicity.14
3. Microbiological characteristics.15
C. Solutions for the OMW problem..16
1. Use of OMW as a natural resource...16
2. Waste water treatment..18
3. Land treatment..21
4. Transformation in liquid fertilizer22
D. Azotobacter vinelandii..25
1. Taxonomy.25
2. Natural habitat...25
3. Description of Azotobacter vinelandii Lipman.27
4. Use of phenolic compounds..29
5. Nitrogen fixation...32
EXPERIMENTS.38
A. First batch experiment...38
B. Second batch experiment: nitrogenase activity.44
C. Third batch experiment: glucose consumption..48
D. Fourth batch experiment: influence of OMW...51
E. Continuous flow reactor....53
GENERAL CONCLUSION..66
APPENDICES.68
WORKS CITED..69
BONDIGE NEDERLANDSE VERTALING.79

I.

INTRODUCTION

**********************************
An old myth tells us that once upon a time, Poseidon and Athena disputed the guardianship
over a town in Attica. They decided that each would give a present to this town and that the
one whose gift was appreciated most would be the winner. Poseidon hit his trident on the
rock of the acropolis of the town and immediately, water came welling up. Athena stamped
her foot on the ground and an olive tree sprouted from the soil. The citizens judged that
Athena's present was the most valuable, and since that day this city proudly carries the name
Athens.

This story depicts well how important the olive culture is for the people in the Mediterranean
area. Since these ancient times, it has kept on growing to the level of a huge industry. But
nowadays people all over the world are getting aware of the price that has been paid for
economical welfare. The environment has reached the limits of its resilience, and Poseidon's
water threatens to become rare.

Indeed, the olive oil production as well demands vast

volumes of water and yields huge quantities of waste water that can no longer be discharged
as people used to do.

This work first gives an overview of the olive mill waste problem and the solutions that have
been proposed up to now. In a second part, you will find a description of experiments that
have been carried out in the framework of one of these solutions.

II. STUDY OF LITERATURE

*******************************************

A. THE OLIVE CULTURE

1. The olive tree
The olive tree (Olea europaea) forms part of the order of Ligustrales and the family of
Oleaceae. This family constitutes of 30 genera and 60 species. Within this family the tree
forms part of the genus Olea, constituting 30 different species all over the world. The species
Olea europaea represents two subspecies; only the subspecies sativa is cultivated (Cifferi,
1950).

2. History
An overview of the history of the olive culture and its spreading is described by MahjoubBoujnah (1997). The place of origin of the olive tree is, up to now, not clear. There are
certain indications that allow scientists to reconstruct the history of the olive culturing, such as
pits and pollen in earth layers, monuments that refer to the olive culture, etc.. Most of these
indications have been found in excavations in the eastern Mediterranean area.
The most common hypothesis is that the origin of this culture was located in the zone that
stretches from the south of the Caucasus, on the Iranian tablelands, to the Mediterranean
coasts of Syria and Palestine (Cifferi, 1950). From this area, the olive culture has extended to
the west. In the Mediterranean area, its extension was started by the Phoenicians and was
fulfilled by the Romans, who brought the olive tree to all the regions having borders with the
Mediterranean Sea (Fig. 1A).

Nowadays, the olive culture is still concentrated around the Mediterranean, but, to a smaller
extent, can also be found in other areas spread over the five continents.

Fig. Error!Argumento de modificador desconocido.: spreading and present distribution of

the olive tree (Loussert and Brousse, 1978).

3. Geographic distribution
The olive is a typical Mediterranean culture, and about 98 % of the population of olive trees
in the world is situated in the Mediterranean (table 1). Today, the culture is concentrated
mostly on the northern side of this region, especially in Spain, Italy and Greece. On the
southern coasts, the olive plantations are localised mostly in the Magreb countries (fig. 1B).

7
Table Error!Argumento de modificador desconocido.: the distribution of the olive tree per
country (C.O.I., 1988)
Country
Algeria
Angola
Egypt
Libya
Morocco
South-Africa
Tunisia
Argentina
Brazil
Chile
Mexico
Peru
U.S.A
Afghanistan
China
Cyprus
Iran
Iraq
Israel
Jordan
Lebanon
Syria
Turkey
Albania
France
Greece
Italy
Malta
Portugal
Spain
Yugoslavia
Australia
Global
Mediterranean area

number of trees (x 1000)

16,430
40
1,650
4,000
33,000
300
55,227
5,000
84
275
480
560
1,750
1,000
20,000
1,290
750
750
1,520
2,650
6,000
36,000
83,000
5,500
5,000
120,000
125,000
23
49,496
167,000
4,104
208
748,423
714,240

surface (ha)
162,800
400
10,500
100,000
330,000
2,500
1,400,000
50,000
840
3,070
6,000
5,603
14,500
128,000
6,880
10,000
10,000
12,600
16,360
32,000
327,037
820,000
39,300
44,600
758,100
1,176,556
200
1,114,000
1,087,000
26,960
2,000
8,701,697
8,451,533

4. Importance of the olive culture

a. in the world
The latest reliable figures about the olive production were published in 1995 on the
International Olive Oil Council. They regarded the situation between 1981 and 1992. The
average annual olive oil production amounted to 1,700,000 tons in this period. 78 % of this

8
quantity was produced in the EC. Spain was, and is, the leading nation in the olive culture
(figures kindly provided by Dr. Zervakis, NAGREF Institute, Kalamata, Greece).
Olive oil is the most important product from the olive culture: more than 91 % of the olives
produced in the world goes to the olivepresses. However, olive oil covers only 4 % of the
global production of vegetable oils and 2.25 % of the global market of oils and fats for the
food industry. On the local markets, on the other hand, the situation is quite different.
Several countries in the Mediterranean area do not produce any other vegetable oil than olive
oil; on average, 47 % of the total quantity of vegetable oil in this region is olive oil (Luchetti,
1993). None of the olive producing countries can cover its own total need of oil. Not only
does this mean that they must import other vegetable oils, but also that they do not export big
shares of their production: 90 % of the olive oil is consumed domestically.
In most of the Mediterranean countries, the olive culture is a rather traditional matter,
demanding a lot of labour (Trigui, 1987). Therefore, it delivers 200 million days of work
annually, or jobs for about one million people. This labour-intensity of the culture is also the
weakness of the resulting product: high production costs per unit of olive oil make this
product expensive, compared to other vegetable oils. It has been estimated that 42% of the
total production cost of olive oil consisted of the cost of labour (principally of harvesting)
(Papageorgiou, 1987).
Nevertheless, the production of olive oil increased 1.6 % every year in the period from 1981
to 1992.

The increase of the demand for olive oil can be explained by the growing

consciousness of people about their health. Olive oil is known to contain a large percentage
of unsaturated fatty acids and vitamins, and is highly resistant to oxidation in heating and
cooking.

b. Greece
As a mountainous country surrounded by the sea, Greece is characterised by a variety of
climatic and soil conditions. Olive trees are best adapted to the conditions prevailing in the
central and southern parts of the country, not too far from the sea, and on the islands. They
can grow on poor, depleted or rocky soils, where the generally poor annual rainfall is
concentrated in a few months and where summer temperatures are high (Papageorgiou, 1987).

Greece used to be the third producer in the world, with an annual production of 270,000 tons
of oil every year. 70,000 tons of this quantity were exported (figures kindly provided by Dr.

9
Zervakis). Due to the important growth in this industry (2.3 % every year) it has recently won
the second position on the worlds market.
Apart from olive oil, the olive orchards yielded 100,000 tons of table olives annually.
The estimated amount of olive trees in Greece was 130 million, grown on a surface of about
7600 square kilometres; compare this with the total surface of Greece, 133,000 km2, and
Belgium, 30,488 km2. In 1984, olive trees occupied 21.2 % of the total cultivated land of the
country.

To illustrate the importance of the olive growing for certain regions in Greece, we will give
some figures about Messinia, the first prefecture in Greece in olive tree cultivation.
Messinia is a rather rural area, located in the south-west of the Peloponnesos peninsula. The
number of inhabitants is 170,000, which must correspond to about 50,000 families (figures
kindly procured by Mr. Karavitis, NAGREF Institute of Kalamata, Greece).

The total

number of olive trees in this region is 13 million. The amount of olive oil produced annually
varies from 25,000 to 45,000 tons, the table olive production from 6,000 to 10,000 tons. 75 %
of the harvest is exported. In Messinia, 350 olive oil mills are in operation during the
harvesting season, as well as 4 olive pomace plants and 30 to 40 plants for the processing and
packaging of olive oil and olives. Currently there are 5 laboratories for analysis of olive oil.
All this provides the region with a gross total income of approx. 60 billion drachmas (9 billion
Belgian franks) annually. 3500 of the families in this area are financially dependent on the
olive tree cultivation. But many more families, from Messinia as well as from other regions,
have an extra income from their own orchards in this prefecture. Sometimes these consist of
only a few trees, but often they are quite vast. This is possible because the harvesting season
is long, giving thus the possibility to the farming family to utilise its own labour, including
children and the aged. Moreover, the peak of work requirements is in the winter, a valuable
complementary with either other crops or other activities such as tourist business.

5. Olive culture by-products

Both olive tree culture and the olive oil industry produce large amounts of by-products. It has
been estimated that pruning alone produces 25 kg of by-products (twigs and leaves) per tree
annually. It must also be considered that leaves represent 5 % of the weight of olives in oil
extraction. On the other hand, the olive oil industry produces 35 kg of solid waste (crude

10
olive cake) and 100 l of liquid waste (olive mill waste water, OMW) per 100 kg of treated
olives (Molina Alcaide and Nefzaoui, 1996).
The twigs and branches are usually burnt. The olives themselves are pressed in either threephase systems or the more modern two-phase systems. In the latter, no OMW is produced;
presses working on this system only yield olive oil and the wet solid residue, called alpeorujo
in Spanish. Hence the name "two phase system". In the more traditional three-phase system,
the amount of waste water produced is considerable (see fig. 2). Paredes et al. (1996)
estimated the amount produced in the Mediterranean area on 30 million m3 per year.
Moreover, it must be considered that this waste is produced in a relatively short period, being
the harvesting season that lasts from November to March.

Fig. Error!Argumento de modificador desconocido.: byproducts of the olive tree culture and olive oil industry

11

Olive mill waste water is generally named alpechin. The etymology of this word is uncertain;
it seems that it is derived form the Mozarabic al-pechin, and this from the Latin faecinus (of
the faeces), because of the characteristic foetid smell of this waste water (Moreno, E. et al.,
1990). A typical property of OMW is its recalcitrant black colour.
The discharge of OMW in the sewage system is illegal, since it is a corrosive liquid.
Dumping the waste in rivers is forbidden as well, given its polluting properties. Nevertheless,
both these ways of getting rid of the waste are very common.
In a second chapter, we will take a closer look at this olive mill waste water.

12

B. CHARACTERISTICS OF OMW
1. Physico-chemical properties
It is known that the properties of OMW vary considerably, and depend on the olives used, the
date they are picked, the way they are pressed, and the age of the OMW. Mouncif et al.
(1993) compared the properties of the waste water from 3 different types of mills in Morocco,
and found huge differences in all physico-chemical properties of the liquor. Fiestas Ros de
Ursinos and Borja-Padilla (1996) studied the composition of the waste water from one
specific olive mill. The results of their analysis are given in fig. 3.

Fig. 3: chemical composition of OMW (Fiestas Ros de

Ursinos & Borja-Padilla, 1996)

13
The organic load of this waste is extremely high: Lopez and Ramos-Cormenzana (1996)
reported values of 230 and 150 g/l for COD and BOD respectively. The pH oscillates around
the 4 to 5 interval and its capability as a plug is very notorious (Moreno, E. et al., 1990).
A very important characteristic of OMW is its high content in phenolic acids. Extensive
studies on the composition of the phenolic fraction of the waste water have been carried out
(Balice & Cera, 1984, Knupp et al., 1996). Some of the components are shown in fig. 4.

Fig. 4: Lewis-structures of some phenolic compounds in OMW.

The typical black colour is due to the formation of polymers of polyphenolic compounds that
get linked to the sugared remainders, proteins, and fatty acids. Saiz Jimenez et al. (1986)
propose the following as a possible explanation: in the olive pulp a large quantity of enzymes
have been found (catecholases, cresolases, peroxidases, etc.); these enzymes could be released
when the olives are crushed and come in contact with various polyphenols, thus forming the
polymer.

When proteins and amino acids are present, the phenols join these through

nucleophilic channels and aminophenols result from this union. Other compounds such as
polysaccharides, fatty acids, metals, etc. are also present. These join the polymer through
reaction or absorption to the matrix. The authors prove a similarity of the alpechin pigment to
lignin or related polymers. This explains its resistance against degradation.

14
2. Toxicity
Capasso et al. (1992) isolated catechol, 4-methylcatechol, tyrosol and hydroxytyrosol, four
derivatives of phenol, from OMW. They tested their phytotoxic effects on tomato leaves and
vegetable marrow leaves. In general, the most harmful components are catechol and 4methylcatechol. At the same time, however, these researchers surveyed the phytotoxicity of
the waste water that was deprived of its phenolic compounds by repeated extraction with
organic solvents. They still observed phytotoxicity, unfortunately without mentioning how
much. They concluded that other, unknown substances attribute to the general phytotoxicity
of OMW.

The OMW is not only toxic to plants. Bacteria suffer from it as well. It is certain that a major
part of this property of OMW is, again, due to the phenolic content.
Capasso et al. (1995) proved that four naturally occurring polyphenols, methylcatechol,
catechol, hydroxytyrosol and tyrosol, are highly toxic to phytopathogens that cause knot
disease and other diseases.

It can thus be admitted that the olive tree produces these

substances to protect itself against the agents of these diseases.

