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Enhanced Biosurfactant Production Using Synchronous Cultures of Corynebacterium alkanokyticum

Department of Chernical Engineering McGilf University, Montreai

March, 1998

A thesis submitted to the Faculty of Graduate Studies and Research in partial hilfilment of the re-quirements of the degree of Master of Engineering

O John Crosman 1998

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Abstract

A technique using fluorescence spectroscopy was developed to measure the

concentration of phospholipid biosurfactant produceci by C o r y n e b a ~ t ~ u m

alkanolyticum. Synchronous cultures of this organism were grown in a self-cycling

fermentor (SCF). Application of a standard cycling scheme to the fe~nentor allowed for

production of between 0.82 and 1.10 g/L of bionirfactant, depending on the rate of

aeration to the systern. Asynchronous cultures grown under the same operathg conditions

produced oniy 0.75 g/L of phospholipid.

Extension of isolated fennentor cyctes allowed for fürther increases in

biosurfactant production by the synchronous culture- Extending a cycle for ninety

minutes increased the phospholipid concentration to 1-44 a, without requiring

additional hydrocarbon substrate. Placing broth harvested h m standard SCF cycles in

separate vessels and ailowing the fermentation to continue ailowed concentrations to rise

to 1.89 g/L without additional hydrocarbon addition, and to 2.92 g/L with extra

hexadecane substrate.

Continual addition of hexadecane to the SCF caused biosurfactant production to

cease, suggesting that the microorganism does not produce biosurfactant to increase the

availabilty of insoluble hydrocarbon substrates.

Résumé

Des cultures synchronisées de Corynebacterium alkanoCyticum, cultivées dans un

fermenteur auto-cyclique ('AC), a produit entre 0,82 et 1,10 g/L de biosurfactant. La

concentration du biosurfactant (mesurée en utilisant une technique de fluorescence

spectroscopique) dans les cycles réguliers avait une dépendance sur la vitesse de

gazéification du réacteur. En exposant les cultures non-synchronisées aux mêmes

paramètres d'opération, elles n'ont produit que 0,75 g/L du même biodactant.

L'extension du cycle fermentatif d'une durée de 90 minutes a augmenté la

concentration du biosurfactant à 1,44 g/L, sans addition du substrat supplémentaire. Les

concentrations entre 1,89 et 2,92 g/L étaient réalisées par une fermentation en deux

étapes, où la fiachon du bouillon de culture prise du FAC a la fin du cycle a été mise dans

un fermenteur indépendant.

L'addition continuelle de substrat au fermenteur a cessé la production du

biosurfactant par l'organisme. Ce résultat propose que la production du composé

t ensioac tif n'est pas associée à un besoin des microbes l'accessibilité des hydrocarbures

insolubles.

Acknowledgements

1 would like to thank the faculty and staff of McGilI Chernical Engineering for ail of their assistance during the last two years. Special thanks to Barbara Hanley, Jeanne Terrasi, Anne Prihoda, Louise Miller, and Michaet Harrigan for ai1 of your help and some good conversations.

My thanks also go out to ai i members of the research group, past, present, and future-Bill McCdEey, Fabien Marino, François Godin, Gregan Dunn, Jeff Barriga, Jeff Karp, Jimmy Gartshore, Mike May, Mike Silverberg, Rob Pinchuk, Scot Hughes, Wayne Brown, and Xiaohong Wang-for showing me the ropes, for good advice, and for putthg up with the not-so-rare bouts of often-extreme moodiness.

I also want to thank other graduate students in the department-Coha Domnik, Jennifer Peters, Shau. Sidwall, Praveen Prasanna, and Karen Sum. Whether 1 needed information or gossip or just wanted to vent, 1 couldn't have sunlved without you.

To Treena Wong, 1 express my sincerest thanks for al1 of your support and a sympathetic ear. 1 must also acknowledge the fnendsbip of Kathy & Gus over the past six years. My voices of sober second thought and my indirect co~ect ion to the homeland, 1 thank you for al1 you've done, right £?om the very first day 1 arrived.

Je voudrais remercier les hommes de ma vie-Jocelyn et Stéphane-pour l'amitié, l'aventure, le drame, et des sessions de psychanalyse gratuites. Mes remerciements aussi sont accordés à Jean-Guy. Après presque trois ans, je comprends mieux, grâce à toi, la vie, i'amour, i'homêteté, l'angoisse, la perte, et, surtout, moi-même. Que tu sois heureux, c'est le vœu que je tiens toujours à cœur pour toi.

It is for mom and dad, for al1 their support over not just the 1st two years, but the Iast two decades, that 1 reserve my wamiest thanks and love.

Finally, 1 express m y sincerest gratitude to Dr. Cooper. Not only did he provide with this fantastic opportunity and put up with a lot of grief, but he pushed me fùrther than I had ever thought 1 could go.

Table of Contents

1 Introduction 1 1.1 Biodâctants 1

1.1.1 Biodactaut Stnictures and Properties 1 1.1.2 Biodactant Production and the Problem of Low Yields 4 1.1.3 Biosuffactant Production and the Problem of

Residual Hydrocarbons 1.2 Self-Cycling Fermentation

2 Objectives

3 Materials and Methoch CuIture Maintenance

Medium and Growth Conditions

B iomass Measuremeat

Cell Counts

Hexadecane Measurement

Surface Tension Measurement

Phospholipid Isolation 3.7.1 Extraction of Total Lipids fiom Broth 3.7.2 Separation of Phospholipids and Neutrd Lipids 3.7.3 Fluorescence Measurements

Experimental Apparatus 3 -8.1 Apparatus for Standard SCF Experiments 3 -8.2 Apparatus for Contirnous Hydrocarbon Addition

Experirnents

4 Results 4.1 Phospholipid Analysis

4.1.1 Determination of Fluorescence Ptoperties of the Phospholipid

4.1.2 Correlation of Phospholipid Concentration with Fluorescence

4.2 Continuous Hydrocatbon Addition Experiments

4.3 Biodactant Production Using the SCF Technique 4.3.1 SCF Dissolved Oxygen Profiles 4.3 -2 Isolateci Extended Cycles 4-.3 -3 Sequence of Extended Cycles 4.3 -4 Two-Stage Fermentations 4.3.5 Hydrocarbon Meastuement

5 Discussion 60 5.1 Fluorescence Analysis of the Phospholipid Biosdactant 60

5 -2 Biosurfactant Production 64 5.2.1 Influence of the Hydrocarbon on Biosurfàctant Production 64 5.22 Biosmfactant Production Using the SCF Technique 67

6 Conciasions 75

7 References 76

Appendix 1 82

Table 3.2.1 Table 3.5.1

Table 4.1.1

Table 4.1 -2

Table 4.3-1

Table 4.3.2

Table 5.2.1

Table 5.2.2

Composition of Growth Medium Operating Parameters for the Gas Chromatograph

Characteristics of Emission Spectra for Various Cultures of C. alkanoIyticunt 26

Fluorescence Measurements fiorn Flasks of CI alhnoiyticum Grown on Mineral Salts Medium 27

Phospholipid Concentration Changes During the Second Stage of a Two-Stage Fermentation 57

Average End-oGCycle Hexadecane Concentrations 58

Cornparison of Extended Cycle and Shake Flask Experirnent Phospholipid Concentrations 72

Hydrocarbon Use Durhg Extended Cycle and Shake Flask

Figure 1.2.1

Figure 3 5 1 Figure 3.8.1 Figure 3 -8.2

Fi,oure 4- 1.1 Figure 4.1 -2 Figure 4.1.3

Figure 4.2.1

Figure 4.2.2

Figure 4.2.3

Figure 4.3.1 Figure 4.3.2 Figure 4.3.3

Figure 4.3 -4

Figure 4.3.5

Figure 4.3.6

Figure 4.3.7

Figure 4.3.8

Figure 4.3.9

Figure 4.3.10

List of Figures

Idealized Biomass Concentration, Limiting Substrate Concentration, and Dissolved Oxygen Concentration Profiles in the SCF I 1

Calibration Curve for the Gas Chromatograph 18 Standard Self-Cycling Fermentation Apparatus 24 Modified Self-Cycling Fermentation Apparatus for

Continuous Hydrocarbon Addition Experiments 25

Emission Spectnim for the Phospholipid Biosurfactant 28 Phospholipid-Fluorescence Caiibration Curve 29 Variation of Surface Tension with Phospholipid Concentration 30

End-of-Cycle Biornass Concentration, Hexadecane Concentration, and Surface Tension Values for Continuous Addition of Hexadecane Experiments 33

Dissolved Oxygen Profiles for Continuous Hexadecane Addition Experiments 34

Intracycle Surface Tension Values and Hexadecane Concentrations for Continuous Hexadecane Addition Experiments

Dissolveâ Oxygen Profile for a Series of Standard SCF Cycles Ce11 Counts for a Standard SCF Cycle Cycle Length, End-of-Cycle Biomass Concentration, and

End-of-Cycle Phospholipid Concentration for Standard Cycles (60 mL 0Jmi.n; 6g/L (NH,),S04; 3g/L N]H4N03)

