the influence of olive mill wastewater on the growth and nitrogen fixation of azotobacter vinelandii

7/21/2019 THE INFLUENCE OF OLIVE MILL WASTEWATER ON THE GROWTH AND NITROGEN FIXATION OF AZOTOBACTER VINELANDII

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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



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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 ariseduring 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.



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CONTENTS

I. INTRODUCTION……………………………………………………………………4

II. STUDY OF LITERATURE………………………………………………………….5

A. The olive culture………………………………………………………….5

1. The olive tree……………………………………………………..5

2. History……………………………………………………………5

3. Geographic distribution…………………………………………..6

4. Importance of the olive culture…………………………………..7

5. olive culture by-products…………………………………………9

B. Characteristics of olive mill wastewater………………………………...12

1. Physico-chemical properties…………………………………….12

2. Toxicity………………………………………………………….143. 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 fertilizer………………………………22

D. Azotobacter vinelandii…………………………………………………..25

1. Taxonomy……………………………………………………….252. Natural habitat…………………………………………………...25

3. Description of Azotobacter vinelandii Lipman………………….27

4. Use of phenolic compounds……………………………………..29

5. Nitrogen fixation………………………………………………...32

III. 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

IV. GENERAL CONCLUSION………………………………………………………..66

V. APPENDICES…………………………………………………………………….…68

VI. WORKS CITED……………………………………………………………………..69

VII. BONDIGE NEDERLANDSE VERTALING………….……………………………79



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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.



5

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 Mahjoub-

Boujnah (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.



6

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 number of trees (x 1000) surface (ha)

Algeria 16,430 162,800

Angola 40 400

Egypt 1,650 10,500

Libya 4,000 100,000

Morocco 33,000 330,000

South-Africa 300 2,500

Tunisia 55,227 1,400,000

Argentina 5,000 50,000

Brazil 84 840

Chile 275 3,070

Mexico 480 6,000

Peru 560 5,603

U.S.A 1,750 14,500Afghanistan 1,000 -

China 20,000 128,000

Cyprus 1,290 6,880

Iran 750 10,000

Iraq 750 10,000

Israel 1,520 12,600

Jordan 2,650 16,360

Lebanon 6,000 32,000

Syria 36,000 327,037

Turkey 83,000 820,000

Albania 5,500 39,300France 5,000 44,600

Greece 120,000 758,100

Italy 125,000 1,176,556

Malta 23 200

Portugal 49,496 1,114,000

Spain 167,000 1,087,000

Yugoslavia 4,104 26,960

Australia 208 2,000

Global 748,423 8,701,697

Mediterranean area 714,240 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



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Zervakis). Due to the important growth in this industry (2.3 % every year) it has recently won

the second position on the world’s 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



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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 three-

phase 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 m 3 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.: by-

products 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.



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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)



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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.

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.

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



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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 4-

methylcatechol. 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.

Tunçel 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.



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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 (PHA’s) from

reactors where Azotobacter chroococcum was grown in diluted OMW. PHA’s 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 µmole Cd 2+ and 5.5 µmole Pb2+ per

µmole CPS, which corresponds to 4.29 g of Cd 2+ 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 meansFlouri 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 wasobtained 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 questionwhether 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 micro-

organisms 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 soil-

borne 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 N 2-

fixation 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 + λ)kt]e-kt

where λ, 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 10

5

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 micro-

organisms 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. A ZOTOBACTER 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 µm or

less, sometimes however much longer, of ten 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 at mospheric nitrogen and can

therefore compete with other micro-organisms on such media. This is the basis

of methods for obta ining pure cultures and studying impure cultures. Pure

cultures grow on a variety of media, but best on those that are nitrogen deficient.

Optimum temperature abo ut 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 micro-

organisms 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 µm long and

1.5 to 2.4 µm 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 µm

in wavelength and 0.39 to 0.55 µm in amplitude. In 2-day and

older cultures the cells (which may then be ellipsoidal) containnumerous sudanophilic and metachromatic granules in random

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 µm. Large cells seem

to occur only in the logarithmic growth phase, the small form is the more natural.

Fig. 6: A. vinelandii.

The bar represents 4

µm (Vela & Rosenthal,

1972).



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, daylight-

visible 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 optimaltemperature 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, p-

hydroxybenzoic 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

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.

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 ββββ-ketoadipate-enol-lactone, which is further degraded to ββββ-

ketoadipate and finally ends up as succinyl CoA and acetyl CoA.



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 4-

hydroxybenzoate 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 p-

hydroxybenzoic 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 earth’s

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 1-

electron 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.