Tunel and Nergiz (1993) determined the minimum inhibitory concentration of several
phenolic acids and found out that caffeic acid is the most effective of them.
Gonzalez et al. (1990) tested the antibacterial activity of phenolic acids against Bacillus
megaterium and a collection of bacteria isolated from unpolluted soil and alpechin polluted
soil. Upon comparing the antibacterial activity of alpechin with that of the phenolic acids,
they observed that these do not agree, since only a mix of the phenolic acids at a 5 times
higher concentration gives rise to an inhibition equivalent to that of the alpechin. Strains
sensitive to the inhibitory effect of the phenolic acids are rare, and many of them belong to the
genus Bacillus, which are especially sensitive to the inhibitory effect of the phenolic
compounds of alpechin. Other possible inhibitors are free fatty acids, either alone or in
synergy with other compounds. The evidence of this is not given; on the contrary, it is proven
that the residual oil that exists in the alpechin does not influence its antibacterial effect.
Similar results were obtained by Perez et al. (1992). They compared the antibacterial effect
of waste waters from a modern mill, a traditional mill and an evaporation pool. The phenolic
content of evaporated waste is considerably lower than this of the other types of waste water.
This can be explained in several ways, polymerisation being prominent among them. The
phenolic polymeric fraction of these wastes does not have antibacterial activity. But still, this

15
waste is toxic for Bacillus megaterium.

The persistence of antibacterial effects in

concentrated evaporated wastes suggests the possible existence of at least two types of
substances responsible for this effect: the biodegradable one, present in fresh wastes, and the
more recalcitrant one, present in both fresh and evaporated wastes.

3. Microbiological characteristics
Mouncif et al. (1993) studied the naturally occurring microbial population in the waste water.
Yeasts, moulds and lactic acid bacteria were the main micro-organisms found in the OMW.
These micro-organisms are resistant to unfavourable environmental conditions such as acidic
pH, high salt concentrations and low nutritional compounds. Penicillium sp., Geotrichum
candidum and Aspergillus sp. were the most frequent moulds, Debaryomyces and Pichia the
most common yeasts.

Perez et al. did a similar study. They isolated 38 bacterial strains from OMW in chemically
defined media containing the pigment of these wastes as a sole carbon source. Most of the
organisms were Pseudomonas sp.. 4 of the 6 phenolic acids associated with polymeric
pigments in OMW were used by 3 strains. Only 1 Pseudomonas strain was able to use the 6
acids.

16

C. SOLUTIONS FOR THE OMW PROBLEM

1. Use of OMW as natural resource
Given its particular composition, many scientists consider OMW as a raw material rather than
as a waste. Numerous research programs are running to investigate whether it is feasible to
use olive mill by-products in industrial or agronomic applications. We will give a brief
overview of some of these initiatives, but it must be mentioned that to our opinion, these
applications will never operate on such a scale that they will deal with the whole quantity of
by-products. The reasons why this is so unlikely are that:
the amount of olive mill wastes produced every year is too huge, and
these wastes are produced in a relatively short course of time, and are difficult to store, so
industrial or agro-industrial equipment to process them would only be operational during a
few months. This would make it more difficult to get the investment repaid.
a. production of biopolymers
Up to now, only a few biopolymers are produced commercially on a large scale. Most of them
are exopolysaccharides (EPS), and one of the best known among these is xanthan, produced
by Xanthomonas campestris (Sutherland, 1996). Xanthan is widely used in food, cosmetics,
pharmaceuticals, paper, paint, textiles and adhesives. One of the greatest factors limiting the
production in large-scale fermentation processes is the cost of production when compared
with similar polymers from algae and plants. To reduce this cost, one could try to use less
expensive feedstock (Lopez and Ramos-Cormenzana, 1996). OMW has a high C/N ratio and
contains 4-5 % of free sugars. These are optimal conditions for xanthan-production, and
experiments were carried out by Lopez and Ramos-Cormenzana (1996) to investigate if X.
campestris brought in good quantities of xanthan when grown in OMW. Although the yield
was quite good, the results were only satisfactory for concentrations that did not exceed 30 %
(v/v).
The major drawback however was that the product kept the typical black colour of the
medium it was grown in, due to the numerous chemical compounds that remained in the
polymer. This impurity of the final product may limit its application. Olive mill waste
contains a whole spectrum of chemical compounds, so if one desired a high-quality product,
he would need an extensive (and expensive) purification-process (Sutherland, 1996).

17

The same consideration can be made for the experiments carried out by Gonzales et al.
(1996).

They investigated the possibility to win polyhydroxyalkanoates (PHAs) from

reactors where Azotobacter chroococcum was grown in diluted OMW. PHAs are especially
interesting because these biodegradable plastics have physical properties that resemble those
of polypropylene. The yields reached in this experiments were 3.15 g/l. after 48 hours in
alpechin enriched with ammonium acetate, while 4.43 g/l in a chemically defined growth
medium.

Pasetti et al. (1996) made A. vinelandii grow in diluted OMW, and used the polysaccharides
that the bacteria produced to recover heavy metals, in casu lead and cadmium. They found a
production of up to 2 mg of capsular polysaccharides (CPS) and 0.5 mg of exopolysaccharides
(EPS) per ml of culture medium after two weeks in steady state conditions. The efficiency of
CPS in removing metals from 10-4 M solutions was 3.8 mole Cd2+ and 5.5 mole Pb2+ per
mole CPS, which corresponds to 4.29 g of Cd2+ and 11.4 g of Pb2+ per kg of CPS.

2. natural antioxidants
Visioli et al. (1995) considered that, since olives and olive oil are rich in natural antioxidants,
the waste water might contain these substances as well. Interest in natural antioxidants is
increasing because of growing evidence indicating the involvement of oxygen-derived free
radicals in several pathogenic processes, such as cancer and arteriosclerosis. Visioli and his
colleagues claimed that their results demonstrate that waste water extracts, obtained with
ethylacetate, have a powerful antioxidant activity and might therefore represent a cheap, as yet
unused source of natural antioxidants.

3. animal food
Olive mill by-products could be used as animal food in both direct and indirect ways. Molina
Alcaide and Nefzaoui (1996) did research work on the digestibility of solid wastes of the olive
cultivation, but found that only fresh leaves, fresh twigs, olive pulp and extracted olive pulp
are digestible for more than 50 %. To our knowledge, no research has been done in this field
that concerns OMW.
On the other hand, one could use OMW as a medium to grow organisms that can than be used
as animal fodder. Sanchez Villasclaras et al. (1996) have done experiments with Chlorella

18

pyrenoidosa and Scenedesmus obliquus, both green algae.

In an aerated and strongly

illuminated (20-25 W/m2) bioreactor that contained 15 % (v/v) OMW, they reached a
maximum biomass production of only 1 g/m3.h.
Other micro-organisms that can be grown with OMW are fungi. Zervakis et al. (1996)
reported that some Pleurotus-species not only formed edible fruitbodies when grown on olive
press cake, but also were capable of forming mycelium when grown in OMW. These fungi
have the disposal of the enzyme laccase, that allows them to degrade lignin and related
products. By doing so they can detoxify OMW as well, and moreover their mycelium might
be used as animal fodder.
2. Waste water treatment
In general, the research carried out nowadays that concerns the OMW problem aims for
treating this waste so that it can safely be dumped either in natural watercourses or on lands.
The challenge in both cases is to detoxify this waste, which means breaking down the
polyphenols present in it and lowering its BOD and COD.

a. physico-chemical treatment

distillation and evaporation processes (Rozzi & Malpei, 1996)

These processes concentrate the organic and inorganic contents of OMW by
evaporation of the water. The main drawbacks of these processes are related to the
post-treatment and disposal of the produced emissions. A first problem can be the
disposal of the concentrated "paste". Its use as animal feed is limited by the very high
concentration of potassium.

Otherwise it can be burnt to feed the boiler which

provides the thermal energy to the distillation plant, but its combustion induces air
pollution which has to be dealt with by post-treatment of the gases. A second problem
is related to the condensate. The distillate is not made of pure water but carries away
an appreciable fraction of volatile compounds found in OMW such as volatile acids
and alcohols. These compounds are the cause of the high COD of the condensate,
which can reach 3 g COD per litre, and make necessary an additional treatment of the
distillate prior to discharge or reuse.

19

Common evaporation ponds do not have this problem, but Cabrera et al. (1996)
reported that serious negative environmental impacts on nearby areas are caused due to
the foul odours, insect proliferation, leakage and infiltration.

flocculation-clarification (Rozzi & Malpei, 1996)

This process is not very efficient in reducing the concentration of pollutants in OMW
because most organics found in these waste waters are difficult to precipitate (e.g.
sugars and volatile acids). Tests have been performed which show that the removal
efficiency of heavy liming is in the order of 40 to 50 % (Mendia & Porcino, 1964).

membrane processes (Rozzi & Malpei, 1996)

Membrane processes are not suitable for the treatment of OMW because of the limited
concentration factor that can be obtained with this method.

Moreover, the final

products (the retentate and the permeate) have to be processed prior to disposal. The
former is a liquid without much use and the latter must be post-treated because it still
contains organics of which the COD is far from negligible.

decolorization by chemical means

Flouri et al. (1996) tested the possibility of treating OMW with chemicals and fungi of
the Pleurotus genus in order to decolorize the liquid. Their motivation was that the
colour is due to polymers of low molecular weight phenolics, so decolorization is an
important objective in the search for a method to eliminate its pollutant properties,
whereas colour alone is a monitor of the level of pollution. Hydrogen peroxide turned
out to be the most effective chemical substance for reducing the colour of the waste.
However, what if one takes a closer look at this method? 50 % decolorization was
obtained after treating one litre of OMW with 45 g of H2O2. To decolorize the total
amount of waste water produced annually, one would need 1.35 million tons of H2O2.
Given the costs of the hydrogen peroxide itself and the consequences of producing,
transporting and applying it, the question rises whether the remedy isn't worse than the
disease. Moreover, Flouri et al. (1996) do not explain what happens to the waste after
treatment with hydrogen peroxide. Beilstein (1923) writes that hydrogen peroxide
oxidizes phenolic substances to quinones. We did not find an answer to the question
whether this is the final product of this waste water treatment.

20

b. anaerobic treatment
Fiestas Ros de Ursinos and Borja-Padilla (1996) studied the possibilities to digest OMW
anaerobically and gain methane gas from this process. They suppose that 80 % of organic
components are biomethanizable. This would produce a yield of 37 m3 of methane per m3 of
vegetation water, with an energy output of 325 kWh. The main problem in this method is the
toxicity of polyphenols towards the anaerobic bacteria in the reactor. Therefore, a 3-stage
system is proposed.
In a first stage, most of the polyphenols are degraded aerobically. The most efficient microorganisms for this job are Aspergillus and Azotobacter species. Also in this stage, the COD is
reduced by 40 to 50 %, and suspended solids, colloidal substances and some of the mineral
salts are eliminated.
In the second stage, the actual biomethanization takes place. The hydraulic residence time in
this unit is 5 days, the purification efficiency up to 80 %.
In the third stage, the COD is further reduced in another aerobic reactor. The waste water can
eventually be applied on lands as a natural fertilizer.
The researchers calculated that this installation would be economically profitable.

Hamdi (1992) found that the darkly coloured pigments in OMW reduce the speed of its
biodegradation, whereas the long chain fatty acids, tannins and simple phenolic compounds
are responsible for its toxicity for methanogenic bacteria.

Hamdi et al. (1995) pre-treated the waste with Aspergillus niger, and found that this enhances
the anaerobic biodegradation. A similar inquiry was done by Borja et al. (1995), who used
Aspergillus terreus and obtained similar results.

Zouari and Elouz (1996) proposed to pre-treat the waste water with resin before applying
anaerobic digestion, in order to adsorb the coloured olive compounds.
c. aerobic treatment

21

Benitez et al. (1997) treated OMW in an aerobic batch reactor using activated sludge from a
municipal waste water treatment plant. They developed a kinetic expression, based on the
Contois-model, to predict the degradation of the waste in the reactor. In experiments where
the initial COD was varied, a direct effect of this variable on the total COD removal was
deduced: an increment of initial COD leads to a diminution of the COD conversion.
The total phenolic content was diminished more than 90 % after seven days through every
experiment.
Ramos-Cormenzana et al. (1996) did tests with Bacillus pumilus and found that this organism
breaks down the phenolic components of OMW.

The ideal concentrations for this

degradation are 40 and 100 vol. % of OMW.

3. Land treatment
The idea of fertilising land with OMW is not new: two centuries BC, Marcus Porcius Cato
advised supplying OMW to improve the fertility of his contemporary farmers' lands (Tomati
and Galli, 1992).
The advantages of the agronomic use can be summarized as follows:

an economical way to dispose waste water,

an effective use of plant nutrients contained in the waste,

a supply of organic matter to improve soil fertility.

The disadvantages are:

the possible polluting load,

the high content of mineral salts,

the presence of organic phytotoxic compounds,

the difficulty in storing and distributing the large quantity of liquid waste, produced during
a short rainy period.

The crops that benefit most from OMW treatment are olive trees and vineyards. Potatoes
react badly to it. The soil itself might degrade in the long term due to the breakdown of its
structure and salinization. The pH of the soil drops initially but tends to return to the initial
value. The number of micro-organisms decreases first, increases to a higher value than before
the treatment and finally returns to its original level. Especially the increase in nitrogen fixers
is very important, not only because of the positive influence on the nitrogen-level in the soil,
but also because these bacteria are good producers of growth-regulating substances, which
play a fundamental role in plant metabolism. On the other hand, some root pathogens, in

22

particular oomycetes, are strongly suppressed when OMW is applied (Tomati and Galli,
1992).
Cabrera et al. (1996) did analogue research and concluded that OMW can be applied at 600
litres per m2 per year on calcareous soils with low permeability, otherwise the risk for
pollution of the water table is too high.

4. Transformation in liquid fertilizer

This work concerns the method as proposed by Chatjipavlidis et al. (1997). OMW is first
treated aerobically and afterwards applied as a liquid fertilizer.
The idea of this method is that OMW constitutes a rich and highly selective nutrient medium
favouring the proliferation of free living N2-fixing bacteria of the genus Azotobacter.
After a pre-treatment with 1 % calcium hydroxide to increase the pH to 11 or 12 and a
subsequent pre-treatment with hydrogen peroxide (up to 1 %), the mixture is transferred into
the bio-reactor where an enriched population of Azotobacter vinelandii has already been
established. The bacterial strain "A" of Azotobacter vinelandii has been isolated from soil
repeatedly treated with OMW.
During this second stage:
a strong N2-fixing activity is manifested,
the OMW phytotoxic constituents are biodegraded;
microbial exopolysaccharides are produced in large amounts;
the micro-organisms produce growth-promoting factors (auxins, etc.);
the microbial populations and their metabolites enhance soil suppressiveness against soilborne fungal root pathogens such as Pythium and Phytophthora species.