Intracycle Hydrocarbon and Phospholipid concentrations for Standard Cycles (60 mL O Jmin; 6g/L W&SO4; 3 a WNO3)

Cycle Length, End-of-Cycle Biomass Concentration, and End-&Cycle Phospholipid Concentration for Standard Cycles (120 mL O,/min; 6g/L (NH4),S0,; 3g/L NKNO,)

Intracycle Hydrocarbon and Phospholipid Concentrations for Standard Cycles (1 20 mL O,/min; 6 g L (NH,)tSO,; 3ga NH4NO3)

Intracycle Hydrocarbon and Phospholipid Concentrations for Standard Cycles (120 mL O-; 3g/L (NHJ2S04; 1 Sg/L MI,N03)

Dissolved Oxygen Profile for a Senes of Standard SCF Cycles FoUowed by a Single Extended Cycle

ïntracycle Hydrocarbon and Phospholipid Concentrations for an Isolateci Extended Cycle (120 mC 02/min; 3d.L (NH,),so4; 1.5gIL NKNO,)

intracycle Hydrocarbon and Phospholipid Concentrations for an

Isolated Extended Cycle (120 mL OJmin; (NH,&m; 3dL NH4No3) 50

Figure 4.3.1 1 Dissolved Oxygen Rome for a Series of Extendeci Cycles 53 Figure 4.3.12 Cell Counts for One in a Series of Extended Cycles 54 Figure 4.3.1 3 Intracycle Hydrocarbon and Phospholipid Concentrations for

One in a Series of Extended Cycles (Cycle Extension of 30 minutes) 55

Figure 4.3.14 Intracycle Hydrocarbon and Phospholipid Concentrations for One in a Series of Extended Cycles (Cycle Extension of 90 minutes) 56

Figure 4.3.15 Standard Deviation of Hydrocacbon Concentration Measurements v e r w the Actual Reporteci Concentrations 59

Figure Al. 1 Fluorescence Emission Spectra for Uninoculated Nutrient Broth 83 Figure A1.2 Fluorescence Emission Spectra for Inoculated Nutrient Broth 83 Figure A1 -3 Fluorescence Emission Spectra for Uninoculated Mineral Salts

Medium 84 Figure A1.4 Fluorescence Emission Spectra for Inoculated Minerai Salts

Medium 84 Figure A1.5 Fluorescence Emission Spectra for the Supernatant of a

Centrihged Sample h m a Culture Gmwing on Mineral Salts Medium 85

Figure A1.6 Fluorescence Emission Spectra for the Resuspended Peilet of a Centrifhged Sample h m a Culture Growing on MSM 85

Figure A1 -7 Fluorescence Emission Spectra for the Organic Phase of a Sample Extracteci Using Chloro form 86

1 Introduction

1.1.1 Biosurfactait Structures and Properties

Biosutfactants are microbialiy-produced surfze active compounds. They are

generaüy less toxic in nature and more bidegradable than synthetic Sllffactants,

charactenstics that make them desirable for use in many industrial applications.<lu")

Biosurfactants have diverse properties, many of which are specific to temperature, pH,

and salinity of the system in which they are used?) These have led to a broad range of

suggested applications in the cosmetics, phannacology, and food processing fields."" The

surface-active nature of these compounds dlows for some solubilization in imrniscîble

oil-water mixtures. This property has generated much interest in the application of

biosurfactants in the areas of oil recovery and in the remediation of oil and chernical

spills in water and ~ o i l < ' ~ - ' ~ ~ ~ ' )

Biosurfactants are produced chiefly by organisms grown on hydrocarbon

substrates such as straight-challr &anes and o~~s~"-'3*"2'-"26~an~55~ althou& s0me

work reported production using simpler, water-soluble ~ubstrates.'"~*~*'~) Other special

constraints on the growth medium composition have also been reported. Frequentiy,

biosurfactants are produced when the organism is subj ected to nitrogen-limiting growth

COnditioilS.(7.8.10.~ 1.121 B- iosurfactant production by Arthrobacter parafineus grown on

sucrose has been linked to the concentration of cupric (Cu2') ions in the rnedi~m."~) ~ e ~ +

levels affectecl the production of a glycoprotein by PseudomonasfIuorescens while

EDTA concentration was shown to be a factor in f-entations with Pseudomonas N-1

on hydrocarb~ns."~ Thiobaciïlas thiooxidans produced phospholipid biosdactants using

sulphur-based, rather than carbon-baseci, energy so~rces.""'~

There is no single chemical structure that characterizes biosurfactants. However,

the most commonly seen biosurfactants in femientation processes fa11 into one of four

categ~ries?*'~-'" Glycoiipids, the most commonly-produced biosdiactantsp) con& of a

carbohydrate unit bonded to a glyceride backbone. Trehdose glycolipids have been

produced by Rhodococcus ery~hropolis>('~) Arthrobacter p~rafinars,(~*"*'~ and by species

of Brevibacteria, Cogmebacteria, and Mi~rococcii.'~ Pseudomonadr are known to

produce rhamnose glycolipids, or rhamn~lipids.<'~~'~) Sophorose lipids are commonly

produced by among Torulopsis ~pecies.'~)

Amino acid bionirfactants are the second comrnonly occuring class of

biosurfactants. These biosurfactants are characterized by the inclusion of one or more

proteins in their chemical structures. Lipopeptides have been produced by Pseudomonas

rubescens'") and Agrobacterium tumefaciens."' Pseudomonas N-1 has been s h o w to

produce ornithine lipids when grown on various straight-chah hydmcarbons."')

Biosurfactants of this type are among the most effective reported in the Literature.""

Corynebacterium nlknnolyticum)" ntiobaciiius thio~xidans,<'"~~ and

Micrococcus c e n f c a n ~ ~ ' ~ ) have al1 been shown to produce the third class of

biosurfactants, phosphoiipids. Suc h compounds con& of a glycerol backbone with two

estenfied fatty acids and a phosphate group. The variation in surface activity among

phospholipids is due to the different structurai units substituted onto the phosphate

group. Phospholipids are present in ali organisms as a component of the celluiar

membrane. Few organisms have been shown to produce such compounds extracellularly,

although the addition of antibiotics or other materials to the culture medium can induce

excretion of these lipids into the fermentation broth?"')

Fatty acid and neutral lipid biosurfhctants round out the classification scheme.

These compounds, !ike phospholipids, are common in al1 organisms, although, unlike

phospholipids, they are offen extracellular in n a t ~ r e . ~ . ' ~ Alcohols, esters, glycerides, and

carboxyllic acids are dl examples of compounds that have shown some SUff'e-active

properties. These secondary metabolites have nomalIy been produced during

fermentations using hydrocarbon substrates as the chief carbon source."')

The surface activity of many biosurfàctants has been reported in previous studies.

The rharnnolipid produced by Pseudomonas fluorescence 15453 had critical micelle

concentrations of between only 10 and 30 mg/L in distilled water? while the rhamnose

lipid produced by Pseudomonas sp. MUB could have a criticai micelle concentration as

low as 5 mg& depending on the pH of the s o l u t i ~ n . ~ The glycolipid pmduced by

Pseudomonas aeruginosa 44T1 lowered the sdace tension of water to a minimum of 25

mN/m (f?om a value of 72 r n ~ / r n ) . = ~ The interfacial tension between water and n-

hexadecane was lowered to 2 mN/m by the glycolipid of Arthobacter parafFneus 15591

and 4 mN/m by the sophorose lipid of Torulopsis bombicola (fiom a value of 40

m~/m)." M y small quantities of biosurfactant have been necessary to significantly

alter the surface properties of many solutions.

Biosurfactants have dso been shown, in some cases, to have excellent

ernulsification/demuisification properties."-IM It is the ability of such biosurfactants to

create stable hydrocarbon-water emdsions (and thus increase the surface area of the

hydrocarbon-water interface) that has driva research into the area of enhanceci

bioremediation of oil spi Il^.''.'^) It is believed that more organisms can attach to this larger

interfaciai area and thus degrade the spilled oïl at a faster rate.

1.1.2 Biosurfactant Production and the Problem of Low Yields

A key problem in biosuffactant production using straight-chah hydrocarbon

substrates has always been low product concentrations at the end of the fermentation."'

Microgram or rnilligram amounts of biosurfactant per litre of fermentation broth make

downstream separation of the product very costly. While concentrations of biosurfactant

have occasionally surpassai 1 g/L, large initial concentrations of substrate and long

fermentation times were required. Brevibactenkm species grown on 10% (w/v) para*

produced only about 0.4 g/L of a glycoiipid surfactant,(") while Micrococcus sodoneslr

could only produce 0.1 g& of a glycolipid firom the same initial concentration."" The

products of Corynebacterium KY4309 and Corynebocterïum KY4336 averaged higher

product concentrations (about 1 .O fi), but still required at least 10 % (wlv) alkanes to

attain this level."') Micromolar amounts of a phospholipid were reportecl produced fiom

1% (w/v) tetradecane, pentadecane, hexadecane, and heptadecane by Micrococw~

cenifians .(ZZIZ4)

Production bas been even lower on simpler substrates. Norcardia butanka

yielded only 400 mg/L of a giycoiipid h m an unidentifid concentration of sucrose

s~bstrate,'~ while Brevibacterium species produced a mere 250 m@L?