HC≡CH + 2 e- → H2C=CH2 + 2 e- (slow)→ H3C CH3

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 cell’s 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 cell’s 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 (Linkerhägner 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 (Ysubstrate

max) than during growth under

conditions of oxygen limitation.

In these reactions and due to leakage of electrons from the respiratory chain, oxygen-

radicals 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 thecytoplasm.

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 vinelandii’s 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 Pk a 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 break-

down 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 Azotobacter ’s 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 replaceglucose 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 µ,

• 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 population’s

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.

0,0E+00

2,0E+07

4,0E+07

6,0E+07

8,0E+07

1,0E+08

1,2E+08

1,4E+08

1,6E+08

1,8E+08

0 10 20 30 40 50 60

time (hours)

# c . f . u . p e r m l .

0

0,2

0,4

0,6

0,8

1

O . D .

# c.f.u.

O.D.

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



41

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

logaritmic scale.

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

0,0E+00

2,0E+07

4,0E+07

6,0E+07

8,0E+07

1,0E+08

1,2E+08

1,4E+08

0 10 20 30 40 50 60

time (hours)

# c . f . u . p e r m l

0

0,2

0,4

0,6

0,8

1

O . D .

# c.f.u.

O.D.

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60

time (hours)

l o g # c . f . u . p e r m l

0

0,2

0,4

0,6

0,8

1

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

O.D.



42

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

In theory, the function describing bacterial growth is:

dC / dt = µ C

where C is the concentration of bacteria and t is the time. µ 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 = µmax C

integration gives:

ln C = t.µmax + constant

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

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

0

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50

time (hours )

l o g # c . f . u . p e r m l

0

0,2

0,4

0,6

0,8

1

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

O.D.

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

scale.



43

If a population grows from C1 to C2, where C2 = 2.C1, in time interval τd , being t2-t1, one can

write:

ln 2 = µmax . τd

τd 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: µmax = 0.2462 hour -1

Co = 1443 cells/ml

τd = 2.82 hours

(R 2 for these values was 0.9564)

for Burk: µmax = 0.2381 hour -1

Co = 2362 cells/ml

τd = 2.91 hours

(R 2 for these values was 0.9875)

In our experiments no lag-phase was observed. This was to be expected since the micro-

organisms 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.



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 10³ pas . 10-6 m³) / (8.31 J/mole°K . 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.


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.

Graph ¡Error!Argumento de modificador desconocido.: growth of A. vinelandii in batch6

6,2

6,4

6,6

6,8

7

7,2

7,4

7,6

7,8

8

0 10 20 30 40 50

t ime (hours)

l o g # c . f . u . p e r m l

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



47

Another remark to be made is that the µ 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.

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).

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

0

1E-14

2E-14

3E-14

4E-14

5E-14

6E-14

7E-14

8E-14

9E-14

1E-13

0 10 20 30 40 50

time (hours)

m o l e C 2

H 4 / ( m i n . c . f . u . )



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.


a. cultures

The batch culture used in this experiment was a 1-litre conical flask with 400 ml of nitrogen-

free 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 themedium. 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

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.

Fig. 9: anthrone



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.


The population of Azotobacter vinelandii and the glucose concentration in the reactor are

represented in graph 7.

The exponential growth started with a value for µ of 0.22. At the time that µ 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.

4

5

6

7

8

9

0 10 20 30 40 50 60 70 80 90

time (hours)

l o g # c . f . u . p e r m l

0

0,02

0,04

0,06

0,08

0,1

0,12

[ g l u c o

s e ] i n g / l

log # c.f.u.

[glucose]

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



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 . µmax . Cs / (K s + Cs)

where C represents the concentration of micro-organisms and Cs the concentration of limiting

substrate. µmax and K s are constant values that characterize the growth. The physical meaning

of K s 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.

µmax had been determined in former experiments (see A), K s 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.


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 withsterilised 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 µm, 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 µm 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.


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 toolow 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.



54


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 PLG-

Peristaltic 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.



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

integration gives: ln C = -(F/V).t + cte

and finally: C = e-(F.t / V) . C0

with C0 the initial concentration of OMW.



57


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.

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.

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

0,E+00

1,E+08

2,E+08

3,E+08

4,E+08

5,E+08

6,E+08

0 20 40 60 80

residence time (hours)

# c . f . u . p e r m

l .

0

1

2

3

4

5

6

7

8

9

10

[ g l u c o s e ] ( i n g / l ) .

A. vinelandii

contaminant

[glucose]



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 (r s) at a certain growth rate (r x) and cell

concentration (Cx) is therefore:

r s = r x /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:

r x = F.(Cx)/V(2)

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

made for the substrate:

r s = F.(Cs,in - Cs)/V (3)

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

r s = r x,1 /Y1 + m1.Cx1 + r x2 /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:

r s = Cx . F / (Y . V)

The results are represented in graph 9.