Fig. 5: Schematic diagram of the diazotrofic bioremediation of OMW (Chatjipavlidis

et al., 1996).

23

The product obtained is a thick, yellow, non-phytotoxic liquid, with a pH about 7.5 - 8.0. It is
characterized as an 'organic soil-conditioner biofertilizer in liquid form' with the following
characteristics:
The exopolysaccharides that are present favour the formation of stable soil aggregates and
thus contribute to the improvement of soil tilth and structure.
It contains almost all the major and trace plant nutrients that were contained originally in
the olive fruits and passed into the water fraction of the wastes.
It is biologically enriched with organic forms of nitrogen through the mechanism of N2fixation at the expense of the carbon sources of the wastes (sugars, organic acids, phenolic
compounds, etc.), and with plant growth promoting factors (auxins, cytokinins).

It constitutes a soil microbial inoculant that allows the establishment of rhizospheric

micro-organisms favourable to plants.

The biofertilization efficiency of the product was examined in field trials with olives, vines
and potato. When 100 kg of biofertilizer was administered per olive tree, comparable yields
were obtained as with 5.2 kg of a chemical fertilizer. Similar or better results were noted in
the experiments with the other crops.
Therefore, this product is rendered particularly useful for the exhausted Mediterranean soils
and offers perhaps a valuable tool in developing a sustainable agricultural system.

Balis et al. (1996) developed a kinetic model to predict the nitrogen fixation rate in a batch
culture:

y = yo [1 + (1 + )kt]e-kt
where , yo and k are parameters to be determined experimentally and y represents nitrogen
fixation. The equitation turned out to fit nitrogen fixation by A. vinelandii in OMW fairly
well.
From Balis' experiments, it was clear that hydrogen peroxide has an inhibiting effect on the
nitrogenase activity. CaO on the other hand is indispensable for the adjustment of the pH;
Azotobacter's nitrogen fixation activity is much lower in acidic conditions.

24

Papadopoulou et al. (unpublished data) studied the bio-remediation of OMW inoculated with
A. vinelandii in a biowheel type batch reactor under non-sterile conditions. Initially, the waste
was inoculated at 105 cells per ml. 5 days after operation (first cycle), 70 % of the processed
product was removed and was replaced with fresh OMW. Another 5 day long incubation
followed without new inoculation (second cycle). A. vinelandii developed the same patterns
of growth and nitrogen fixation during both consequent cycles which, as the authors expect,
indicates that the process may be well standardised and repeated, at least in a lab scale reactor.
Azotobacter vinelandii was growing at very small rates for the three first days of each cycle
before showing a flush and reaching a peak on day 4 of each cycle. The slow growth of A.
vinelandii observed during the first bio-remediation stages is to be expected since the
concentration of polyphenols in the medium is high. A very similar growth retardation has
been observed for Azotobacter species by Garcia-Barrionuevo et al. (1993).
Initially only few micro-organisms other than A. vinelandii could form colonies in plates
containing nitrogen-free Rennie medium. Direct measurement of total or specific phenolic
content was not performed during bio-remediation, but the proliferation of various microorganisms plated in the same medium four days after the start of each cycle indicates a
significant reduction on polyphenol toxicity allowing many micro-organisms, including fast
growing fungi, to grow. This led to a sharp reduction of A. vinelandii population on day five
of each cycle, apparently due to microbial competition.
Contrary to growth, N2-fixation did not show a lag phase, and increased drastically reaching a
peak during the first 2 days after the start of each cycle, but it was not sustained after day 3 in
both cycles. Comparison of the nitrogen fixation with the population data shows therefore
that the early nitrogen fixation flush is followed by a later population flush in both cycles,
resulting to a dramatic reduction of the nitrogen fixation potential per A. vinelandii cell as the
bio-remediation process proceeds. The data suggests a late shift of metabolism from energy
demanding nitrogen fixation and wasteful respiration (perhaps to achieve micro-aerophilic
conditions favouring nitrogenase activity) to rapid biomass synthesis.
Although the A. vinelandii population pattern against time was similar for both cycles the
absolute population numbers tended to be smaller during the second cycle. This is probably
the result of microbial competition from micro-organisms remaining with the inoculum in the
biowheel after the first cycle. This suggests that perhaps removal of all the bio-remediation
product and addition of new inoculum at the end of each bio-remediation cycle, or even
continuous inoculation may improve bio-remediation efficiency.

25

D. AZOTOBACTER VINELANDII
The idea of using Azotobacter vinelandii for the processing of OMW is barely surprising.
This bacterium unites some interesting qualities that make it almost ideal for this application.

1. Taxonomy (Thompson and Skerman, 1979)

In 1901, Beijerinck isolated and described aerobic, heterotrophic bacteria capable of fixing
nitrogen non-symbiotically for which he created the genus Azotobacter with two species, A.
chroococcum and A. agilis. His definition of the genus was:
Azotobacter. Thick bacteria, in young stages diplococci or short rods, 4-6 m or
less, sometimes however much longer, often containing a vacuole, surrounded by
a slime layer of variable thickness. Young cells are more or less motile, with a
single polar flagellum or bundles of 4-10 flagella also situated at the poles, these
flagella being about as long as the bacteria themselves.

No endospores.

Azotobacter is an oligonitrophile, which means it can grow in nitrogen-deficient

media with suitable sources of carbon; it fixes atmospheric nitrogen and can
therefore compete with other micro-organisms on such media. This is the basis
of methods for obtaining pure cultures and studying impure cultures.

Pure

cultures grow on a variety of media, but best on those that are nitrogen deficient.
Optimum temperature about 28 C.

Two years later, Lipman described a third species of the genus Azotobacter, A. vinelandii,
isolated from a soil in Vineland, New Jersey, U.S.A..

2. Natural habitat (Thompson and Skerman, 1979)

The occurrence of Azotobacter in soils has been extensively studied and was reviewed by
Jensen, H. (1965). However, much of the information probably refers to only one species of
Azotobacter, A. chroococcum. Geographically, Azotobacter is widely spread, apparently only
very rare in polar regions. Many surveys in all continents have detected Azotobacter in 30 to
80 % of soil samples, but cell densities in positive samples are usually less than tens of
thousands per gram of soil. Rarely is Azotobacter present in soil more acidic than pH 6.0 and
this corresponds with the minimal pH value for growth of most species in pure culture.

26
The nutrients that Azotobacter requires for growth are a non-nitrogenous organic substrate,
phosphorus, sulphur, potassium, calcium, magnesium, iron and molybdenum (Jensen, H.,
1954). Attempts to correlate the occurrence of Azotobacter with soil concentrations of these
nutrients have been largely unsuccessful, although some positive correlations with available
phosphorus have been obtained. (Jensen, H., 1965) Under otherwise suitable conditions, the
addition of organic substrates such as simple carbohydrates, organic acids and alcohols to soil
results in rapid multiplication of Azotobacter and the supply of such substrates in soil, most
likely organic acids and alcohols, is probably a major factor determining Azotobacter numbers
(Jensen, H., 1965). Jensen indicated that the moisture tensions required for multiplication of
Azotobacter in soil are of the same order as those required for the growth of higher plants with
an apparent optimum at pF 2.8, but that Azotobacter can survive as cysts in air-dried soil.
He also considered that there must be long periods in cool regions when subminimal
temperatures limit active growth of Azotobacter.
Most soil surveys for Azotobacter have yielded almost exclusively A. chroococcum and rarely
A. beijerinckii and A. vinelandii. Winogradsky (1938) noted the apparent rarity of A.
vinelandii in soil, stating that he had succeeded in isolating it only once during a dozen years
of research. Jensen (1965) stated that A. vinelandii may be relatively common in tropical and
subtropical soils but gave no evidence to support this suggestion.
Even when enrichment media, claimed to be selective for A. vinelandii, have been used, this
species has not been frequently isolated from soil. Azotobacter vinelandii was detected only
in a few garden soils out of a considerable number of cultivated soils (Jensen, V., 1961)
and in 6 of 18 soil or water samples (Claus and Hempel, 1970). Unfortunately, the latter
authors did not present separate results for soil and water samples.
In the rhizosphere, a zone of soil immediately adjacent to plant roots, total numbers of microorganisms are many-fold greater than in root-free soil. Azotobacter numbers, however, are
either unaffected or decreased in the rhizospheres of most plant species (Jensen, H., 1965a).
This general absence of a rizosphere effect on Azotobacter could be because root exudates are
on average quite nitrogenous, favouring other micro-organisms and that many rhizospheres
might be too acidic for Azotobacter. Strzelczyk (1961) has shown that other micro-organisms
in the rhizosphere can antagonize Azotobacter. The only exception to this rule is A. paspali,
which seems to be strictly rhizophylic.
Ruinen (1956) found large populations of saprophytic bacteria and fungi on the leaves of
higher plants in Indonesia and used the term phyllosphere for this habitat. Later, Ruinen
(1961) detected Azotobacter in the phyllospheres (95 % of samples) of natural forests species,

27
and cultivated tree species including citrus and cacao, in Surinam. She claimed that the
species present were A. chroococcum, A. vinelandii, A. beijerinckii, A. agilis, and A. insignis.
It appears that many fresh waters contain one or more species of Azotobacter but little is
known of their distribution in this habitat or of the controlling factors.

In Denmark,

Azotobacter was detected (Jensen, V., 1955) in all except completely stagnant waters, but
whether this is generally true is unknown. In France, Winogradsky (1938) considered that A.
vinelandii could be more readily isolated from fresh, unpolluted water than from soil.
In a review of marine microbiology, Wood (1958) indicated that Azotobacter was apparently
absent from open seas and considered that strains infrequently isolated from estuaries had
arrived there from other habitats. However, one species, A. miscellus, has been found widely
distributed in the Black Sea in surface water, in mud and on the surface of thallophytes
(Pshenin, 1964).

3. Description of Azotobacter vinelandii Lipman.

Thompson and Skerman (1979) summarized the findings of 80 years of research by numerous
scientists and characterized this species thoroughly.

a. cell morphology
Cells are rods, axis straight, ends rounded, occurring singly and
in pairs. The mean cell dimensions are 3.0 to 4.5 m long and
1.5 to 2.4 m wide. Cells are Gram-negative. Cells from 1 to 2
day old cultures are motile with numerous peritrichous flagella of
normal wave-shape, mean flagella dimensions are 2.4 to 2.9 m
in wavelength and 0.39 to 0.55 m in amplitude. In 2-day and
older cultures the cells (which may then be ellipsoidal) contain
numerous sudanophilic and metachromatic granules in random

Fig. 6: A. vinelandii.
The bar represents 4
m (Vela & Rosenthal,
1972).

positions. Microcysts are present in older cultures, but endospores

are not produced.
Gonzalez Lopez an Vela (1981) found that in dialysed soil media, Azotobacter vinelandii cells
are smaller than in Burk nitrogen free medium: their length is only 0.3 m. Large cells seem
to occur only in the logarithmic growth phase, the small form is the more natural.

28
b. colony morphology
The strains grow relatively fast on nitrogen-free glucose agar at 28 C, the colonies becoming
macroscopic after 2 days incubation. One-week-old colonies are generally 2-6 mm diameter,
opaque, low convex or high convex, viscid, glistening and smooth. Variant colony forms,
generally smaller and butyrous, may arise through dissociation in the quantity of extra-cellular
polysaccharides produced.
Non-diffusible pigments are not produced. On iron-deficient media, diffusible, daylightvisible yellow-green pigments and ultraviolet fluorescent, yellow-green pigments are
produced.

c. Requirements for growth

All strains are capable of fixing nitrogen and growing with molecular nitrogen as sole source
of nitrogen. Ammonium and nitrate, but not glutamate, can also be utilized as sole source of
nitrogen for growth.
The minimum is pH 6.0 and the maximum is 10.0.
Out of those temperatures tested the minimum is 14 C and the maximum is 37 C.
The strains are obligatory aerobic although it is known that the efficiency of nitrogen-fixation
(milligrams of nitrogen fixed per gram of carbohydrate consumed) increases with decreasing
p02 values from 0.2 atm to around 0.04 atm and that high aeration rates may inhibit growth in
nitrogen-free media.
Acid is not produced fermentatively from glucose.
Catalase, cytochrome oxidase and peroxidase are produced.
No strain is antagonistic to Gram-positive bacteria.

Kauffman and Toussaint (1951) found that the optimal pH was 7.5 and that the optimal
temperature for growth was a broad interval around 28 C.

29
4. Use of phenolic compounds
In the soil, the natural environment of A. vinelandii, humus is slowly but constantly degraded,
releasing a variety of monomeric components including some phenolic acids (Wu et al.,
1987).

These energy-rich substances are available only to a few members of the

autochthonous flora of the soil and the Azotobacteraceae are prominent among these. They
probably do not have to compete for carbohydrates in nature if an ample supply of utilizable
substrates such as the phenolic acids is available. In Wu's experiments, A. vinelandii grew
well in medium made from soils and distilled water that contained little or no carbohydrates.
Those soils contained syringic, ferulic and other, unidentified aromatic acids.

Nitrogen

fixation, however, was not observed, probably because sufficient mineral nitrogen was
present in the soils.

Moreno, J. et al. (1990) tested growth of A. vinelandii in 4 different media: with ferulic,
vanillic, p-hydroxybenzoic and coumaric acid. They observed that of these substances, phydroxybenzoic acid is the only one that yields good growth.

Chen et al. (1993) surveyed six free-living nitrogen fixing bacteria, among which Azotobacter
vinelandii, for their ability to grow and fix N2 using aromatic compounds as sole carbon and
energy source. They compared 6 media: a mixture of 58.4 mM sucrose as reference, benzoate,
catechol, naphtalene and 4-hydroxybenzoate at 1mM, and protocatechuate and 4-toluate at 2
mM. Protocatechuate and 4-hydroxybenzoate did not significantly inhibit the growth of A.
vinelandii, benzoate and catechol yielded a 60 % growth rate and naphtalene and 4-toluate
only a 40 % growth rate. Nitrogenase activity was substantially higher when the bacterium
was grown with protocatechuate (about 1.5 times more than with sucrose), lower when grown
in 4-hydroxybenzoate and 4-toluate, and similar with the other carbon sources.
Chen and his colleagues isolated the ring-cleavage enzymes from A. vinelandii during its
growth on different media, and concluded that both ortho and meta cleavage are possible for
all substances. Therefore, Azotobacter vinelandii produces both catechol 1,2-dioxygenase and
catechol 2,3-dioxygenase during growth in catechol, naphtalene and benzoate.