A few isolated studies have reported high biosUTfactant yields. Tondopsis

bombicda has been shown to produce up to 80 of a glycolipid in a continuous

fermentation process. The medium, however, required up to 10Y0 D-glucose and the

addition of vegetable oils. A simîlar medium composition yielded 90 g/L of a glycolipid

by Tonrlopsis apicola MET 43747. Such high yields, regardless of the amount and

number of substrates provided, have been rare.'"

High yields have also recently been reportai in genetically engineered organisms.

Strains of Pseudornonas aeruginosa (s& 65E12) grown on 2% glycerol medium

showed accentuated rhamnolipid production when engineered with certain plasmids.Q')

The large amount of antibiotics required to maintain the culture seem to render the

process infeasible economicaliy.

1.1.3 Biosurfactant Production and the Problem of Residual Hydrocarbons

Hydrocarbon is essential for the production of many biosudactants. Components

of the hydrocarbon have been shown to be incorporated into the biosurfactmt structure.

The work of Makula and Finnerty m24) illustrated very well how the structure of the

hydrocarbon substrate influenced the structure of various cellular metabolites, including

biosurfactants, of Micrococcus cenficans.

In using hydrocarbon substrates, the problem that fkquently arises dong with low

product concentrations is that of residual hybcarbon- Biosurfactant-producing

organisms

ofien do not consume all of the hydrocarbon substrate made available to them.

It is widely reported that the presence of hydrocarbon in a fermentation system

induces biosurfactant production in micr~or~anisrns~~~.~~-~~) Biosurfactants are reporteci

to increase the solubility of organic substrates insoluble in water. The method by which

the hydrocarbon is consumed by the organisms is d l the subject of much debate. The

two main consumption mechanisms that have been proposed are (1) that the organism

attaches to large drops of hydrocarbon where it then consumes the substrate, and (2) that

hny droplets of hydrocarbon attach to and are then consumed by the organism. Some

previous work has suggested that it is the second method of hydrocarbon uptake that is

pre ferred b y organism~.~*' 1'0.61)

Regardless of the method of hydrocarbon uptake by the organism, much work has

involved the use of large initial concentrations of hydrocarbon in a fermentation in an

attempt to induce biosurfactant production. Work with Rhodococ~~~ eryrhopoh used 100

glL of straight-chain al.kane~,('~) while Arthorobocter parafineus KY4303 cultures were

subjected to 10% (w/v) paraffin in a fetmentation broth.""*'" Growth limitation by

another medium component vimially ensured that not all of the carbon source could be

consumed by the organisrn and allowed for the presence of residual hydrocarbons in the

broth. A few experiments in which much srnalier, non-limiting concentrations of

hydrocarbon were useci, however, still showed that not alI of the substrate was consumed

during the fe rmenta t i~n . ' '~~~~)

The general concern in most previous biosurfàctant research has ken sïmply

producing the biosurfactant. In many cases, hydrocarbon concentrations have not been

reported. In work that has reporteci such concentrations, however, it is clear that the

presence of residual hydrocarbon reduces the amount of biosurfactant obtainable in a

fermentation and creates an expensive separation problem in product recovery.

Developed by Cooper and Sheppard@I1, selfcyclhg fermentation represented an

improvement over continualiy-phased culture of mi~roorgan i sms .@~~~ Key to the SCF

process is the monitoring of a parameter associated with growth of the microorganism

(usually dissolved oxygen concentration in the fermentor), as changes in that parameter

often reflect changes in the m e t a b o h of tbat organism. An extremum in the measured

parameter signals the end of microbid growth due to depletion of a limiting nutrient in

the fennentor. Upon attainment of this extremum, half of the contents are harvested h m

the bioreactor and replaced with an equivalent volume of h h nutnents. This pmcess of

nutrient replenishment is referred to as cyciing, with the the between successive

replenishment steps hown as a cycle.

While the h t few cycles in a self-cycled f-entor nonnaily show a transient

behaviour, the system attains a regular, stable, periodic behavio~r.(~"" Idealized profiles

for biomass concentration, Limithg substrate concentration, and dissolved oxygen

concentration in the SCF are shown in figure 1.2.1. At the beginnuig of a cycle,

dissolved oxygen and Iimiting substrate concentrations are at their highest levels, and

decrease throughout the cycle as the organism grows. Growth of the organism is reflected

in the increasing biomass concentration profile. At the end of the cycle, the dissolved

oxygen level has reached a minimum and the Limiting substrate has been depleted.

Biomass levels double over the course of the cycle. The system shows good stability and

results have good repeatability.

Carefid examination of actual intracycle biomass concentration pronlcs, such as

those of Brown and Cooper,(3s) show that during the course of one SCF cycle, the biomass

concentration in the fermentor doubles. This suggests that the duration of one SCF cycle

corresponds to the doubling time of the organism king cultivateci.

Biomass production in the SCF is aIso characterized by a lack of lag and

stationary growth phases. Only the exponentiai phase of biomass growth is present. As a

result, growth of microorganisms can be maintaineci for long periods of time at the

maximum growth rate. Time is not lost as organisms acclimatize to their environment in

the lag growth phase, nor is it lost to cIeaning and sterilization steps that accompany

batch fermentations.

Another important aspect of the SCF process is that it supports the maintenance of

synchronous ce11 cultures. At any specific instant of tirne in a synchronous ce11 culture,

the entire ce11 population is at approximately the same point in its growth cycle.

Synchrony in ce11 cultures in the SCF is confirmeci by looking at measurements of the

number of cells in the system. Previous ~ o r k , ( ~ ~ has shown that during most of a cycle, a

constant number of celIs exists in the fermentation broth. As the end of the cycle is

reached, the number of ceils doubles in a step increase. The synchrony of the culture

alIows for easier study of the ce11 cycle. The fermentation cau be studied as representative

of the growth of a single cell since the growth cycles of ail ceils in the reactor coincide.

There have been two key applications of the SCF in previous work The fkst has

been production of various secondary cellular metabolites, such as antibiotics,

sop horolipids, and citinc acid-(3'37*40-J'*43) The second has bbeen the biodegradation of such

cornmon pollutants as hydrocarbons and aromatic cornp~unds.~""~)

Figure 1.2.1 Idealized biomass concentration, limiting substrate concentration, and dissolved oxygen concentration profiles in the self-cyciing fermentor.

2 Objectives

This work was part of a continuing research effort in which the dtimate goal is to

use self-c ycling fermentation technology to achieve enhanceci yields of secondary

microbial metabolites. In worlring towards this goal, the specific objectives of the present

study were:

1. To develop a rapid, diable analytical technique in which smd sample

volumes couid be used to detennine the biodactant concentration in the

fermentation broth.

2. To detennine if the method by which substrate was added to the fmentor

affécted biosurfactant production by the organism.

3. To determine if the biosurfactant production could be maintaineci by an

organism in a system where no excess hyhcarbon substrate was present.

4. To determine if synchronous ce11 cultures would give more biodactant

production than asynchronous cultures.

3 Materials and Methds

3. f Culture Maintenance

Corjmebacterim alkanolyticum (ATCC 2151 1) was the organism used in this

study. Cui?ures were maintained on nutrient agar (Difco Bacto 0001 -14) plates and on

agar stants at 4°C. Pure colonies were transferred to new plates every two weeks. New

slants were inoculated h m pure colonies growing on the older slants monthlyy Frozen

samples of the organisrn were also kept at -70°C in a Revco fkeezer.

3.2 Medium and Gmwfh Condittons

C. aZkanoiytict(m was p w n on the mineral salts medium of Nakao et al.@" with a

few modifications. The medium composition is described in table 3 -2.1 below.

Table 3.2.1 Composition of the medium used for experiments with C. alkanolyticum.

Compound Concentration "/O (w/v)

w-b)2so4 0.6 NKNQ 0.3 K*04 0.25 =%PO4 0.10 MgS04.7H20 0.05 FeSO47.,O 0.005 bf.&o4*7H20 0.002 ZnS02H20 0.00 L C0Cl2~6H20 0.00 1

The cyclone reactor had a wocking volume of 1.0 L. AU media and apparatus were

sterilized in an Amsco autoclave at 121°C and 19 psig. Medium was steriiized in 10 L

volumes for 1.5 bouts, while glassware and fermentor components were sterilized for 2.5

hou. penods.

Inocula for ail experiments came h m shake flasks. The organimi was aîiowed to

grow up first on nutrient broth. It was then transferrPd to a flask containing 1 mL

hexadecane in 100 mL of the mineral salts medium, M e r two transfers in mineral salts

medium, inoculation of the fennentor was then made h m one of these flasks.

The sole carbon source for the expe-ents was n-hexadecane. The substrate was

not included in the mineral salts medium, as it wodd evaporate upon autoclavation.