60

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.

0

0,05

0,1

0,15

0,2

0,25

0 20 40 60 80

residence time

g l u c o s e c o n s u m p t i o n

( g / l . h o u r )

A. vinelandi i

contaminant

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

medium.



61

For the limiting substrate in a culture, 2 more parameters can be calculated: K s and µmax.

They are both parameters of the Monod model for bacterial growth, which says:

dCx/dt = µmax . Cs / (Cs + K s) . Cx

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

other, resulting in:

F.(Cx)/V = µmax . Cs / (Cs + K s) . Cx

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

The reason why K s and µmax 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

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

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)

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

flow reactor with Burk medium.

0,0E+00

5,0E-08

1,0E-07

1,5E-07

2,0E-07

2,5E-07

3,0E-07

3,5E-07

4,0E-07

0 20 40 60 80

residence time (hours)

m o l e C 2 H 4 / m l . m i n

.

0

1

2

3

4

5

6

7

8

9

10

[ g l u c o s e ] ( i n g / l ) .

acetyleneconversion

[glucose]



63

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.

Graph 11: nitrogenase activity per A. vinelandii cell in continuous flow reactor with

Burk medium on logarithmic scale.

1E-17

1E-16

1E-15

1E-14

1E-13

0 10 20 30 40 50 60 70 80

res idence t ime (hours)

m o

l e C 2 H 4 / m i n . c . f . u . .



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.

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

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

0,0E+00

2,0E+07

4,0E+07

6,0E+07

8,0E+07

1,0E+08

1,2E+08

1,4E+08

1,6E+08

1,8E+08

-2 3 8 13 18 23 28 33 38 43 48 53

t ime (hours)

# c . f . u . / m l

0

2

4

6

8

10

12

14

v o l % O M W [A. vine lan dii]

[OMW]

0,E+00

1,E-15

2,E-15

3,E-15

4,E-15

5,E-15

6,E-15

7,E-15

-2 3 8 13 18 23 28 33 38 43 48 53 58

time (hours)

m o l e C 2 H 4 / c . f . u . . m i n

.

-2

0

2

4

6

8

10

12

14

v o l % O M W

.

nitrogenase

activity

[OMW]



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

K 2HPO4 : 0.8 g/l

KH2PO4 : 0.2 g/l

NaCl : 0.1 g/l

Na2FeEDTA : 0.028 g/l

Na2MoO4.2H2O : 0.025 g/lyeast 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)

K 2HPO4 : 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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79

De invloed van afvalwater vanolijfperserijen 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.



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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 geïdentificeerde

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 bacteriën 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.



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Ten tweede is er de anaërobe 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 bacteriën.

Daarom ziet het er naar uit dat een voorbehandeling nodig zal zijn.

Ten derde is er de aërobe 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 aërobe

biologische voorbehandeling te geven en pas daarna als meststof te gebruiken. Het micro-

organisme 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 bacteriën die de voorkeur

geven aan neutrale of alkalische bodems. In tegenstelling tot de meeste andere

stikstoffixerende bacteriën zijn de Azotobacteraceae vrij levende bacteriën; 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

bacteriën van meer dan 4 µm vermelden; anderzijds wordt er gewag gemaakt van een zeer

uitgesproken polymorfisme, in die mate dat ook cellen van 0,3 µm 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 nutriënt 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 beïnvloeding 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.



84

B. EXPERIMENTEN

1. Eerste batch experiment

In een eerste, verkennend onderzoek werden Rennie medium en Burk medium met elkaar

vergeleken en werd de constante µmax, die de exponentiële 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 bacteriële groei duidelijk waargenomen,

en kon de waarde van µmax 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 à 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 bacteriële 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

exponentiële fase en niveaus bereikte van één nanomol ethyleen per minuut per tienduizend

bacteriën. 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 bacteriële 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 exponentiële groei

van A. vinelandii gehalveerd wordt. Deze concentratie is een karakteristiek voor een micro-

organisme dat groeit volgens de Monod-kinetiek en waarbij één substraat limiterend werkt.

Deze concentratie wordt de K s 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 micro-

organismen. 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 geïdentificeerde 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 K s en µmax 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.



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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 detoxifiëren, 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 detoxifiëringsproces moet er bekeken worden met welke

ingrepen een efficiëntere 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 bacteriën 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 remediëren.

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.

the influence of olive mill wastewater on the growth and nitrogen fixation of azotobacter vinelandii

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