4-

Hydroxybenzoate, protocatechuate and 4-toluate induce the production of protocatechuate

3,4-dioxygenase and protocatechuate 4,5-dioxygenase.

30

Fig. 7 and 8: Ortho (above) and meta (right) cleavage pathways

for the degradation of aromatic substances in Azotobacter
(Gibson, 1984).
The final product in the degradation through the ortho pathway
is -ketoadipate-enol-lactone, which is further degraded to ketoadipate and finally ends up as succinyl CoA and acetyl CoA.

In the article, the writers included an important remark: it is known that many cleavage
dioxygenases, particularly meta cleavage enzymes, are encoded on plasmids in a variety of
bacterial species. Many of these plasmids may be transferred between organisms representing
different species or genera, greatly complicating interpretation of dioxygenase expression.

Hardisson et al. (1969) clarified the complete pathways of the degradation of aromatic
substances, as illustrated in fig. 7 and 8.

31
It is worth mentioning that exactly those substances that yielded the lowest bacterial growth in
Chen's experiments, naphtalene and 4-toluate, are not mentioned as naturally occurring in
soils by Gieseking (1975). It is thus not surprising that A. vinelandii does not have the
disposal of very efficient enzymes to degrade those compounds.
However, Balajee and Mahadevan (1990) report that Azotobacter vinelandii is capable of
degrading benzenoid compounds with nitro, amino or halogen substituents such as the
herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The authors have proved that the genes for
the assimilation of 2,4-D are encoded on a 78 kb plasmid.

Moreno and Vargas-Garcia (1995) compared growth and nitrogen fixation of Azotobacter
vinelandii in batch cultures in two different conditions: first in Burk medium containing 28
mM glucose as carbon source.

Then, they grew the bacteria in the same medium

supplemented with the same amount of glucose, but amended with 36 mM p-hydroxybenzoic
acid.
They concluded that glucose is used preferentially. In cultures that contained the mixture of
the 2 carbon sources, this caused a biphasic growth curve: first, glucose was used up, and only
then the bacteria produced the enzymes to degrade 4-hydroxybenzoate. This phenomenon is
called diauxie. In general, the same amounts of bacteria were measured in both conditions.
When it came to nitrogen fixation, however, p-hydroxybenzoic acid turned out to be a better
substrate: the values measured in the cultures that contained both glucose and 4hydroxybenzoate were up to 30 % lower than those in cultures with 4-hydroxybenzoate as the
sole carbon source. As soon as the glucose was used up, the bacteria took advantage of the phydroxybenzoic acid and higher amounts of nitrogen were fixed.
Nevertheless, the oxygen consumption was lower when no glucose was present. This seems
to be contradictory to the theory of respiratory protection of the nitrogenase enzyme: more
nitrogen fixation, but still a lower respiration rate.

32
5. Nitrogen fixation
Every living organism needs nitrogen in its diet to grow and multiply. Plants and many
primitive organisms tend to take up nitrogen as ammonia or nitrates, animals usually live on
amino acids. There is yet another reservoir of nitrogen, huger than any other: the earths
atmosphere. It consists of 79 % N2, the most stable form of nitrogen. Breaking the triple
bond between the two nitrogen atoms requires an energy input of 710 kJ per mole (Hoornaert,
1992). Molecular nitrogen from the atmosphere can be fixed by certain prokaryotes and
incorporated as biomass.

In order to do this, these organisms need an enzyme called

nitrogenase.

a. Nitrogenase
Nitrogen-fixing bacteria can carry out the 6-e reduction of molecular nitrogen to ammonia and
so convert an inert form of nitrogen into one that is readily assimilated by biological systems
(Walsh, 1979).
6 H+ + N2 + 6 e-

2 NH3

Nitrogenase activity is displayed by a two-enzyme complex that has been difficult to purify
and study because of its extreme sensitivity to oxygen-mediated inactivation (substantial to
complete and irreversible inactivation on exposure to air or less than a minute).
The stoichiometry of the nitrogenase from Clostridium pasteurianum and Klebsiella
pneumoniae is the following:
N2 + 3 Fe.dox.(FeII) + 12 ATP nitrogenase 2 NH3 + 3 Fe.dox.(FeIII) + 12 ADP + 12 Pi

Two features of the stoichiometry warrant comment. The electron-donating cosubstrate is

reduced bacterial ferredoxin, which contains two (4 Fe)/(4 S) clusters, each capable of 1electron transfer. Thus 3 ferredoxin-FeII represent six electrons. ATP also is a required
cosubstrate, undergoing enzyme catalyzed hydrolysis to ADP and Pi. A ratio of 12 ATP
molecules per N2 molecule reduced has been obtained consistently. The thermodynamic
driving force is 12 x (-7) = -84 kcal (-352 kJ) per mole of N2 reduced, a costly process on that
basis alone. It could be used in some way to "ratchet down" the potential of redox sinks in
nitrogenase.

33
Purified nitrogenase contains two protein components, termed molybdoferredoxin (MoFd)
and azoferredoxin (azoFd), each containing Fe/S clusters as the names indicate. In addition to
nitrogen, a variety of other substrates can undergo reduction. Acetylene can be reduced by 2
electrons to ethylene and also (less well) by 4 electrons to ethane.
HCCH + 2 e- H2C=CH2 + 2 e- (slow) H3CCH3

Additionally, the two-enzyme complex shows hydrogenase activity: 2-electrone reduction of

2 H2, using one molecule of reduced ferredoxin as electron donor.
Fe.dox.(FeII) + 2 H+ nitrogenase H2 + Fe.dox.(FeIII)

Azotobacter vinelandii can synthesize three different types of nitrogenase which differ in
metal content. One contains iron and molybdenum, a second has iron and vanadium, and the
third has only iron. Proteins that regulate expression of nitrogenase genes (nif genes) sense
environmental stimuli such as ammonia, the metals molybdenum and vanadium, and oxygen.
Nitrogenase requires the products of approximately 20 nif genes for its synthesis and activity.
The nif genes are not expressed if sufficient fixed nitrogen is available in the environment
(Blanco et al., 1993).

b. protection of the nitrogenase

A major difficulty for aerobic nitrogen fixing bacteria is oxygen. Nitrogenase can only
function at low redox potential levels, and is therefore sensitive to molecular oxygen as
present in the atmosphere. Oxygen induces a so called switch-off of the nitrogenase, or might
even irreversibly damage the enzyme. On the other hand, oxygen is of vital interest for the
other cells functions of Azotobacter vinelandii. This bacterium has four different ways to
protect itself against the fatal effect of oxygen on nitrogenase.

High respiration rate.

Consuming the incoming oxygen at a very high rate is the simplest solution. Liu et al.
(1995) showed that under increased oxygen stress, there was a simultaneously increased
production of the non-coupled cytochrome d terminal oxydase.

34
To cope with incoming oxygen, Azotobacter vinelandii must not only have the appropriate
enzymes, there must be a reductans as well. This reductans is the cells carbon and energy
source, typically a sugar. Kuhla and Oelze (1988) showed that the switch-off behaviour
of Azotobacter vinelandii is completely dependent on the rate of supply of the energy and
carbon source.

Enzymatic ways.
Enzymes can remove oxygen in different ways: the nitrogenase enzyme itself can destroy
it in a so-called autoprotection reaction (Linkerhgner and Oelze, 1995):
Nitrogenase + O2 nitrogenase + O-radicals

Another option is that O2 is reduced to H2O2 by cytochrome d (Lemberg and Barrett,

1973).

This coenzyme can deliver electrons rapidly, but this kind of respiration is

uncoupled with ATP formation. It is thus a very energy-inefficient process.

Indeed, Haddock and Jones (1977) ascertained that the growth of Azotobacter under
nitrogen-fixing conditions in the presence of a high pO2 is characterized by a higher
respiratory activity and a lower molar growth yield (Ysubstratemax) than during growth under
conditions of oxygen limitation.

In these reactions and due to leakage of electrons from the respiratory chain, oxygenradicals such as superoxide (O2-) are formed. These radicals are reduced to hydrogen
peroxide by superoxide dismutase (Dixon and Webb, 1979). The hydrogen peroxide
formed is reduced by catalase.
The evidence that superoxide dismutase and catalase are involved in the protection of the
cell under oxygen-stress is given by Dingler and Oelze (1987). Moreover, they found that
little superoxide dismutase is formed when ammonium is present.

This is a clear

indication that the whole system is indeed meant to protect nitrogenase.

Conformational change of nitrogenase

Moshiri et al. (1994) pointed out the mechanism of conformational change of the
conventional molybdenum nitrogenase complex. This way of protecting the enzymes

35
occurs mostly upon energy starvation. Such a conformational change leads to a so-called
"switch off" of the nitrogenase enzyme.
A rather surprising article was published by Thorneley and Ashby (1989). They proved
that oxidized components of Azotobacter nitrogenase associate with an iron-sulphur
protein, the so-called Shetna protein, to form an oxygen-stable ternary protein complex,
that allows the bacterium to keep on fixing nitrogen.

Change of cell morphology

Dingler and Oelze (1987) report that A. vinelandii swells under oxygen stress. In this
way, the cell's surface-volume ratio decreases. This makes it easier for superoxide and
catalase, which are concentrated in the cell membrane, to keep the radicals out of the
cytoplasm.

c. influence of phenolic compounds

A lot of research has been carried out to find the relation between nitrogen fixation and the
metabolism of phenolic compounds. Uzenskaya and Syrtsova (1984) showed that phenolic
acids have a inhibiting effect on Azotobacter vinelandiis nitrogenase. Reduction of the
nitrogenase enzyme requires ATP hydrolysis:

It is known that ATP hydrolysis and nitrogenase reduction are uncoupled to some degree:
even when no Na2S2O4, the electron donor in vitro, is provided, still 30 % ATP hydrolysis is
remaining.
Uzenskaya and Syrtsova found out that hydrophobic phenolic compounds inhibit the
hydrolysis of ATP, so the reduction of nitrogenase is not affected directly. More hydrophilic
phenolics on the other hand, such as pentachlorophenol, inhibited the coupling site between
the two reactions; even when these compounds inhibited the nitrogenase activity fully, ATP
hydrolysis was still observed, although reduced to 30 percent.

The mechanism of this

inhibition was not explained, but it is assumed that a proton from the phenolic compounds
plays a key role. The I50 value, i.e. the concentration at which nitrogenase is reduced to 50 %,

36
of different phenolic substances was compared. It turned out that the lower the Pka value, the
lower the I50 value. So stronger acids are stronger inhibitors.
On the other hand, there is evidence that phenolic components might even enhance nitrogen
fixation (see above: "use of phenolic compounds").

Werner et al. (1982) obtained similar results with a close relative of Azotobacter, Azospirillum
brasiliense. Phenol seemed to give a kind of protection against oxygen, although it was
neither used as a carbon source nor built in the cell.

Balajee and Mahadevan (1990) revealed a relation between nitrogen fixation and the breakdown of phenolic compounds due to an important similarity in the genes that encode the
enzymes for these 2 processes. They stated that the enhancement of nitrogen fixation yields
an enhancement of degradation of phenolic compounds. They proved this by comparing the
degradation of 2,4-D in media with and media without fixed nitrogen.

This could be

explained by the respiratory protection theory: when no nitrogen is present, nitrogenase must
be produced and protected. This protection is achieved by a high respiration rate, requiring a
lot of substrate. Therefore, it is of great interest to the bacterium that phenolic compounds,
very likely to be present in its natural environment, can be degraded quickly.

37
d. influence of OMW
The next logical step to make is only a small one: if we know that this is the effect of phenolic
compounds on Azotobacter vinelandii, then what will be its behaviour in waste water
containing considerable amounts of phenolic substances?
Garcia-Barrionuevo et al. (1993) did research on this topic, but they worked with Azotobacter
chroococcum in both artificial Burk medium and dialyzed soil medium, the latter more
resembling Azotobacters natural environment.

Their experiments led to the following

conclusions:

In chemically defined medium, no nitrogen fixation is present when nitrogen has been
added to the medium. In this case, OMW has no inhibiting effect on the growth of
Azotobacter chroococcum, and 15 % of OMW added to the medium can even replace
glucose as a carbon source.

In the same medium, devoid of nitrogen source other than molecular nitrogen, OMW has
an inhibiting effect on both growth and nitrogen fixation and can not replace glucose as
carbon and energy source.

In dialyzed soil medium provided with 10 or 15 % of OMW the bacteria need an

adaptation time of 24 to 48 hours during which they grow less than in the same medium
without OMW. After this period, however, the waste water seems to have a stimulating
effect on both growth and nitrogen fixation.

These findings do not allow one to draw a simple conclusion. In general it seems that OMW
does not have a positive effect on Azotobacter chroococcum. The stimulation of its growth in
dialyzed soil medium could perhaps be explained by the supplement of minerals or carbon
that is provided with OMW.

Papadelli et al. (1996) concluded from their experiments that the induction of nitrogenase of
Azotobacter vinelandii is observed earlier when the bacteria grow in OMW than in chemically
defined media.

38

III. EXPERIMENTS
*****************************

A. FIRST BATCH EXPERIMENT

1. Goal
In order to prepare ourselves well to the later experiments with A. vinelandii cultures, we did
a preliminary study on its growth in batch cultures first. This had to allow us to

calculate some characteristic numbers of the exponential growth phase as the generation
time and the specific growth rate ,

get an idea of the duration of the lag-phase and the exponential phase,
compare Rennie-medium with Burk-medium.

2. Materials and methods

a. cultures
Two batch reactors were followed up: one was a 2-litre conical flask containing 750 ml
nitrogen-free Rennie medium, the other was a similar flask with 750 ml nitrogen-free Burk
medium. For details about the composition of the media we refer to appendices 1 and 2.
The two reactors with their medium were stoppered with cotton plugs and autoclaved for 20
minutes at 121 C. After having cooled down, they were inoculated with 0.75 ml of the same
medium that contained an overnight culture of Azotobacter vinelandii strain A. The reactors
were continuously shaken at a temperature of 24 C. Samples from the reactors were taken
with sterile pipettes.