Instead, the hexadecane was added to the fermentor separately.

Filtered oxygen was supplied to the fermentor at rates of 60 mU min and 120

mL/min. The temperature rernained constant at 27°C.

3.3 Biornass Measurement

End of cycle biomass rneasurements were made using a standard dryweight

analy~is.'~' Tnplicate 20 mL sarnples were centrifuged at 8000 g for 20 minutes in a

Dupont instruments RC-5 Superspeed Refngerated centrifige. The centrifiige was

operated at a temperature of 4°C in order to separate any hexadecane that remaineci in the

s a ~ n p l e . < ~ ~ The supernatant was decanted and the pellet was washed with and resuspended

in distilled water. The procedure was repeated two more times. The final pellet was

placed in a weighed aluminum pan and left for 24 hom in an oven at 10S°C. Final

biomass concentrations were reported as gram of dry cell m a s p a Litre of fmentation

broth. On average the replicate biomass rneasuements agreed within 6.39% of their

average value.

3.4 Cell Counts

A standard colony count method, based on that outlined by K o ~ h , ' ~ ' was used to

detexmine the number of cells in the férmentor. A 1 mL sample of broth was placed in an

autoclaved Nalgene bottle, diluted to a hnal voIume of 100 mL with steriIized, distilIed

water, and sealed, After tifty inversions, 1 mL of the solution was placed in a second

bottle and diluted to 100 mL with steriiized, distilied water. This solution was also shaken

fifty times. The final dilution consisted of 1 mL o f sample fiom the second dilution and 9

mL of sterile water.

A 0.1 mL sample was taken fiom the nnal dilution bottle, spread on nutrient agar

plates, and aliowed to grow in an incubator for 36 to 48 hours at 27°C. The b a l number

of cells in the fermentor was then calculated using the equation:

where: C = ceil concentration in fennentor (# cells/ml broth)

#COL = number of ceil colonies counted on agar plate.

Three plates were spread for every sample taken, with replicate values agreeing within

12.6% of the average values.

3.5 Hexadecane Measurwnent

Hexadecane concentrations were measured b d on the procedure of Brown and

Cooped3% 2.0 mL samples were taken h m the fermentor and 10.0 pL+ of pentadecane

(intemal standard) were added- The solution was vortexed on high using a Vortex Genie

for one minute. A 5.0 mL volume of iso-octane was added to the sample and the solution

was vortexed on high again for one minute.

Samples were analyzed in a Hewlett-Packard 5890 Series XI gas chromatograph

wiîh a flame ionization detector and HP3395 integrator. The sample volume Uijected into

the chromatograph was 2 PL- Settings on the chromatograph are summarwd in table

3.5.1.

Table 3.5.1 Operating parameters of the Hewlett-Packard 5890 Series II gas chromatograph for hexadecane analysis.

Operating Parameter Vaiue Injection Temperature 200°C Initial Column Temperature 1 OO°C Column Temperature Ramp 1 SCO/min Final Column Temperature 200°C Detector Temperature 250°C Initial Tirne 2.5 min Final Tirne 5.0 min

A calibration cuve was obtained by plotting the hexadecane concentration versus

the area ratio of the chromatogram peaks (ratio of the hexadecane peak area to

pentadecane peak) area using samples of known hexadecane concentration in water. Since

the solutions were made in water and subjected to the same extraction procedure

described above, the cali'bration curve takes into acwunt the fact that not aü of the

hydrocarbon is extracted. The curve is shown in figure 3.5.1 below.

O 1 2 3 4 5 6 7 8 9 10

Hexadecane Concentration (mUL broth)

Figure 3.5.1 Cdibration Curve For Hexadecane Concentration In The SCF Fennentor.

3.6 Surface Tension Measumment

Surface tension measurements were made using the Mode1 215 Autotensiomat

surface tension analyzer (Fisher), which employs the du Nouy method- A platinum-

iridium alloy ~g of circdetence 6.000 cm and R/r value (ratio of the radius of the ring

to the radius of the wire of the ring) of 53.75. AU measurements were made at 21°C.

A 5.0 mL volume of sample was used in each case. Samples were p I d in Petri

dishes of diameter 3.5 cm. The ring was lowered into the sample, and the sample was

then lowered (at speed 0.2 idmin) until the ring broke through the sample-air interface.

the measurements were repeated until a constant surface tension value was obtained. The

ring was cleaned between samples by heating in a Bunsen bumer to burn off any residue.

Measurements were made using samples of the whole broth. It was obsmed over

the course of the experimental work that centrifugation of the samples to remove

particulate matter fkom the samples did not have a significant effect on the surface tension

measurements.

3.7 Phospholipid h o l a t h

3.7.1 Extraction of Tom Lipids fkom Broth

Lipid materials were extracted fiom the fermentation broth ushg a chlorofom-

methanol solvent mixture (2:1 v/v) and potassium chioride according to the methoci of

Folch et al,'-

3.7.2 Separation of Phosphoüpids and Neutrai Lipids

Phospholipids were removed fiom the total lipid extract using acetone

precipitation according to the method of Kated4') A 10% MgC1,a6H2O salt solution in

methanol was used. The acetone solvent was evaporated under a stream of nitrogen at

room temperature and the solid phospholipids were dried in a vacuum dessicator over

potassium hydroxide.

3.7.3 Fluorescence Measuremea ts

Fluorescence of aqueous solutions was measured using a Shimadzu RF4501

spectrofluorophotometer with a xenon arc light bulb (Ushio Inc.). The cuvette was an

artificial quartz cuvette (Shimadzu Corp.) that did not give fluorescence interference at

the wavelengths used, Samples were excited at 283 nm and fluorescence was measured at

445 nm. Samples taken h m the femientor were diluted using distilleci water to l m . of

sample per lOOmt total solution in order to obtain readings that fell on the scaie of the

fluororneter.

3.8.1 Apparatus for Standard SCF Esperimecits

A schematic of the standard cyclone SCF apparatus is s h o w in figure 3.8.1.

Hydrocarbon was added to the fermentor at the beghnbg of each cycle using a syringe

pump (Sage Instnunents, mode1 341B) and 30 cc Iuer-lock glass syringe (Becton,

Dickinson, & Co.).

Dissolved oxygen levels in the fermentor were monitored using an Ingold IL 53 1

polaragraphic oxygen sensor with a Pegasus ampEer. The signal produced was a 4-20

mA curent that was sent to a data acquisition board in the controliing computer.

Upon detection of a minimum in the dissolved oxygen signal, the computer would

tum off the circulating pump, open the harvesting solenoid valve, and drain 500 mL of

broth to an overflow container. Then, the dosing solenoid valve was opened and 500 mL

of fiesh mineral salts medium was added to the fermentor, Volumes were controlled

through measurement of a pressure differentid using an Omega PX170 differential

pressure transducer, with the signal being sent to the data acquisition board of the

controlling computer. The pump was turneci on and the contents allowed to circulate until

the next dissolved oxygen minimum. 250 mL of the harvested broth were allowed to pass

nom the harvesting overflow container to a sample container. The remainder of the

harvested broth was sent to waste.

Temperature control of the fermentor was maintained using a recirculating water

bath (Haake Mode1 FE 2). The computer peripherals were the same as those demibed in

previous work?

3.8.2 Apparatus for Continuois Hydrocubon Addition Experiments

The modifieci SCF cyclone apparatus used for the study of continual hydrocarbon

addition is shown in figure 3.8.2. AU components of the apparatus, except those

associated with the substrate addition, are the same as those descri'bed in the previous

section.

The syringe pump was removed. Hexadecane was kept in a sterile glass ceservou

and circdated with aid of a peristaitic pump (Masterflex ; did setting # 1.5). Tubhg

outside the reactor was Mastdex Tygon fûel and lubricant tubing (6401-13) with 0.8

mm inside diameter. Within the fermentor, platinum-cured Masterflex silicone tubing

with 0.8 mm inside diameter was used. Since iniet and outlet ports of the fennentor had

to be seded to prevent loss of contents and maintain steriiity, tubing on either side of the

reactor was attached to 20 gage cannulae that pierced the septa in the wail of the reactor.

One hundred holes were pierced in the silicone tubing using a metal pin (diameter = 0.42

-1-

air iniet

%

dissolved oxygen probe

J

dosing

h a t exchanger

7

Figure 3.8.1 Schematic of the cyclone SCF mctor, set up for hexadecane addition at the begiMing of every cycle. Bascd on the figure in "Production of Lipase by Conrii'. bombiwh in a SeIf-Cycling Fcnnentd', U y , Ma;ill University, 1997.

air

dosing

*con tubhg

peristaltic pump 1 % pressure tnnsducer

1 -

Figure 3.8.2 Schematic of the cyclone SCF reador, set up for continual addition of haradecane- Baseci on the figure in "Production oflipase by CPnrii'& bombicotrr in a SeffXycling Fennentei', M hky, M m University, 1997.