39
b. population estimation

by optical density
The O.D. was measured with a Hitachi 100-40 spectrophotometer of which the light
source, a regular wolfram lamp, was set at 600 nm. In it, we put three silica sample flasks
of which two contained ca. 2.5 ml fluid from the batch culture and one contained distilled
water. The O.D. of sterile medium was compared with the O.D. of distilled water; for
Rennie medium it remained constant at ca. 0.013, for Burk it was approximately 0.020.

by plate counts
Each time a series of tenfold dilutions in sterile distilled water was made.

From the

dilutions containing a presumable concentration of 300 to 3,000 cells per ml, 0.1 ml was
plated in triplicate in agar plates made with the same medium as the corresponding
sample. The plates were then placed in a Memmert U 50 incubator at 29 C. The colony
forming units (c.f.u.) on the plates were counted 2 or 3 days later, depending on the
colonies' stage of development.

3. Results and discussion

The measurement of the optical density (O.D.) of a sample from a batch culture was by far not
an accurate way to estimate the present population, but it gave an idea of the populations
order of magnitude. This allowed us to determine which dilutions we had to use to make
plate counts.
During the first day, the culture was not very concentrated and moreover the bacteria in it
formed aggregates. The former fact led to a long period of growth in the batch culture where
the population was too small to be estimated well by measurement of the optical density. The
latter caused troubles for the estimation of the population, as well by optical density
measurement as by plating diluted samples of the cultures. The fact was that one aggregate
more or less in a sample being taken from the culture caused a dramatic difference in the final
estimation of the population, especially in the first stages of the experiment when the
population was still quite low.

40
Although strict precautions were taken, contamination of the Rennie-culture was observed
after one day. The number of the contaminants' colony forming units on the plates reached
only 10 % of the number of Azotobacter colonies though, and moreover the contaminants did
not seem to influence the population of Azotobacter seriously. There where a colony of
Azotobacter grew close to a colony of the contaminant, it was the former that overruled the
latter, rather then the other way around. This made us decide to consider the contamination as
not important.
The Burk-culture remained pure at all times.

On graphs 1 and 3 the growth of Azotobacter vinelandii is shown in the Rennie and Burk
reactors respectively. The odd values after 38 and 45 hours must be considered as measuring
faults. Graphs 2 and 4 show the growth on a logarithmic scale. The optical density is plotted
on the same graphs.

1,8E+08
1,6E+08

0,8

1,2E+08
0,6

1,0E+08

O.D.

# c.f.u. per ml .

1,4E+08

8,0E+07

0,4

6,0E+07
4,0E+07

0,2

2,0E+07
0,0E+00

0
0

10

20

30

40

50

60

time (hours)

Graph 1: growth of A. vinelandii in a batch culture with Rennie medium.

# c.f.u.
O.D.

10
9
8
7

1
0,8

6
5
4
3
2
1
0

0,6

O.D.

log # c.f.u. per ml

41

0,4

log # c.f.u.
O.D.

0,2
0
0

10

20

30

40

50

60

time (hours)

Graph 2: growth of A. vinelandii in a batch culture with Rennie medium on

logaritmic scale.

1,4E+08
1,2E+08

0,6

8,0E+07

O.D.

# c.f.u. per ml

0,8
1,0E+08

6,0E+07

0,4

4,0E+07
0,2
2,0E+07
0,0E+00

0
0

10

20

30

40

50

60

time (hours)

Graph 3: growth of A. vinelandii in a batch culture with Burk medium.

# c.f.u.
O.D.

42

0,8

7
6

0,6

O.D.

log # c.f.u. per ml .

0,4

log # c.f.u.
O.D.

3
2

0,2

1
0

0
0

10

20

30

40

50

time (hours)

Graph 4: growth of A. vinelandii in batch culture with Burk medium on logaritmic

scale.

On graphs 2 and 4, the exponential way of growth is clearly visible.

In theory, the function describing bacterial growth is:
dC / dt = C
where C is the concentration of bacteria and t is the time. is a constant for each bacterial
population, and depends on intrinsic factors (qualities of the micro-organism) and external
factors such as the temperature, availability of nutrients and oxygen and the presence of
inhibitors. In the exponential growth phase, when all nutrients and oxygen are abundant, we
can write:
dC / dt = max C
integration gives:
ln C = t.max + constant

This constant is the natural logarithm of the population at time 0.

population Co, one must take the exponent of this constant.

To find the initial

43
If a population grows from C1 to C2, where C2 = 2.C1, in time interval d, being t2-t1, one can
write:
ln 2 = max . d
d is then called the doubling time.
We plotted ln C versus t for the exponential growth phase of our data and found the following
values:
for Rennie:

max = 0.2462 hour-1

Co

= 1443 cells/ml

= 2.82 hours
(R2 for these values was 0.9564)

for Burk:

max = 0.2381 hour-1

Co

= 2362 cells/ml

= 2.91 hours
(R2 for these values was 0.9875)

In our experiments no lag-phase was observed. This was to be expected since the microorganisms came from overnight cultures that were identical environments as the ones in the
batch-reactors. In there they were already growing in an exponential way, as we assume.

The pH of the reactors at the end of the experiment was 7 for the Rennie medium and 6.5 for
the Burk medium. Apparently, it had not or barely changed during the experiment. This is in
agreement with the fact that Azotobacter vinelandii does not produce acids during its growth
on glucose (Thompson and Skerman, 1979).

4. Conclusion
We felt more apt to continue the experiments with Burk medium for these reasons:
contamination seemed less likely to occur in Burk medium, because Rennie does
contain yeast extract and therefore a (small) nitrogen source,
growth in Burk medium was barely slower than in Rennie medium: the difference in
generation time was only 5 minutes.

44

B. SECOND BATCH EXPERIMENT: NITROGENASE ACTIVITY

1. Goal
In a second survey, nitrogenase activity was measured along with bacterial growth. This
allowed us to get familiar with the acetylene reduction assay, described below. Also, we
wanted to get an idea of the intensity of the nitrogenase activity in the different phases of
growth.

2. Materials and methods

a. cultures
The batch cultures used in this experiment were 1-litre conical flasks with 400 ml medium.
Burk medium was chosen because of the good results obtained with it in the first experiment.
Also, we expected that Burk medium would yield higher values of nitrogenase activity
because it contains no nitrogen at all. Higher measurements make the results more adequate
because the relative error becomes smaller.

Moreover, most scientists doing surveys on A.

vinelandii use Burk, and comparing results with their findings is easier when the same
medium is used.
We did measurements on 2 identical reactors inoculated with 12 hours difference. The
measurements from both reactors linked up perfectly. In order to have acetylene reduction
values, high enough to be measured accurately, an inoculum of 4 ml from a culture of
Azotobacter vinelandii in the stationary phase was taken. This culture was tested on purity of
the population first. All other manipulations and properties of the reactors were identical as in
the first batch experiment.

b. population estimation
The concentration of bacteria was measured in the same way as in the first batch experiment.

c. nitrogenase activity
The most suitable method for the measurement of nitrogen fixation is based on the capability
of the nitrogenase enzyme to reduce acetylene to ethylene. Hardy et al. (1968) were the first
to use this property to quantify the nitrogenase activity.

45
The nitrogen fixing bacterium is incubated with a known quantity of acetylene. After a
certain time interval, the composition of the gas is determined by gas chromatography.
Neither ethane nor methane are detected, since their formation is much slower than the
formation of ethylene from acetylene. The rate of acetylene reduction is constant up to 18 to
20 hours for Azotobacter, which means that the duration of the incubation is not important, as
long as it remains shorter than 18 hours and, of course, as long as it is known exactly.
Addition of NH4Cl decreases the rate of acetylene reduction, just as it decreases the rate of
nitrogen fixation. A linear relationship exists between cell number and acetylene reduction.
The ratio of moles of N2 fixed to moles of C2H4 formed is 3 to 4.5.
Hardy et al. described this method as to be applied in complete absence of nitrogen. Later,
however, other scientists (Moreno & Vargas-Garcia, 1995, Balis, 1996) have used variants of
this method, where nitrogen was present together with acetylene. We do not know whether
the nitrogen fixation in the absence of acetylene can be calculated from such experiments, but
it is clear that it allows one to compare the intensity of the nitrogenase activity under different
circumstances.

In our experiments, 2 ml from the reactor content were taken and incubated in gas-tight test
tubes of 14.3 0.1 ml. 1 ml of the atmosphere above the liquid was replaced by pure
acetylene, and the tubes were then incubated diagonally in a rotary shaker (BK Model 620)
for one hour and a half. Then, 1 ml of the gas above the samples was taken and injected in a
Fisons GC 8060 gas chromatograph.

The GS-Q column in the chromatograph was

manufactured by J&W, was 30 m long, had an internal diameter of 0.53 mm and was clothed
with a "microns" film. The components were quantified by a Fisons EL 980 flame-ionisation
detector as "units", corresponding to quantities.
The number of moles of ethylene formed per ml was calculated as follows.
The number of moles of acetylene injected in the tube before incubation was calculated with
the universal gas law:
P.V /R.T = n
(101.3 x 10 pas . 10-6 m) / (8.31 J/moleK . 298 K) = 4.1 x 10-3 mole

The number of units of ethylene and acetylene detected after incubation were added up. This
corresponds to the total quantity of acetylene injected in the test tube before incubation. The

46
units of ethylene divided by this total represents the share of ethylene in the test-tube. The
number of moles of ethylene formed is therefore 4.1 x 10-3 times this share.
It must be remarked that the quantity of ethylene formed was always much smaller than the
quantity of acetylene remaining in the test tube; therefore, the formation of ethylene was
never limited by a lack of acetylene.
At least three replicates of each sample were measured. The means, standard deviations and
studentized residuals of the results were calculated. The values with studentized residuals
higher than 1.5 were considered as outliers. The means of the other values were used for data
processing.

3. Results and discussion

a. growth
The growth curve is plotted on logarithmic scale in graph 5. It is clearly visible that in this
experiment, there was a lag-phase of about 20 hours. This can easily be explained: the cells
used for the inoculation of the reactors were not in the exponential growth phase, and
therefore needed some time to start growing exponentially.

8
7,8

log #c.f.u. per ml .

7,6
7,4
7,2
7
6,8
6,6
6,4
6,2

Graph Error!Argumento de modificador desconocido.: growth of A. vinelandii in batch

culture6 with Burk medium
0

10

20

30

40

tim e (hours)

Graph 5: growth of A. vinelandii in batch culture with Burk medium.

50

47
Another remark to be made is that the value that characterizes the following exponential
growth phase was considerably smaller in this experiment: 0.15, whereas it was 0.24 in the
preceding batch experiment. An unequivocal explanation for that is hard to give. Perhaps the
different reactor size caused a different aeration level, and therefore limited growth.
The fact that growth was slower in this experiment is an indication that one must be careful
when he determines the exponential growth rate: it seems that it is not easy to reproduce
identical circumstances of growth, even in an identical medium and with an identical ambient
temperature.

b. nitrogen fixation
The course of the nitrogenase activity per cell is represented in graph 6.
1E-13

mole C2H4 / (min . c.f.u.)

9E-14
8E-14
7E-14
6E-14
5E-14
4E-14
3E-14
2E-14
1E-14
0
0

10

20

30

40

50

time (hours)

Graph 6: nitrogenase activity of A. vinelandii in a batch culture with Burk medium.

The fact that the maximum level of nitrogenase activity per cell occurs in the very beginning
of the exponential growth phase is in agreement with the observations of Papadopoulou et al.
(unpublished data).

48

C. THIRD BATCH EXPERIMENT: GLUCOSE CONSUMPTION

1. Goal
In a third survey, the glucose consumption was followed up along with bacterial growth. This
allowed us to have an idea of the energy requirements of Azotobacter vinelandii.

2. Materials and methods

a. cultures
The batch culture used in this experiment was a 1-litre conical flask with 400 ml of nitrogenfree Burk medium, inoculated with 2 ml of a 3-day-old culture of Azotobacter vinelandii.

b. population estimation
The population density was measured in the same way as in the first batch experiment.

c. glucose content
Samples to determine the glucose content of the medium in the reactor were first centrifuged
at 12,000 rounds per minute in order to precipitate the cells from the
medium. The supernatant was kept in a freezer at 20 C until
measurement.
The glucose content of the samples was determined with a
colorimetric method, described by Ehaliotis (1996), in which
anthrone is used.

The chemical structure of this product is

Fig. 9: anthrone

represented in fig 9. The reagent itself is an aqueous solution of 76 % (volume per volume)
H2SO4, in which 0.1 gram anthrone is solved per litre. 4 ml of this reagent was poured in a
test tube, and 1 ml of the solution to be examined was layered on top of it. The test tube was
then shaken vigorously, in order to mix reagent and sample well. Afterwards, this mixture
was heated in a bath at 100 C for 10 minutes. When this time had expired, the test tubes
were immediately cooled down in water at ambient temperature, in order to stop the reaction.
The absorption was then measured in the spectrophotometer, mentioned in chapter A.2.b, of
which the wavelength was set at 625 nm.
At the same time, a calibration curve was made. It allowed us to know the relation between
concentration of carbohydrates and absorption.

49
The measurement of glucose with anthrone is very sensitive. The light absorption by the
sample is only a linear function of the glucose concentration in the interval from 0 g/l to 0.1
g/l.

Therefore, we had to dilute the glucose samples 100 times before they could be

measured. In order to have a good dilution we made 2 succeeding tenfold dilutions in

distilled water.

For every sample, 3 replicates were measured.

3. Results and discussion

The population of Azotobacter vinelandii and the glucose concentration in the reactor are
represented in graph 7.

0,12

log # c.f.u. per ml

0,08
7
0,06
6
0,04
5

[glucose] in g/l

0,1

log # c.f.u.
[glucos e]

0,02

0
0

10

20

30

40

50

60

70

80

90

time (hours)

Graph 7: growth of A. vinelandii and glucose concentration in a batch culture with

Burk medium.

The exponential growth started with a value for of 0.22. At the time that had reached a
value of 0.11, the concentration of glucose in the reactor had dropped from 10 to 4.5 grams
per litre. Further in the experiment, the glucose consumption seemed to have diminished.
This could be an indication that it is mostly multiplication of A. vinelandii that requires the
energy source, and not so much maintenance of existing cells.