4 Results

4.1 Phospholipid AnaIysis

4.1.1 Determination of Fluorescence Properties of tbe Phosphoiipid

Table 4.1 -1 summarizes the main features of the emission spectra detected in

samples obtained fÏom C. ulkanol)ticum ATCC 21511. Emission wavelenghs due to

solvent or Raman effects are not listed The phosphoiipids were isolated h m the

organism using the methods of Folch et al and Kates mentioned in section 3.7.

Table 4.1.1 Characteristics of fluorescence emission spectra for various cultures of Corynebacten-um alkanolyticum ATCC 21 5 1 1

SampIe Properties of Emission S pectrum Nutrient Broth

uninoculated flash only solvent and Raman effects seen inoculated flasks only solvent and Raman effects seen

Dehed Medium uninocdated flasks only solvent and Raman effects seen inoculated flasks

whole sarnple hCXQmtiOII=285 nm L-m=447 nm supernatant of centrifùged sample ka~dm=263 mn XanimoII-AAA nm pellet of centriftged sample resuspended ka6<i-=285 n m Lcnnram=446 nm in distilled water sample extracteci using chlomform hw=283 X-=443 nm

isolated phospholip

Table 4.1.2 shows actual fluorescence readings h m two flasks of C.

alkanoZyticurn grown on the mineral salts medium. Al1 samples were diluted twenty-fold

to obtain fluorescence readings that fell withui the range of the fluororneter. The results

hclude readings fiom whole broth and the fbctions obtsined after extraction with

chioroform.

Table 4.1.2 mineral

Fluorescence Measurements h m flasks of calknnolyiinm grown on salts medium

FIask 1 Flask 2 Biomass in Hask (g/L) 2.899 2.479 Fluorescence of M o l e 73 -620 67.253 Broth Fluorescence of Supernatant 63.498 59.786 Fluorescence of Chloroform 16.843 16.249 Extract Fluorescence of Aqueous 50.149 45.222 Extract Partition Coefficient 0.790 0.756 Distribution Coefficient 2.977 2.783

Figure 4.1.1 shows a fluorescence emission spectrum for the isolated

phospholipid in distilled water, excited at a wavelength of 283 m. The three emission

peaks occurred at 283 am (light scattered by solvent molecules), 3 13 nm (Light due to

Raman scattering), and 445 nm (light due to the fluorescence of the phospholipid). The

fluorescence intensity measured at 445 nm was used to prepare the correlation between

fluorescence intensity and phospholipid concentration. Typical emission spectra fkom

other sampies are shown in Appendix 1.

Figure 4.1.1 Emission spechum for 2.78 g/L phosphoüpid biodactant, excited with 283 nm iight.

4.1.2 Correlation of Phosphoüpid Concentration with Fïuoreseence

Known amounts of the dry, extracted phospholipid were ciissolved in distiiied

water, and fluorescence readings were taken for each sample. The fluorescence-

concentration data allowed for construction of the calibration curve shown in figure 4.1.2.

nie curve was quite iinear, with an R2 value of 0.997. This c w e was used in conversion

of aü fluorescence meaçurments to phospholipid concentration vaiues in this study.

90

Figure 4.1.2 Calibration c w e to detemine phospholipid concentration in the fermentation broth. Fluorescence was measured at hm--445nrn. The excitation wavelength was h,=283nm.

Variation of the d i c e tension of samples of the whole bruth (c aZkzznoZyticum

in mineral salts medium) with phospholipid concentration is shown in figure 4.1.3.

Results show that the correlation is poor, especiaiiy below 1.5 g/L of phospholipid. Over

the course of the experimental work, centrifbgation of some samples showed that the

presence of particdates in the broth did not affect the surface tension measurement.

Figure 4-1.3 Variation of surface tension with phospholipid concentration for cultures of C. alkanoi'yticunz grown using the traditional self-cycling fmentation technique.

Hexadecane was added continuously to the SCF reactor at a rate o f 0.1 14 rnI,

C,&,IUh using pierced silicon tubing. The endof-cycle hydrocarbon cG@&r&od

biomass level, and surface tension are shown in figure 4.2.1. Dissolved ~ x y ~ e h profile

are shown in figure 4.21. The average end-of-cycle biomass level was 0.257 b i a ~ s s / L

broth. The end-of-cycle hydrocarbon was negligiible, at 0.01 7 mL hexad-w mm.

The surface tension at the end of each cycle was approximately constant at 69,9 d e d c m .

Sarnples taken at the end of several cycles did not exhibit any detectable pbi]rs ofl the

fluororneter.

Intracycie data for the continuous-addition experiments are shown in &fi& 4-2.3-

Hydrocarbon concentration and surface tension were monitored as functions ~&+k 'Ihe

hydrocarbon level remained constant throughout the cycle at approximately 0,005 nilL/t,

showing that the presence of a thin layer of biomass on the tubing did bot ,pttevent

hydrocarbon corn difising into the reactor. Surface tension also remained CO@@ ai 67

dynedcm, very close to the swface tension of the uninoculated mineral salts %w&u (70

dynedcm) and of distilleci water (72 dyndcm). The high surface tension IS hftI-16

indication that no significant levels of biosurfactant were present-

The fact that the hydrocarbon level retllained constant showed & m~st

hexadecane was consumeci by the organism as it was put into the fermentoi. admge,

0.176 g hexadecane were added to the fennentor during a two hour cycle. AppfPWately

fifty percent of the biomass produced was carbon. With the amounts of )it@ecaae

added, most of the substrate wouid have been used ditectly to synthesize bia@&, *iii

iittle Ieft over to manufacture other products. Biomass leveIs should have increased in the

reactor linearly over the course of a cycle, until a lirniting substrate was exhausted.

Udortunately, it was not possiile to c o d i m this with biomass measurements, as it

would not be possible to maintain the stabiiity of the system if the large samples rquired

to follow the biomass levels were removed.

Figure 4.2.1 End of cycle biomass concentration (*), hexadecane concentration 0, and surface tension (A) vaiues for the continuous substrate addition experirnents.

Figure 4.2.3 Intracycle surfàce tension (A) and hydrocarbon concentration results for a contlliuous substrate addition experiment.

4.3 8iosurf;actant Producdon Using the SCF Twhnique

The control parameter used in a i i fermentations was dissolved oxygen. Each

different type of cyclùig scheme descnbed below is labeled by the type of dissolved

oxygen profile obtained.

4.3.1 SCF Dissolved Oxygen Pronles

The standard dissolved oxygen profile shown in figure 4.3.1 was similar to those

obtained in previous experiments using sekycling fnmentation. Cyciing of the

fermentor occurred once the dissolved oxygen concentration reached a minimum.

Experiments were carried out with different aeration rates and different concentrations of

ammonium compounds in the growth medium. 1.1 mL of hexadecane was added to the

fementor at the beginning of every cycle.

Fermentations of C. alknnolytinun with dissolved oxygen profiles like that shown

in figure 4.3.1 were shown to contain synchronous cell populations. Ceil synchrony in the

culture was confirmecl by intracycle ce11 count measurements, shown in figure 4.3.2. The

number of cells in the fermentor remaineci constant at approximately 16x106 CFU/ml

broth throughout most of the cycle, jumping to 35x10~ W / m l when the minimum

dissolved oxygen level was reached. This doubling of the ce11 number at the end of a

cycle (near the dissolved oxygen minimum concentration), was a phenornenon also seen

in previous SCF work.

O 20 40 60 80 100 120 140

Tim (min)

Figure 4 3 3 Ce11 counts for a standard SCF cycle. Aeration rate: 120 mL OJmh, 6 g L m4)2so4,3 g/L mm-

When the cyclone was aerated at 60 mL OJmin and contained the ammonium ion

concentrations indicated in section 32, standard SCF cycles yielded 0.804 g

phospholipid/ g biomass. The average cycle t h e was 155 minutes and 1.019 g biomassR.

were produced. At the end of the cycies, phospholipid concentrations averaged 0.819

gL. Representative end-of cycle biomass concentrations, end-of-cycle phosphoüpid

concentrations, anci cycle times are shown in figure 4.3.3.

Intracycle redts for the synchronous culture on the original medium can be seen

in figure 4.3.4. Hydrocarbon profiles were scattered at the beginning of the cycle when

the system was highly non-homogeneous and difncult to sample accurately, wbile a

clearer decreasing trend became apparent at the end of the cycle. Phospholipid production

began a short time after the beginning of the cycle, and leveled off near the end of the ceil

growth cycle.

Figure 4.3.3 Cycle length (a), end of cycle biomass (*) , and nid of cycle phospholipid concentration for standard cycles. Aeraîion rate: 60 mL OJmin, 6 g/L (NHJ2S0,, 3 g/L NH,NO,.

Figure 4.3.4 Intracycle hydrocarbon (*) and phospholipid concentrations for standard SCF cycles. Aeration rate: 60 mL OJmh, 6 @ @H4)2S04, 3 g/L m 4 N 0 3 .