50
The Monod model for bacterial growth describes the relation between the concentration of the
limiting substrate and the exponential growth rate of a micro-organism. The equitation of the
model is:

dC / dt = C . max . Cs / (Ks + Cs)

where C represents the concentration of micro-organisms and Cs the concentration of limiting
substrate. max and Ks are constant values that characterize the growth. The physical meaning
of Ks is the substrate concentration at which the exponential growth rate is only half of its
maximum value. In our experiments, we assumed that glucose was the limiting substrate.
max had been determined in former experiments (see A), Ks was calculated here as 4.5 g/l.
This seemed to be a high value; this observation corresponded to the basic idea of the
respiratory protection of the nitrogenase enzyme. On the other hand, such a high value would
mean that the growth rate at the initial glucose concentration of 10 g/l was only 2/3 of the
maximum growth rate, according to the Monod model.

51

D. FOURTH BATCH EXPERIMENT: INFLUENCE OF OMW

1. Goal
In a fourth batch experiment, nitrogenase activity and bacterial growth were measured in a
batch culture that, apart from the usual Burk medium, contained 10 % of non-sterilised OMW.
The aim of this survey was to get a view on the influence of OMW on Azotobacter, and in
particular on the inhibition or retardation effect on the nitrogenase activity.

2. Materials and methods

a. inoculation culture
The cells of A. vinelandii for this experiment were taken from a smaller culture. This culture
was grown in Burk medium, based on a solution of 10 % OMW.
The OMW used was collected from a factory in Kalamata where olive mill waste is
processed. It was kept in a plain freezer in expectation of its use. After being thawed out, it
was neutralized with calcium oxide, passed over a fibreglass filter and centrifuged three times.
Both the precipitate and the oily upper layer were removed each time. Afterwards, the liquid
was autoclaved, filtered again and autoclaved a second time. Finally, it was mixed with
sterilised water to a final concentration of 10 % (v/v). The concentrations of all compounds
of the Burk medium added stayed identical as in the former experiments.
This mixture was inoculated with Azotobacter vinelandii cells from agar plates.

b. batch reactor
In this experiment as well, the reactors used were conical 1-litre flasks with 400 ml of
medium. The medium of the reactor was identical to the one of the inoculation cultures,
except that the OMW used was not autoclaved. The reason why we did not do this is that
heating OMW may change its composition considerably. We tried to pass the liquid through
a microbiological filter with a pore diameter of 0.45 m, but the pores were clogged as soon
as a few drops of the liquid had passed through the filter. The explanation for this must lie in
the medley of polymers present in OMW. Besides, 0.45 m would probably not have been
small enough to keep out contaminants, since even Azotobacter itself is known to form cells
smaller than that (Gonzalez Lopez an Vela, 1981).

52
Therefore, we took the decision not to filter the medium and rely on the dominance of A.
vinelandii in OMW.
We inoculated the reactors with 4 ml of the 5-day-old inoculation culture. Further treatment
and measurements were carried out identically as in the former batch experiments.

3. Results and discussion

From all the plates made during the five days that this experiment ran, none was suitable for
the counting of Azotobacter vinelandii, since all of them contained far too many colonies of
numerous other micro-organisms. Moreover, not the slightest nitrogen fixation was measured
throughout the experiment.
The pH of the reactors was measured; it was found that it was only 3. This value is much too
low for growth of Azotobacter.

On the first sight, this is contradictory with reports from several scientists (Chatjipavlidis et
al., 1996, Papadopoulou et al., unpublished data). However, it must not be forgotten that the
experiment described here was carried out with diluted OMW, while Chatjipavlidis and
Papadopoulou had used pure OMW. This might be the reason why in our experiments, the
growth of micro-organisms present in the medium was not inhibited strongly, and therefore
why A. vinelandii was not competitive.
Another explanation might be that the aeration, provided by simply shaking the reactor, is not
sufficient for A. vinelandii to compete with other micro-organisms. The fact is that in the
bioreactors used by Chatjipavlidis and Papadopoulou there was a system that provided forced
aeration to the reactor. It can thus be admitted that oxygen and medium were mixed much
better in their system.

53

E. CONTINUOUS FLOW REACTOR

1. Introduction
The main objective of this research was to find the optimal flow rate through a bioreactor
containing a chemically defined medium with a pure culture of Azotobacter vinelandii. The
definition of this optimum is not obvious. Is it the flow rate at which we have the largest
population, the flow rate at which the nitrogen fixation reaches its highest level, or the one at
which the biotoxicity of the outflow has become acceptable? Clearly, the latter seems to be
the most interesting when we consider the bioreactor as a plant to clean waste water. But this
is not the only consideration to be made here. We are trying to obtain a biofertilizer, so we
must not only have the toxic compounds in the water broken down; we must also have a
considerable amount of potassium, phosphorus and nitrogen in it. The amounts of potassium
and phosphorus can not be risen in a biological process, but the amount of nitrogen can be.
Therefore, we must enhance nitrogen fixation.

The questions to be answered are then:

1. How does A. vinelandii grow in continuous reactor with a well-defined medium?
2. How fast does it fix nitrogen in these circumstances?
3. How is the bacterial growth altered by addition of OMW?
4. How is the nitrogen fixation altered?
5. What is the quality of the outflow?
6. How competitive is Azotobacter vinelandii when it has to face the presence of other microorganisms, and how efficiently does it work then?

Our intention is to answer the first four questions. Answering the fifth question requires
analogous experiments combined with biotoxicity-tests. The sixth question can only be
answered well when a pilot plant is studied in real life circumstances. This has been done
in the experiments of Papadopoulou et al..

54
2. Materials and methods
a. reactor
The reactor used was a New Brunswick Scientific BioFlo Model C-30, with the following
properties and settings:

agitation: 300 rounds per minute

aeration: 0.5 l per minute bubbled through the culture. The reaction vessel was provided
with baffles to promote the dispersion of the air in the culture,

temperature: the thermostatic system of the apparatus failed, so maintaining a stable

temperature in the reactor was not possible. It was therefore dependent of the ambient
temperature, and varied diurnally from 26 to 29 C,

volume: 400 ml. The liquid volume when aeration and agitation were activated was 385
ml. It is the latter volume that we considered when making calculations,

sample port: the sample flask was connected to the sample port on one side and to a
rubber balloon on the other side. When the balloon was squeezed, air was removed from
it; when it was loosened again, a vacuum resulted and liquid from the reaction vessel was
sucked into the sample flask. The sample flask could then be unscrewed from the reactor
and the sample was poured into a sterilised test tube.

The reaction vessel was fed with Burk medium from an 11-litre reservoir by a Desaga PLGPeristaltic Pump. The pumping speed of this device could be controlled with a stepless
switch. The minimum flow rate was 0.088 ml per minute.

The medium used was nitrogen-free Burk medium.

55

Fig. 10: schematic presentation of

the continuous reactor.

11-litre reservoir with

sterilized medium

peristaltic pump
air pump
cotton air filter
reaction vessel
port
sample port
sample flask
sample pump

b. population estimation
Countings were done as described in chapter A.2.b.

c. nitrogenase activity
This was measured with the acetylene reduction assay as described in chapter B.2.c.
d. glucose measurement
The measurement of the glucose concentration was carried out as described in chapter C.2.c.

56
e. strategy
Three days after the setting of a particular flow rate a sample was taken and the measurements
were carried out.

In the first phase of the experiment, A. vinelandii was followed in a reactor with pure Burk
medium. The measurements consisted of:

counting bacteria,

measuring nitrogen fixation,

measuring glucose content.

In the second phase of the survey, we tested the influence of adding 10 % of OMW at the
flow rate corresponding to a residence time of 15 hours. At this stage, we only measured the
population size and nitrogen fixation.
A known quantity of the culture liquid (43.5 ml the first time, 41.5 ml the second time) was
removed from the reaction vessel and replaced by sterilised OMW, treated as described in
chapter D.2.a.

The OMW was not added continuously, so its concentration lowered

continuously. A calculation of this concentration at any time could be made as follows:

if C was the concentration of OMW in the reaction vessel, F the flow rate, V the reactor
volume and t time, one can write:

dC / dt = -F.C.dt / V.dt

this yields:

integration gives:

and finally:

dC / C = -(F/V).dt

ln C = -(F/V).t + cte
C = e-(F.t / V) . C0

with C0 the initial concentration of OMW.

57
3. Results and discussion
a. population
After a short time, apart from Azotobacter vinelandii, an unknown bacterium was observed in
the reactor. The bacterium was identified as Bacillus sp., and its growth is compared with that
of A. vinelandii in graph 8.

6,E+08

10
9
8
7

4,E+08

6
3,E+08

5
4

2,E+08

3
2

1,E+08

[glucose] (in g/l) .

# c.f.u. per ml .

5,E+08

A. vinelandii
contaminant
[glucose]

1
0,E+00

0
0

20

40

60

80

residence time (hours)

Graph 8: growth of A. vinelandii and contaminant in continuous flow reactor with

Burk medium.
How the contamination by Bacillus sp., detected after a few weeks of operation, has occurred
is not certain. The most plausible explanation seems to us that the contaminant entered the
reaction vessel through the sample port. It is a fact that, while one pumped to create the
vacuum necessary to suck up sample liquid, the air from the sample balloon bubbled through
the culture. To avoid further contamination, we therefore forced the air to leave via the
sample flask holder in later samplings.

The contaminant was tested on its nitrogen requirements. It was found that the bacterium did
not grow well in nitrogen-free Burk medium by itself, but did grow well when ammonium
chloride was added to the Burk medium. Also, we found that it did not convert acetylene to
ethylene.

58
In the situation that two micro-organisms compete for the same limiting substrate in a
bioreactor, this must lead to the elimination on the least competitive of them in the long term.
This, however, did not occur in the continuous reactor described above. The two organisms
coexisted, and their population ratio was dependent of the residence time. This suggests that
at least for the contaminant, not glucose was the limiting substrate.
The tests we carried out with pure cultures of the contaminant indicated that it could not live
without a nitrogen source in the medium. Therefore, we assume that this bacterium was
dependent on Azotobacter for its nitrogen supply.
This can be confirmed by the relatively low Bacillus population at short residence times:
Azotobacter's metabolites, containing the much-needed nitrogen, are washed out more
quickly, actually lowering the concentration of the contaminant's limiting substrate.

It is clearly visible in graph 8 how the ratio of A. vinelandii and the contaminant increased
with a decreasing residence time.

The population drop of Azotobacter vinelandii at a

residence time of 72 hours may have been due to the fact that the glucose concentration at this
point was too low to maintain growth. The contaminant seemed not to suffer from this low
substrate concentration, and reached its highest population level at this residence time, thus
doing even more harm to Azotobacter by consuming even more glucose.

The relation between bacterial growth and substrate utilization is characterized by 2

parameters: Y, the yield factor, that tells how much substrate is used per new cell formed, and
m, the maintenance factor, that represents the amount of substrate used per cell in a certain
time interval for maintenance purposes.

The general equitation for the substrate utilization rate (rs) at a certain growth rate (rx) and cell
concentration (Cx) is therefore:

rs = rx /Y + m.Cx

(1)

59
In a steady state situation, the same amount of cells is washed out of the reactor as is formed
in it. Therefore:

rx = F.(Cx)/V

(2)

where F represents the flow rate and V the reactor volume. An analogous reasoning can be
made for the substrate:

rs = F.(Cs,in - Cs)/V

(3)

When 2 micro-organisms are involved, however, equitation (1) must be extended:

rs = rx,1 /Y1 + m1.Cx1 + rx2 /Y2 + m2.Cx2

(4)

where the indices 1 and 2 refer to A. vinelandii and the contaminant respectively.

On the basis of our measurements, the parameters Y1, Y2, m1 and m2 were calculated. This,
however, yielded senseless values for Y1 and Y2 and very small values for m1 and m2.
The parameters Y1 and Y2 were then recalculated with the assumption that m1 and m2 were
negligible. This yielded the values:
Y1 = 74 x 109 cells formed per gram glucose consumed
Y2 = 357 x 109 cells formed per gram glucose consumed
The rate of substrate utilization of each population of micro-organisms can then be calculated
from equitations (1) and (2) using the yield factors:

rs = Cx . F / (Y . V)
The results are represented in graph 9.

60

glucose consumption (g/l.hour)

0,25

0,2

0,15
A. vinelandii
contam inant

0,1

0,05

0
0

20

40

60

80

residence time

Graph 9: glucose consumption per population in continuous culture with Burk

medium.
It can be assumed that the contaminant did harm to Azotobacter vinelandii by consuming
glucose and other medium components. On graph 9 however, it can be seen that the share of
the glucose "stolen" by the contaminant was rather small.

61
For the limiting substrate in a culture, 2 more parameters can be calculated: Ks and max.
They are both parameters of the Monod model for bacterial growth, which says:

dCx/dt = max . Cs / (Cs + Ks) . Cx

In a continuous culture in steady state, dilution of the culture and its growth compensate each
other, resulting in:

F.(Cx)/V = max . Cs / (Cs + Ks) . Cx

On the basis of our measurements, the parameters max and Ks have been calculated. This,
however, yielded senseless values again.

The reason why Ks and max could not be calculated is not clear. It is possible that the
measurements were not carried out at a steady state situation.
Another possibility is that glucose was not the limiting substrate in this medium. This,
however, seems unlikely since many scientists use the same medium, and none of the
consulted works report other substantial needs of A. vinelandii, although sometimes sodium
chloride is added to the medium (Becking, 1961).
A third possibility is that the micro-organism does not grow on the kinetic theory of Monod.
In any of these cases, more extensive research is necessary to modelize A. vinelandii's growth.

62
b. nitrogenase activity

4,0E-07

10

3,5E-07

9
8

3,0E-07

2,5E-07

2,0E-07

1,5E-07

4
3

1,0E-07

5,0E-08

[glucose] (in g/l) .

mole C2H4 / ml . min

The nitrogenase activity per ml of culture is represented in graph 10.

acetylene
conversion
[glucose]

0,0E+00

0
0

20

40

60

80

residence time (hours)

Graph 10: nitrogenase activity of A. vinelandii per ml culture in continuous

flow reactor with Burk medium.

It is remarkable that at high flow rates, although the population density is smaller, high rates
of nitrogenase activity are observed. This is very clear when the nitrogenase activity per cell
is represented, as in graph 11. Note that the nitrogen fixation is presented on logarithmic
scale.