A second set of synchronous fmentations was carried out using the same growth

medium but with a higher aeration rate. Oxygen was supplied to the system at a rate of

120 mL OJmin. End of cycle results are shown in figure 4.3.5. The time required for the

organkm to complete one growth cycle decreased slightly h m the previous system to

about 139 minutes, showing that the growth rate was now limiteci by the tramfer of

oxygen and not hydrocarbon to the cells, as was the case in the continuous addition

experiments. The amount of biomass in the femientor, as expected, remained at

approximately 0.849 g biomass/L broth- However, the phospholipid yield increased

significantly, up fiom 0.804 g/g in the previous experiments to 1.30 g/g with the

increased oxygen rate. The average end-O f-cycle phospholipid concentration was 1.1 0

Intracycle data for the cycles with increased aeration are shown in figure 4.3.6. As

in previous experiments, the hydrocarbon results were varieci. Phospholipid

concentrations continued to increase with the increasing biomass levels throughout the

course of the growth cycle, and becarne roughly constant toward the end of the cycle.

A final set of synchronous fermentations were performed using the increased

aeration rate (120 mL OJmin) and haIf the ammonium ion concentrations in the original

medium. The resulting concentrations were 3 g (NH&SOJ L and 1.5 g NH,NOJL at the

beginning of each cycle. Cycle times increased slightly h m the previous two cases to

160 minutes/cycle (see figure 43.7). The product yield per unit biomass (Y,& was 0.977

g phospholipid/ g biomass. Actual endo f-cycle phospholipid concentrations were about

1-11 g/L.

90 i 1 6 1 i

5 1 52 53 54 55 56

Cyck Numkr

Figure 4.3.5 Cycle length (a), end of cycle biomass (*) , and end of cycle phospholipid concentration 0 for staudard cycles. Aeration rate: 120 mL OJmin, 6 g L (NH,)2S04, 3 g/L NWO, .

Timm (min)

Figure 43.6 Intracycle hydrocarbon (*) and phosphoiipid concentrations for standard SCF cycles. Aeration rate: 120 mL O ~miÜ, 6 g/L (NHJ2SOJ, 3 g/L NH,NO,.

50 t 00 150

Tim (min)

Figure 4.3.7 htracycle hydrocarbon (*) and phospholipid concentrations for standard SCF cycles. Aeration rate: 120 mL OJmin, 3 gR (NH4)$04, 1 .S g/L NH,NO,.

4.3.2, Isolated Extended Cycles

Following a senes of standard cycles (as d e s c r i i above), a single cycle was

prevented fiom cycling once the minimum dissolved oxygen level was attaineed. Instead,

cycling was initiated at a Gxed time &er the minimum DO level occurred- This type of

cycle was termed an isolated extended cycle, and demonstrateci the e E i t s of nutrient

deprivation on the synchronous ceU culture. A typical dissolved oxygen profile for the

isolated extended cycle is shown in figure 4.3.8.

Figure 4.3 -9 shows intracycle analysis for an isolated extended cycle carried out

with an aeration rate of 120 mL 02/min and with initial concentrations of 1.5 g

NH,NO,/L and 3.0 g (MI,),SOJL in the medium. The dissolved oxygen minimum

occurred d e r 1 77 minutes, at which point there were 1 .O3 g phospholipidL in the broth.

The cycle was extenàed for ninety minutes before cycling. At the end of the cycle, the

phospholipid concentration had increased to 1.44 g/L, while the hexadecane

concentration had decreased by nearly an order of magnitude. The end of cycle biomass

was 1.205 g/L, simila. to the values at the end of non-extended cycles nui under the same

conditions (see figure 4.3.5).

A cycle with the original concentration of nitmgen compounds and an increased

oxygen flow rate of 120 mL OJmin was aIso subjected to a delay before cycling. Figure

4.3.10 shows the intracycie behaviour observed. The dissolved oxygen minimum

occurred after 215 minutes, at which point there were 0.99 g biosurfactanfi present in

the broth. Hydrocarbon concentration r d t s were scattered, although they seem to

suggest a decreasing trend. The cycle was extended for 125 minutes, with the

phospholipid concentration increasing to 1.375 g/L after 85 minutes and remaining

constant for the duration of the cycle. The hydrocarbon concentration fefl below O 2

rnW]L.

Once the phospholipid concentration leveled off, an additional 0.23 mL C , a M

were added to the reactor. Over a period of appmximately 80 minutes, the biosurfactant

concentration increased to 1.563 g/L. A ha1 pulse of 0.44 mL hexadecane was added to

the broth, increasing the biosufactant concentration to 1.875 g/L after two hours-

The additional hydrocarbon did result in some additional biomass being produced.

For the regular cycles, the average biomass concentration at the end of the cycle, as

reported in section 4.3, was 0.804 g biomass5,. At the end of this cycle, there were 1.462

g/L of biomass.

I ! 0.ooo O 50 100 150 200 250

n- (min)

Figure 4.3.9 Intracycle hydrocarbon (*) and phospholipid concentrations for an isolated extended cycle. Aeration rate: 120 mL OJmin, 3 g/L ~ . & S 0 4 , 1.5 g L NKNO,.

Figure 4.3.10 htracycle hydrocarbon (*) and phospholipid concentrations for an isolated extended cycle. Aeration rate: 120 mL OJmia 6 gfL (NH,),SO,, 3 g/L m 4 N 0 3 .

4.3.3. Sequence of Ertended Cycies

A thirty-minute delay between the detection of the minimum dissolved oxygen

concentration and the point at which the reactor was cycled was added for a sequence of

cycles. The dissolved oxygen profile for this group of cycles is shown in figure 4.3.1 1.

Such a series of cycles d t e d in a non-synchronous ce11 culture (figure 4.3.12). The

culture began a cycle with approximately 11x106 viable ceWmL bm* the number of

ceus increasing to a point corresponding to the minimum dissolved oxygen level in the

fermentor. Afterwards, the broth maintained a constant number of cells, roughly 28xlO6

cells/mL broth. The synchrony seen in the standard SCF cycles had been lost.

Hydrocarbon and phospholipid concentration profiles for the series of extended

cycles are shown in figure 4.3.13. The average end of batch biomass concentration was

0.798 g b i o m a s d broth.

The phospholipid concentration profile showed a large increase in biodactant

Ievels d u ~ g the ! k t half of the cycle. However, the levels dropped to approximately

0.75 g/L (roughly double the initial concentration) when the minimum dissolved oxygen

level was reached and remained approximately constant untii the fementor was cycled.

A second series of extended cycles was executed, this time with a two-hour delay

between the point of minimum dissolved oxygen concentration and cycling. A typical

hydrocarbon and phospholipid profile for this series of cycles is shown in figure 43-14.

The hexadecane levels showed a much clearer decreasing trend throughout a cycle than

was seen in the shorter cycles, although results were still slightly scattered in the non-

homogeneous system- The amount o f biomass produced during the fermentation was

approximately the same at 0.814 g biomasdL broth.

The phospholipid concentration increased during the fint çixty to seventy

minutes, much as it did during the fermentations extended for only thirty minutes.

However, with the longer fermentation times, there was no subsequent concentration

decrease. The biosurfactaut concentration remained constant for the remainder of the

cycle at 0.75 g/L. This was the same concentration as was seen in the fe~tlentations with

only haif-hour extensions, but no peak in phospholipid concentration occurred.

Figure 4.3.11 Dissolvecl oxygai p d e for 8 d e s of aactrdd S a cycles.

O 20 40 60 80 100 1 20 140 160

Tïnn (min)

Figure 4.3.12 Ce11 counts for one cycle in a sequence of extendeci cycles.

limo (min)

Figure 4.3.13 Intracycle hydrocarbon (*) and phospholipid concentrations for one cycle in a senes of extended cycles. The cycle was extended for thirh, minutes.

ïïnn (min)

Figure 4.3.14 Intracycle hydrocahon (*) and phospholipid concentrations for one cycle in a series of extended cycles. the cycle was extended for ninety minutes.

4.3.4. Two-Stage Fermentations

In the two stage fermentations, the initial stage was a standard cyde with no

irnposed cyciing delay. Upon cycling, a portion of the harvested reactor contents was

added to 500 mL shake flasks in which the second phase of the fermentation occurred-

Table 4.3.1 shows the phospholipid concentrations in the flash measured after twenty-

four and/or thïrty-seven hours. Addition o f hexadecane to some flasks o f the second stage

of the fermentation gave additional phospholipid production. As more hexadecane was

added to the flask, a fûrther increase in phospholipid concentration was observeci- In the

shake flasks, phospholipid concentrations increased to nearly 3 g/L after 37 hours.

Table 4.3.1 Phosphoiïpid concentration changes during the second stage o f a two-stage fermentation.

C,& Added Initial Final Percent Length of (mW100mL) Pbospboüpid Phospholipid Increase Experiment

Concentration Concentration (%) (ho-)

4.3.5 Hydrocarbon Measurement

Average end-of-cycle hydroçarbn concentrations are summarized in table 4.3.2.

During this experimental work, samples in which the hexadecane concentration was

below approximately 0.600 mL C,$134/mL broth seemed to give fairly reliable results.

Figure 4.3.15 shows the standard deviation for end of cycle hydrocarbon concentrations

as a huiction of the average value reporteci. Below 0.600 mL/L, the standard deviation in

results remained below 0.030 mLn, roughly withui the detectability lie& of the gas

chromatograph.. Above 0.600 mWL, however, most results showed wide variations about

the mean.

Table 4.3.2 Average end-of-cycle hexadecane concentration in the SCF reactor for different types of cycles.

Type of Cycle Average End-of-Cycle Hexadecane Concentration

Standard SCF (60 mL/min O,; 0.44_+0.03 6 (NH,),SO,; 3 glL NH~NO?) Standard SCF (1 20 Wmin O*; 0.3=.03 6 gfL (NH,),so4; 3 fl NH,NO,) Standard SCF (120 W m i n O,; 0.41H.04 3 (NN,),S04; 1 -5 g/L NH4N0,) Isolated Extended Cycle 0.23M.O 1 Isolated Extended Cycle O. 13M.O 1 (with hydrocarbon addition during extension) Series of Extendeci Cycles 0.28M.02 (30 minute delay) Series of Extendeci Cycles O. 1 W.02 (90 minute delay)

Figure 4.3.15 Standard deviation of hydrocarôon concentration measurements versus the actual reported concentration values.

5. Discussion

in order to study the effects of diffkrent operating methods of the fermentor on the

amounts of phospholipid produced, an appropriate technique to quanti@ the phospholipid

concentration had to be found The most commonly used methods to quanti@

biosurfactants in the past have been relateci to surface phenornena, namely

surface/interfacial tension measurements and the critical micelie con~entration."-'~*~~

The variation of surface or interfacial tension with biosurfâctant concentration wiU

depend on the type of biosurfactant produced. This variation will not always be large, and

samples with very different biosurfactant concentrations may have surfafe tension values

that are very close to each other."" Surface tension measurements may also be

influenced by biomass and materials other than the biosurfactant in the broth."")

Measurement of the surface tension does not allow for direct quantitative

measurements when the critical micelle concentration has been exceeded, since the

surface tension remains constant beyond this level. Measurements of surface tension,

taken alone, serve o d y as an indication of the presence of bio~urfactants?~) The variation

of surface tension with phospholipid concentration for C. alknnolyticum cultures used in

these experiments (figure 4.1 -3) showed ho w unreliable surface tension measurements

could be as a quantitative analytical tool.

Dilution of broth to the critical micelle concentration has been used to quantifi.

biosurfactant production. '2925"95''55m Samples of the fermentation broth are diluted in

water and the surface tension of each solution is measured, Surface tension is then

plotted against the log of the percent dilution, Usually, the value of the percent dilution

read at the inflection point of the curve is assumed to be the inverse of the critical micelle

concentration. BioSUffactant concentrations are reported as multiples of the criticai

micel le c o n ~ e n t r a t i o n , ~ ~ ~ ~ ' - ~ ~ ~ so solutions of the pure biosurfactant with known

surface tension values are required to translate these results into grams-per-litre vdues.

As a great ded of previous work in the biodactant field has been reported without

knowing the actual composition of the surfactant, it is fkequently ody the relative

biosurfactant concentrations that have been r e p ~ r t e d . ~ = ~ ~ ~ ' " ~ ~ This method dso

requires large sarnples and can be quite t h e consuming. For organisms with shorter

growth cycles, the length of tirne needed to complete the CMC analysis at each sampling

interval may allow changes in the biosurfactaut concentrations. Cellular production or

consumption of biosurfactant may continue outside of the fmentor, introducing mors in

the results.

Emulsification tests are unreliable, as a dac tant , while present in the broth, may

not have a stabilizing effect on an exnulsion.'*) Demulsification pmperties of the

fermentation broth, based on its ability to break oil-in-water emulsions, aiso iadicate only

the presence of surfactant, not the surfactant concentrati~n.'~~) High performance Liquid

chromatography<'" and thin layer chromatography<') methods have aiso been reported

previously, but these too are tirne consuming and somewhat complicated. A colorimetric

anaiysis for phospholipids using absorbance also seemed promising,(S8) but derivatization

of the lipids involved several steps and took over one hour to complete.

Fluorescence of derivathxi iipids has been documented in the literat~re!'~'

Fluorescence measurements were first obtained for cultures of C. alkanolyticurn grown on

nutrient broth. The orgauism produced no measurable biodactant when p w n on

nutrient broth (with au average surface tension value of the whole broth of 70 dynedcm)

so any fluorescence produced by cultures grown on this medium would indicate the

presence of another fluorescent cellular component that could potentially cause

interference with biosurfactant anaiysis.

The iininoculated nutrient broth medium was shown not to have fluorescence

charactenstics (see table 4.1.1)- Furthennore, there were no fluorescent compounds

present in the inoculated broth. The only light detected was due to solvent and Raman

s c a t t e ~ g of the exciting light.

Cultures grown on the mineral salts medium showed fluorescence at 445 m.

Presence of the biosuffactant was confhmed by a measured surface tension value of 44.6

dyndcm. Since no fluorescence peaks were detected for the uninoculated medium, the

fluorescence was likely due to the biosurfactant produced by the organism.

Samples of the culture were then subjected to a chloroform extraction.

Phospholipids have solubility in both organic and aqueous phases, so detection of similar

excitation and emission fluorescence peaks in chloroform would signal that the

fluorescence was likely due to the biosurfactant. Similar peaks were obtained (Xm=283

nm, h,=443 nm). The fact that significant, measurable quantities of the fluorescing

compound were present in both the aqueous and organic phases (see table 4.1.2) fbrther

indicated that the biosurfactant was the detected compound.

The finai test was to isolate the phospholipid using the methods in the

literat~re(~*~~) and test for fluorescence. A typical emission spectnim for the isolated

phospholipid is shown in figure 4.1 -1. The results matched those obtained for the whole

broth (table 4.1.1). The resuits proved that the phospholipid gave a fluorescence signal

and that the fluorescence intensity of the compound could be correlated directly to

concentration (figure 4.1.2). Only small sarnples were r e q d for use in the fluororneter

(less than 2 ML), so kqueat sampiïng during the organism's p w t h cycle was possible

without removing volumes large enough to r n d Q the growth cycle itself. Resuits were

also immediate. Samples that are left for long periods of t h e before undagoing Lengthy

analytical procedures c m be altered due to evaporation and growth of the organism.

Fluorescence spectroscopy eliminated this errer-

5.2.1 Influence of the Hydrocrrbon on Biosurfactant Production

In tnditional fermentations, the growth rate of the organism is w t lirnited by the

availability of the hycirocarbon substrate to the cells. An excess amount of hydrocarbon

made available to an organism in the bioreactor at the beginning of the fermentation

allows that organihn to grow at its maximum growth rate. While microbial growth may

be limited by the rate at which hydrocarbon cm enter the cells (whether by diffusion,

facilitated transport, or some other mechanism), excess hydrocarbon in the broth ensures

that rate of substrate transport across the celi membrane is not compromised.

By continually adding very small amounts of hexadecane to the fermentor using

the pierced silicon tubing, the growth rate was limited by the hydrocarbon available in

the vessel. Intracycle hydrocarbon concentrations in the fermentor throughout a ceU cycle

(see figure 4.2.3) remaineci essentially zero. Since hydrocarbon did not accumulate in the

fermentor, it was being consumed by the organism as quickly as it was added to the

fermentor. Therefore, the rate of hexadecane transport across the cell membraue was

lirnited by the rate of hexadecane addition to the femientor. This hrher suggests that the

growth rate of the organism was, consequently, limited by the rate of hexadecane addition

to the vessel and was less than the maximum growth rate (& of the organism. Had the

rate of hexadecane addition been such that transport of the substrate across the ceii

membrane occurred at the fastest possible rate, the growth rate of the organism would

have also attained a maximum and hexadecane would have accumulated in the system.

Despite the fact that biomass production was limited by the hydrocarbon

availability, the hexadecane was stilI the sole avaïiable carbon source. A widely-accepted

theory on biosurfactant production is that the production is initiated by the organism in an

attempt to make the immiscible hydrocarbon substrates more accessible to the ~ e l l s . ( ' ~ . ' ~

There is speculation that, in lowering the interfacial tension between the hydrocatbon and

the fermentation broth, a larger contact area between the two phases results. This would

facilitate hydrocarbon consumption by the organism. Whether the hydrocafbon substrate

is added in excess at the beginning of a fermentation or continually over the dwation of

the fermentation, biosurfactant production should be induced in the organism.

Because the hydrocarbon concentration in the fe~nentor using the silicon tubing

would be lower than when excess substrate was added at the beginning of the

fermentation, it was postulateci that biosurfactant production may even be enhanced. Just

as siderophore production (a class of compounds responsible for transport of iron across

an organism's outer membrane) by organisms increases with lower iron concentrations in

the ceIl environment in an effort to retrieve more iron for the cel1,'4') lower hexadecane

concentrations in the fermentor could increase biodactant production by the ce11 in an

effort to obtain the less-abundant substrate. The effect has been seen in the biosurfactant

work of Persson et al., who found that elimlliating an iron supplement from their growth

medium increased yields of the glycoprotein biosurf'tant produced by Pseudomonas

fluorescens 378 by 120%."