The fact that higher flow rates yielded higher nitrogenase activities didn't come as a surprise
after the batch experiments. Indeed, the glucose concentration at high flowrates is relatively
high; therefore, the cells grow fast, just as in the early exponential growth phase in a batch
culture. And it is in this early exponential phase that nitrogenase activity reaches its highest
level. Besides, the values for nitrogenase activity at the shortest residence times match the
maximum values in batch cultures pretty well: 5.1 x 10-14 moles ethylene formed per minute
per c.f.u. in the continuous reactor, 8.6 x 10-14 in the batch reactor (see III.B)

63

mole C2H4 / min . c.f.u. .

1E-13

1E-14

1E-15

1E-16

1E-17
0

10

20

30

40

50

60

70

80

residence tim e (hours)

Graph 11: nitrogenase activity per A. vinelandii cell in continuous flow reactor with
Burk medium on logarithmic scale.

It was strange however, that even at very high flow rates, close to the wash-out rate, the
highest total levels of nitrogenase activity were measured.
There seem to be two possibilities.
The first is again that a steady state situation was not obtained at the time of the sampling.
However, this does not explain the high levels of nitrogenase activity per cell, and moreover
at exactly those highest flow rates, the adaptation time was manifold higher than the residence
time, suggesting that the reactor did have enough time to come to a steady state.
A second possibility is that these observations match reality, and that indeed very high flow
rates are required for optimum nitrogenase activity. This may mean that high substrate levels
are necessary, but it is also possible that the small amounts of nitrogen containing metabolites,
produced by Azotobacter vinelandii, inhibit the nitrogenase activity.

Especially at these high flow rates, more testing seems desirable.

64

c. influence of OMW
During the tests with OMW, no contamination of the reactor occurred. The effect of OMW is
shown in graph 12 (influence on the population) and graph 13 (influence on nitrogenase
activity). Time 0 is the moment of the first addition of OMW.

14

1,8E+08
1,6E+08

12

1,4E+08

# c.f.u. / ml .

1,0E+08

8,0E+07

6,0E+07

vol% OMW .

10

1,2E+08

[A. vinelandii]
[OMW]

4,0E+07
2

2,0E+07
0,0E+00

0
-2

13

18

23

28

33

38

43

48

53

tim e (hours)

Graph 12: influence of OMW on the A. vinelandii population.

14

6,E-15

12

7,E-15

10
.

8
4,E-15
6
3,E-15
4

vol% OMW

mole C2H4 / c.f.u. . min

5,E-15

nitrogenas e
activity
[OMW]

2,E-15

1,E-15

0,E+00

-2
-2

13

18

23

28

33

38

43

48

53

58

time (hours)

Graph 13: influence of OMW on A. vinelandii's nitrogenase activity.

65

On graph 13, it can be seen that after the first addition of OMW (at time 0), there was an
adaptation time before nitrogen fixation jumped to its highest value.

After the second

addition (after 48 hours), this was not the case any more. An analogous phenomenon could
be observed with the population size, although less pronounced.
Apparently, Azotobacter vinelandii does respond well to OMW in the medium, at least at
these concentrations and in a pure culture of A. vinelandii.

To quantify the influence of OMW on Azotobacter vinelandii, more extensive measurements

should be done with a continuous supply of OMW in order to keep the concentration stable
throughout the experiment.

d. pH
The pH of the solution in the reactor was measured regularly and fluctuated between 6.5 and
7.

66

IV. GENERAL CONCLUSION

*******************************************
The production of OMW is a challenge for environmental engineers for 3 reasons:

the huge quantities produced in the regions involved,

the short time in which they are produced,

the toxic substances in this waste and the high BOD value.

Also, it is a fact that the wastes are produced in regions that do not have problems with
hypertrophy.
These observations lead to the conclusion that aerobic treatment with land application of the
treated waste water is an interesting, if not the most interesting solution to this problem.

Whether Azotobacter vinelandii is the appropriate micro-organism to deal with this waste is
another question. It is true that this micro-organism has the ideal enzymatic means to cope
with OMW. However, the experiments of scientists doing research in this field and our own
experiments give indications that A. vinelandii is not very competitive in waste water
treatment plants. Moreover, it has been observed that the toxic components in OMW can be
degraded by several other micro-organisms, even those present in activated sludge of common
waste water treatment plants. Also, it must not be forgotten that apart from the polyphenols,
there is still this mysterious other toxic fraction of the waste; as long as that one has not been
identified, it can not be said whether or not Azotobacter can degrade it.
Therefore, the question forces itself whether it is A. vinelandii that "does the job" in the plants
that are operational nowadays. It might be interesting to make a qualitative and quantitative
study of the micro-organisms present in a plant where OMW is treated and of which the
bacterial population has not been forced to a certain composition.

If it turns out that Azotobacter vinelandii is indeed the dominant bacterium that degrades the
polyphenols in the waste water, some improvements to the waste water treatment installations
can be made to optimize its efficiency. Although the experiments described in part III do not
allow to draw solid conclusions, some interesting observations, together with research results
of scientists, may help in this matter.

67

An important fact is that high flow rates promote nitrogen fixation and substrate consumption.
A second observation is that in a solution with 10 % OMW, other micro-organisms overrule
Azotobacter. Thirdly, it has been proven that high aeration rates enhance substrate utilization
considerably. Fourthly, Azotobacter species require a relatively high pH level.

Therefore, we propose to extend the waste water treatment plants as they exist today with one
or more basins, in order to make a cascade system of continuous reactors.
A problem with an ordinary well-mixed continuous reactor is that the liquid in the reactor has
the same composition as the outflow. This means that for an output product with a low
toxicity, the liquid in the reactor as well will only be a little bit toxic or not at all. This
favours the growth of micro-organisms that have nothing to do with the degradation of
phenolic compounds, and may deteriorate the growth circumstances for A. vinelandii, for
instance by lowering the pH.
In a cascade system of continuous reactors, the first reactor should be small enough to keep
the residence time short, so that only a small share of the polyphenols are degraded. In such
an environment, Azotobacter vinelandii would be more competitive, especially if this reactor
were well aerated. Also, since Azotobacter does not affect the pH of its environment much,
the pH in the reactor would remain stable at 8, the common level of OMW neutralized with
calcium oxide and likewise the ideal pH for Azotobacter.
This basin could function as a nursery for Azotobacter, constantly inoculating the reactors
downstream of this first unit. In those downstream tanks, further degradation by Azotobacter
and other organisms could be obtained, eventually with a less intensive aeration to reduce the
operation costs.
Another advantage of this system would be that the residence time in the first tanks could
more easily be controlled if the last reactor is concipiated as a storage tank of biofertilizer.

For the design of such a system, it is necessary to know the optimum concentration of OMW
for growth and nitrogen fixation of Azotobacter vinelandii. This as well could be the subject
of further research.

68

V. APPENDICES
*******************************
APPENDIX 1: COMPOUNDS OF NITROGEN-FREE RENNIE MEDIUM

K2HPO4

: 0.8 g/l

KH2PO4

: 0.2 g/l

NaCl

: 0.1 g/l

Na2FeEDTA

: 0.028 g/l

Na2MoO4.2H2O

: 0.025 g/l

yeast extract

: 0.1 g/l

glucose*

: 10 g/l

NaLactose

: 0.5 %Vol

MgSO4.7H2O

: 0.2 g/l

CaCl2

: 0.06 g/l

APPENDIX 2: COMPOUNDS OF NITROGEN-FREE BURK MEDIUM

as described by Page and Sadoff (1976)

K2HPO4

: 0.3828 g/l

KH2PO4

: 0.380 g/l

pH is then brought at 7.1 with NaOH

Na2MoO4.2H2O

: 0.242 mg/l

MgSO4.7H2O

: 0.2 g/l

CaSO4.2H2O

: 0.1 g/l

FeSO4.7H2O

: 0.005 g/l

glucose*

: 10 g/l

* Glucose is

sterilised seperately to avoid complexation with salts.

69

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De invloed van afvalwater van

olijfperserijen op de groei en stikstoffixatie
van Azotobacter vinelandii.
*********************************************

A. LITERATUURSTUDIE
1. De olijfteelt
De verbouwing van de olijfboom, Olea europaea, is al sinds de oudheid een van de
belangrijkste landbouwactiviteiten in het gebied rond de Middellandse Zee. Tot op de dag
van vandaag gebeurt 98 % van de olijfteelt ter wereld in deze regio. Het belangrijkste product
van de olijfteelt is de olie. In het voorbije decennium bedroeg de totale productie van olijfolie
gemiddeld 1,7 miljoen ton per jaar.

De olijfteelt is dan ook een belangrijke bron van

inkomsten voor duizenden mensen in Zuid-Europa en Noord-Afrika. Met name het landschap
van zuidelijk Griekenland wordt volledig beheerst door de olijfgaarden.

De verbouwing van olijven brengt echter een belangrijk milieuprobleem met zich mee. Bij
het plukken en persen van de olijven worden er namelijk grote hoeveelheden vast en vloeibaar
afval gevormd. Het afvalwater, in het Spaans alpechin genaamd, in het Grieks katsigaros en
in het Engels gewoonlijk afgekort als OMW (olive mill waste water), is een belangrijk
afvalproduct van de zogenaamde drie-fase systemen voor het persen van olijven.

De

hoeveelheid die jaarlijks vrijkomt in het gebied rond de Middellandse Zee wordt geschat op
niet minder dan dertig miljoen kubieke meter.
Het onderzoek van dit eindwerk heeft betrekking op het vinden van een oplossing voor dit
afvalwater. Er dient opgemerkt te worden dat bij modernere twee-fase systemen praktisch
geen afvalwater meer geproduceerd wordt. Daar beperkt het probleem zich tot de vaste
substantie die achterblijft na het persen, en in het Spaans alpeorujo heet.

80
2. Het afvalwater
Het afvalwater van de olijfperserijen is nogal variabel van samenstelling. De pH ligt rond de
4,5 en de COD bedraagt 230 gram per liter. Een uitgebreid overzicht van de samenstelling is
gegeven in figuur 1.
Een belangrijke eigenschap van het afvalwater is zijn hoog gehalte aan polyfenolen. De
typische gitzwarte kleur is trouwens een gevolg van polymerisatie van deze stoffen. Door de
aanwezigheid van deze fenolische componenten en andere, totnogtoe niet gedentificeerde
stoffen, is dit afvalwater toxisch voor planten en micro-organismen.

Daarom wordt

momenteel veel onderzoek uitgevoerd naar de mogelijkheden om dit water te behandelen.

3. oplossingen voor het afvalprobleem

a. gebruik als grondstof
Verscheidene wetenschappers hebben onderzoek gedaan naar de mogelijkheid om de
specifieke eigenschappen van het afvalwater van olijfperserijen uit te buiten. Zo zijn er
publicaties

over

de

mogelijkheid

om

xanthaan,

andere

polysacchariden

en

polyhydroxyalkanoaten te winnen uit culturen van bacterin die groeien in het afvalwater.
Andere onderzoekers hebben gesuggereerd om de natuurlijk voorkomende antioxidantia uit
het afvalwater te recupereren. Nog andere stelden voor om er algen of fungi in te kweken die
dan als veevoeder gebruikt kunnen worden.

b. afvalwater zuivering
Wat de technieken betreft om op grote schaal afvalwater te zuiveren, daarin zijn er drie
belangrijke groepen te onderscheiden.

Ten eerste de fysico-chemische behandelingen.

Er zijn experimenten uitgevoerd met

installaties om het afvalwater uit te dampen en met omgekeerde osmose, maar deze leverden
geen positieve resultaten op. Ook het doen neerslaan van organische stoffen in het afvalwater
door behandeling met calciumoxide was niet veelbelovend. Een techniek die al lang wordt
toegepast is het toevoegen van waterstofperoxide. Het juiste effect van die behandeling is ons
niet duidelijk.

81
Ten tweede is er de anarobe biologische behandeling. Het onderzoek naar deze technieken is
al vrij ver gevorderd, en de hoop bestaat dat deze zelfs een netto winst zullen kunnen
opleveren doordat bij deze behandeling biogas gevormd wordt. Het belangrijkste probleem is
echter dat de fenolische stoffen in dit afvalwater giftig zijn voor methanogene bacterin.
Daarom ziet het er naar uit dat een voorbehandeling nodig zal zijn.

Ten derde is er de arobe biologische behandeling.

Hierover is niet veel literatuur

gepubliceerd, maar wat we erover gevonden hebben is wel heel erg veelbelovend. Afvalwater
van olijfperserijen zou goed afbreekbaar zijn in beluchte zuiveringsinstallaties.

c. gebruik als meststof

Een derde mogelijkheid is het gebruiken van het afvalwater als meststof en bodemverbeteraar.
Onbehandeld kan het afvalwater bij een beperkt aantal teelten ingezet worden, maar er blijven
problemen bestaan met verzouting van de bodem en vooral ook met het toxische karakter van
het afvalwater.
Daarom hebben Chatjipavlidis et al. (1997) voorgesteld het afvalwater eerst een arobe
biologische voorbehandeling te geven en pas daarna als meststof te gebruiken. Het microorganisme dat een sleutelrol zou spelen in de afbraak van de toxische stoffen in het afvalwater
is Azotobacter vinelandii. Deze bacterie is niet alleen in staat de fenolen in het afvalwater af
te breken, ze rijkt het ook aan met stikstof die ze uit de atmosfeer gefixeerd heeft. Bovendien
produceert A. vinelandii exopolysacchariden. Deze stoffen hebben een gunstige invloed op de
textuur van de bodem. Er worden daarnaast ook stoffen afgescheiden die de groei van
bepaalde pathogene fungi onderdrukken.
De proeven die tot nu toe uitgevoerd zijn met dit systeem zijn erg veelbelovend.

De

behandeling bestaat eruit dat het afvalwater eerst met waterstofperoxide en calciumoxide
wordt voorbehandeld en daarna in de bioreactor als substraat voor Azotobacter dient.
Het belangrijkste obstakel is echter dat Azotobacter vinelandii niet noodzakelijk competitief is
in een dergelijke bioreactor. Verschillende onderzoekers hebben een aanzienlijke groei en
stikstoffixatie vastgesteld in batch-reactoren met afvalwater van olijfperserijen in de eerste
dagen na het enten met een (zeer groot) inoculum van Azotobacter vinelandii. Er zijn nog
geen experimenten uitgevoerd om de overlevingskansen van het micro-organisme op langere
termijn na te gaan.

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4. Azotobacter vinelandii
De familie der Azotobacteraceae, waarvan A. vinelandii deel uitmaakt, is in het begin van
deze eeuw ontdekt. Alle vertegenwoordigers zijn stikstoffixerende bacterin die de voorkeur
geven aan neutrale of alkalische bodems.

In tegenstelling tot de meeste andere

stikstoffixerende bacterin zijn de Azotobacteraceae vrij levende bacterin; zij komen dus
zelden voor in de rizosfeer. Ze verkiezen naast de bodemlaag ook de strooisellaag en zoet
water als habitat. Azotobacter komt praktisch overal ter wereld voor.

Het opvallendste morfologisch kenmerk van A. vinelandii is de grootte: er zijn publicaties die
bacterin van meer dan 4 m vermelden; anderzijds wordt er gewag gemaakt van een zeer
uitgesproken polymorfisme, in die mate dat ook cellen van 0,3 m gevonden zijn. De cellen
bezitten flagellen en zijn Gram-negatief.

Azotobacter vinelandii beschikt over de genen om dioxygenases en andere enzymen aan te

maken waarmee het aromatische stoffen kan degraderen.

Vermoed wordt dat het deze

gebruikt om componenten van humus als nutrint te kunnen gebruiken. Wanneer echter
glucose aanwezig is in het medium wordt dit bij voorkeur geconsumeerd.

Zoals eerder vermeld is Azotobacter een stikstoffixerende bacterie.

Deze stikstoffixatie

gebeurt met het nitrogenase enzym. Bij A. vinelandii zijn niet minder dan 3 verschillende
soorten nitrogenasen gevonden, die allemaal een ander metaalion in hun kern hebben. Voor
het enzymatische proces zijn 12 ATP-moleculen nodig per gefixeerde stikstofmolecule.
Nitrogenase zet niet alleen moleculair stikstof om: ook verschillende andere verbindingen,
waaronder ethyn, kunnen gereduceerd worden. Ook protonen kunnen omgezet worden in
waterstofgas. Om energie te sparen blijft de expressie van de nif-genen, die coderen voor
nitrogenase, uit wanneer er genoeg minerale stikstof aanwezig is in het milieu.

Een belangrijk probleem waarmee Azotobacter te kampen heeft is dat het enerzijds zuurstof
broodnodig heeft om te overleven, maar anderzijds dat nitrogenase wordt gedeactiveerd of
zelfs vernietigd door diezelfde zuurstof. De bacterie beschikt daarom over een heel arsenaal
aan mechanismen die de schadelijke invloed van zuurstof binnen in de cel moeten beperken.
De voornaamste is de zogenaamde bescherming door ademhaling.

In omstandigheden

waarbij stikstoffixatie nodig is voor de cel maar er ook een grote zuurstof aanvoer is worden

83
er meer oxidasen gevormd en wordt er meer substraat verbruikt. Met de elektronen die op die
manier beschikbaar gesteld worden, wordt zuurstof dan gereduceerd.
Een andere mogelijkheid is dat zuurstof enzymatisch omgezet wordt in radicalen. Met deze
wordt dan afgerekend door superoxide dismutase en catalase.
Verder ondergaat ook het nitrogenase zelf een verandering van structuur zodat het
gedeactiveerd wordt of simpelweg actief kan blijven zonder schade te ondervinden van
zuurstof.
Een laatste aanpassing van de cel is dat diens volume vergroot, zodanig dat relatief gezien een
kleiner celoppervlak overblijft waarlangs zuurstof kan binnendringen.

Verscheidene auteurs maken melding van benvloeding van de stikstoffixatie activiteit door
aromatische stoffen. Enerzijds zijn er aanwijzingen dat nitrogenase geschaad wordt door deze
substanties, anderzijds blijkt er meer expressie van de genen voor stikstoffixatie op te treden.
Een genetische verklaring voor dit fenomeen is gegeven door Balajee en Mahadevan (1990);
in afwezigheid van stikstof komen niet enkel de nif-genen tot expressie maar ook de genen die
coderen voor de enzymen waarmee aromatische componenten afgebroken worden. Op die
manier is er genoeg substraat voorhanden om de bescherming van nitrogenase door
ademhaling te garanderen.

Ook het afvalwater van olijfperserijen brengt dit positieve effect met zich mee, al is het
minder duidelijk of de groei en stikstoffixatie van Azotobacter in dit substraat ook niet
bevorderd worden door de aanwezigheid van mineralen en andere voedingsstoffen.

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B. EXPERIMENTEN
1. Eerste batch experiment
In een eerste, verkennend onderzoek werden Rennie medium en Burk medium met elkaar
vergeleken en werd de constante max, die de exponentile groei karakteriseert, bepaald. De
groei werd gevolgd met behulp van metingen van optische densiteit en met plaattellingen.

In beide media werd het exponentieel verloop van de bacterile groei duidelijk waargenomen,
en kon de waarde van max vastgesteld worden op ongeveer 0.24 uur-1. In Rennie media trad
echter besmetting op. De pH op het einde van de experimenten was onveranderd: 6,5 7.

Omdat Rennie medium gistextract bevat en daardoor een stikstofbron, vermoedden we dat dit
medium minder selectief was voor A. vinelandii en daarom meer risico op besmetting met
zich mee brengt. Bovendien was het niet ondenkbaar dat dit de nitrogenase-activiteit zou
onderdrukken.

Overigens valt het op dat ook de meeste wetenschappers die onderzoek

uitvoeren naar Azotobacter vinelandii Burk medium gebruiken. Daarom werd beslist met
Burk medium te werken in de volgende experimenten.

2. Tweede batch experiment: nitrogenase activiteit

In een tweede experiment werd synchroon met de bacterile groei ook de stikstoffixatie
opgevolgd. Dit gebeurde met de zogenaamde acetyleen reductie methode, die gebaseerd is op
het vermogen van nitrogenase om acetyleen om te zetten in ethyleen. De hoeveelheid etheen
geproduceerd in een zekere tijdspanne is een maat voor de nitrogenase activiteit. Deze
hoeveelheid werd gaschromatografisch bepaald.

Er werd gevonden dat de nitrogenase activiteit het hoogst was in het begin van de
exponentile fase en niveaus bereikte van n nanomol ethyleen per minuut per tienduizend
bacterin. Deze resultaten zijn in goede overeenstemming met wat in de literatuur gevonden
kan worden.

85
3. Derde batch experiment: glucose consumptie
In een derde experiment werd gelijktijdig met de bacterile groei ook het glucoseverbruik
opgevolgd. Dit gebeurde door stalen van de batchreactor met een anthron oplossing te
behandelen en dan de lichtabsorptie van de vloeistof te meten. Op deze manier kon een
benadering gevonden worden van de concentratie van glucose waarbij de exponentile groei
van A. vinelandii gehalveerd wordt. Deze concentratie is een karakteristiek voor een microorganisme dat groeit volgens de Monod-kinetiek en waarbij n substraat limiterend werkt.
Deze concentratie wordt de Ks waarde genoemd.
Deze concentratie was opvallend hoog: 4,5 gram per liter. Misschien kan deze hoge waarde
toegeschreven worden aan de bescherming van nitrogenase door ademhaling.

4. Vierde batch experiment: effect van afvalwater van olijfperserijen

In een vierde proef werd bij het Burk medium tien volumeprocent afvalwater gevoegd. Om
de samenstelling van dit afvalwater niet te veranderen werd dit niet gesteriliseerd. Wel werd
het geneutraliseerd met calciumoxide, gefilterd en gecentrifugeerd om de vaste substantie
eruit te halen.
In dit experiment werd Azotobacter vinelandii volledig overgroeid door andere microorganismen. Van meetbare nitrogenase-activiteit was geen sprake. De pH in de reactoren
was gezakt naar 3, een waarde die geen groei van Azotobacter vinelandii toelaat.

5. Experimenten met continue reactor

Om de optimale verblijftijd van medium in een bioreactor met Azotobacter vinelandii te
bepalen werden er proeven gedaan met een continue reactor, gevoed met Burk medium.
Verschillende verblijftijden werden ingesteld en voor elk van deze situaties werd de grootte
van de populatie, de nitrogenase activiteit en het glucose-gehalte in de reactor gemeten.

Het belangrijkste probleem dat is opgetreden bij deze proeven is dat de reactor besmet is
geraakt met een niet nader gedentificeerde soort Bacillus. De invloed van deze bacterie op
de populatie is in de mate van het mogelijke ingeschat. Vermoedelijk was de impact ervan op
de groei van Azotobacter vinelandii enkel van belang bij zeer lange verblijftijden. Ook kan
aangenomen worden dat deze bacterie afhankelijk was van Azotobacter vinelandii wat betreft
zijn stikstofbron, want ze was niet in staat zelf stikstof te fixeren en kon ook niet groeien in
het stikstofvrij medium dat in deze proeven gebruikt werd wanneer Azotobacter afwezig was.

86
De yieldfactor voor substraatverbruik van zowel A. vinelandii als de besmetting zijn
berekend. Voor Azotobacter werd de volgende waarde gevonden: 74 x 109 cellen gevormd
per gram glucose geconsumeerd.

Het berekenen van de kinetische constanten Ks en max aan de hand van de gegevens die
verzameld werden in dit experiment leverde echter betekenisloze waarden op. Dit kan erop
wijzen dat de verschillende staalnames genomen zijn op een moment dat de populaties in de
reactor nog niet in evenwicht waren. Een andere mogelijkheid is dat er een ander substraat
dan glucose limiterend werkte.

Wat betreft de stikstoffixatie werden eigenaardige resultaten geboekt: Azotobacter vinelandii

bleek in staat een zeer grote nitrogenase activiteit te ontplooien bij zeer korte residentietijden.
Zo bleek de vorming van etheen per cel bij een verblijftijd van 6 uur duizend maal groter te
zijn dan bij een verblijftijd van 46 uur. De activiteit van het nitrogenase enzym liep dan op tot
0,23 nanomol ethyn gereduceerd per tienduizend Azotobacter vinelandii-cellen.

De laatste proef die werd uitgevoerd was het onderzoeken van het effect van gesteriliseerd
afvalwater van olijfperserijen op de groei en stikstoffixatie van Azotobacter vinelandii. De
proeven waren vrij beperkt van opzet, maar er werden duidelijke indicaties gevonden dat het
effect gunstig is: zowel de groei als de stikstoffixatie werden bevorderd, zij het na een korte
aanpassingstijd.

87

C. ALGEMENE CONCLUSIE
Wanneer de resultaten van het hierboven samengevatte onderzoek worden samen gelegd met
de bevindingen die in de literatuurstudie worden beschreven, kunnen enkele besluiten
getrokken worden die eventueel kunnen leiden naar een verbeterde methode om het probleem
van het afvalwater van olijfperserijen aan te pakken.

Het afvalwater wordt geproduceerd op zeer grote schaal, in relatief korte tijd, het is toxisch en
heeft een zeer hoog gehalte aan organische stof, en het wordt geproduceerd in een regio die
niet te lijden heeft van overbemesting, integendeel.

Dit zijn goede argumenten om het

gebruik van dit afvalwater als meststof te verdedigen.

Dit wil echter nog niet zeggen dat Azotobacter vinelandii het geschikte organisme is voor de
voorbehandeling van dit afval.

Het is een feit dat A. vinelandii over de enzymatische

middelen beschikt om dit afval te detoxifiren, maar er zijn verschillende aanwijzingen dat
deze weinig competitief is ten opzichte van andere micro-organismen in bioreactoren.
Bovendien is het lang niet zo dat alleen Azotobacter de giftige stoffen in het afvalwater aan
kan. De vraag dringt zich dan ook op of er geen bijkomend en kritisch onderzoek nodig is
naar de samenstelling van de natuurlijk voorkomende microflora in deze bioreactoren.

Indien inderdaad zou blijken dat Azotobacter vinelandii als dominante bacterie
hoofdverantwoordelijke is voor het detoxifiringsproces moet er bekeken worden met welke
ingrepen een efficintere werking van de bioreactor kan bekomen worden.

Laten we hiervoor enkele belangrijke vaststellingen op een rijtje zetten. We weten dat
Azotobacter een hoge pH vereist. We weten ook dat bij lage concentraties aan afvalwater
andere bacterin de A. vinelandii-populatie overgroeien. Daarnaast hebben verschillende
wetenschappers aangetoond dat hoge zuurstofgehaltes de substraatconsumptie van
Azotobacter doen verhogen. En tenslotte blijkt een korte residentietijd in een bioreactor de
stikstoffixatie van Azotobacter vinelandii te bevorderen.

88
Vandaar ons voorstel om in de plaats van een gewone continue reactor zoals deze vandaag in
werking is een cascade van twee of meer bioreactoren te gebruiken.
Het probleem met een gewone continue reactor is namelijk dat de vloeistof in de reactor
dezelfde samenstelling heeft als het eindproduct. Doordat de eigenschap van dit eindproduct
nu juist is dat het weinig of niet toxisch is impliceert dit dat de groei van micro-organismen in
de bioreactor niet of nauwelijks geremd wordt, zelfs als deze organismen met de afbraak van
fenolen niets te maken hebben.

Dit kan alleen maar een negatieve invloed hebben op

Azotobacter, bijvoorbeeld door verlaging van de pH. Wanneer daarentegen een opeenvolging
van bioreactoren gebruikt wordt, kan in de eerste reactor een relatief korte residentietijd
gehandhaafd worden om de groei van en stikstoffixatie door A. vinelandii te bevorderen.
Immers, als er nog niet veel afbraak van fenolen gebeurt zal dit medium veel selectiever zijn
voor microben zoals Azotobacter vinelandii, zeker als deze reactor dan nog goed belucht
wordt. Bovendien zal ook de pH van het afvalwater hier bewaard blijven, en deze licht
alkalische waarde is nu precies ideaal voor Azotobacter.
Deze eerste eenheid kan dan functioneren als kweekbak voor Azotobacter-cellen die
stroomafwaarts in het systeem het afvalwater verder remediren.
Wanneer het laatste bassin van het geheel als opslagtank opgevat wordt kan men ook de
verblijftijd in de reactoren beter controleren.

Om een dergelijk systeem te ontwerpen is het wel nodig de ideale concentratie aan afvalwater
te kennen in een reactor. Ook dit kan het onderwerp uitmaken van verder onderzoek.