Fluorescence analysis of samples fiom the reactor showed no detectable

concentration of biosufactant (section 4.2). The negligible hydrocarbon concentrations

and 0.257 g/L of end-ofcycle biomass showed that hexadecane was consumeci by the

cells. However, biosurîactant production was not neces- to facilitate the consumption

by C. alkanolyticum, and the lower concentrations did not induce enhanceci production of

biosurfac tant.

The results also seem to suggest that a certain amount of excess hydrocarbon is

requïred in biosurfactaut production- At no point in the continual addition experïments

was there any appreciable concentration of hexadecane. There was also no biosurfactant.

In the traditional selfcycling férmentation experiments, phospholipid production did

occur, and there was always excess hydrocarbon in the bioreactor (see section 4.3).

Regardless of the cyciing scheme employed, phospholipid was always accompanied at

the end of a fermentation cycle by çome unused hexadecane.

While the excess hexadecane in the reactor may act as an extracthg agent to

remove phospholipid fiom the cells, it stï i l remains unclear as to why the consumption of

the hexadecane and the growth of the organism stops once a certain concentration level of

hexadecane is reached- If the hexadecane acts oniy as an extracting agent, perhaps

concentrations of hexadecane below this minimum cannot further extract any

phospholipid fkom the cells. However, the organism should still be able to consume this

remaining hexadecane, even if biodactant production no longer occurs.

5.2.2 Biosuiiactant Production Using the SCF Technique

Previous work with Corynebacten-um alkanoiyticum produced Iow concentrations

of phospholipid biosurfactant Nakao et al. were able to obtain 0.5 15 g/L of phospholipid

fiom a production medium containing a large excess (10% (wfv)) of hexadecane. Only

3.7% of this product was extracellular, the remainder had to be extracted fkom the cells.

Use of an agent such as peuiciUin to increase the permeability of the ceU membranes

showed a slightly increased concentration of phospholipid to 0.694 g/L. An appreciable

quantity (nearly 42%) still had to be extracted fiom within the ceiI after the fermentation.

The final concentrations were srnaLi, and downstream separations would be made

significantly easier if these values could be increased. Getting as much of the

biosurfactant into the broth (fiee h m the cells) as possible wouid also be beneficial,

eliminating another separation step. Previous studies using self-cycling fkrmentation

showed that the technique could give high production levels. Tetracycline production by

Strepromyces aureofaciens was greater in SCF experiments îhan in traditional batches.

Citric acid production by Candida hpolytica was also slightly improved using the SCF

technique. It was hoped that sekycling fermentation applied to C. alkanoiyticum could

increase the concentrations of phospholipid obtained as weU.

Higher concentrations of phospholipid were obtained in regular SCF experiments

as compared to the results of Nakao et al (figures 4.3.2 to 4.3.5 and 4.3.7). On average,

0.8 19 g/L of phospholipid were obtained fiom a reguiar cycling scheme in the fmentor.

Concentrations showed a dependence on the oxygen levels in the fermentor. Doubling the

oxygen flow rate to the fermentor caused a 34% increase in the final phospholipid

concentration obtained. Changes in concentration of the ammonium salts available did

not affect the phospholipid concentrations obtained. Therefore, p w t h of the organism

and production of the phospholipid biosurfactant was Limited by the amount of oxygen

available. Frequently, biodactant production occurs under conditions of nitmgen

limitation; oxygen iimitation is not cited in the fiterature as an influencing factor.

While there seemed to be an oxygen Limituig effect in the fermentor, this was not

the main reason why concentrations of phospholipid obtained were higher than the batch

results from the Literature. This is clearly seen when comparing the results h m the

standard SCF cycles against the r e d t s h m the experiments in which several extended

cycles were nin in seris. Running the series of extended cycles imitated a batch

fermentation (without the initial lag phase), as cycling of the reactor was not related to the

cycle of the celi. Phospholipid concentrations obtained were in the 0.75g/L-0.80 g/L

range (see figures 4.3.12 and 4-3-13}, very near the values Nakao obtained in his batch

experiments despite the more advantageous oxygen transfer properties of the SCF

cyclone fennentor over those of shake flasks.

For the standard cycles performed with the same oxygen and ammonium levels as

the senes of extended cycles, (aeration at 120 mL O& and ammonium compound

concentrations at the levels of the original medium (see section 3)), the end of cycle

phospholipid concentration was, on average 1.10 g/L (figure 4.3.5). Since the operating

parameters of the fermentor were unchangeci in al1 of these experiments, the differences

in the production had to be attributable to the ceil culture itself. Figure 4.3.12 shows the

asynchronous nature of the cultures in the series of extended cycles. Induchg spchrony

in the cells for the regular SCF cycles (figure 4.3.6) showed a marked increase in

phospholipid concentration. This niggests that it was the synchrony of the ceus and not

the oxygen transfer properties of the reactor that brought about the increased

concentrations of biosurfactant.

While synchrony did enhance biosurfactant concentrations in the broth, it did not

eliminate the residual hydrocarbon problem seen fkquently in the past. For regular

synchronous cultures, there was always approximately 0.4 mUL hexadecane left

uuconsumed at the end of the cycle (figures 4.3.3,4.3.5, and 4.3.7).

To M e r increase the phospholipid concentrations and attempt to reduce the

residual hydrocarbon present, the growth cycle of the organism was periodicaiiy

extended. In such cases, cycling was prevented h m occurring at the minimum dissolved

oxygen concentration. In effect, the cells were ailowed to double, as usual, by the tirne of

minimum D.O. concentration, but the fementor was not then cycleci, It was hoped that,

given this extra tirne, the celis would consume the residual hydrocarbon Ievels present in

the fermentor at the point of minimum D.O. and continue converthg substrate to

phospholipid.

Synchronous cultures, whose tirne in the fermentation broth was extended,

showed continueci phospholipid production (figures 4.3.9 and 4.3.10). The organism also

consumed portions of the residual hydrocarbon left at the minimum D.O. concentration.

Despite the fact additional hydrocarbon added to the fermentor was also completely

consumed, hexadecane levels never feil below -0.2 mUL. This again suggests that there

exists a minimum concentration of hexadecane below which bioswfactant production

does not occur.

The loss of synchrony in the ceii culture removed the enhanced biosurfiactaut

concentrations seen with synchronous cultures. This was evident when series of extendd

cycles were nui in the fermentor (figure 4.3.12). The resuits made evident the advantage

of maintaining synchrony in the culture. Unfortunately, the results also made clear the

fact that it would be impossible to extend every cycle in the fermentation to take

advantage of the enhanced phospholipid concentrations and reduced amounts of residuai

hydrocarbon that were demonstrateci.

The two stage fermentation aUowed for maintenance of a synchronous, productive

ceil culture in the main fermentor vessel while allowing for every cycle to be extended in

an independent vessel. In this manner, every cycle could be extended to obtain the higher

phospholipid concentrations seen earlier without sacrificing the synchrony of the main

culture. Phosphohpid concentrations could be increased by an average of 86% without

a d b g additional hydrocarbon in the second stage. Larger concentrations resulted when

additional hexadecane was added to the flasks.

A cornparison of extended cycle experiments carried out in the cyclone reactor

and two stage fermentation experiments is shown in table 5.2.1. It is interesthg to note

that while the productivity of the ceiis was nearly an order of magnitude higher in the

cyclone reactor than in the shake flasks, the final phospholipid concentrations attainable

in the shake flasks were considerably higher. Lookuig at figures 4.3.9 and 4.3.10,

phospholipid production eventually leveled off in the extended cycles. In the shake flasks.

phospholipid concentrations were still increasing d e r 37 hours (table 4.3.1). The

improved oxygen îransfer properties and the better mixing of the oil and water phases in

the cyclone fermentor codd explain the higher productivity levels seen during the

extended cycles.

Hydrocarbon use in each experiment is o u h e d in table 5.2.2. The organism was

able to use more hexadecane in the shake flasks than in the cyclone fermentor. The

leveling off of phospholipid and hexadecane concentrations seen in the extended cycles in

the fermentor was not seen in the flasks. Hexadecaue concentrations did in fact fa11 below

the 0.2 mL/L minimum level seen in the cyclone.

Increased oxygen levels were shown in section 4.3 to increase the amount of

phospholipid produced (doubhg the aeration rate of the reactor increased the

phospholipid concentrations h m 0.82 g/L to 1.10 fi. Oxygen transfer in shake flasks is

known to be lower than it is in the SCF apparatus due to the smaller gas-liquid interface

in the flasks. The fact that phospholipid concentration increases in the shake flasks were

higher than those seen in the SCF is not paradoxical, though, due to the Iowa

productivity of the cells in the shake flasks. Since the cells produce the biosurfactant at a

slower rate in the shake flasks than in the cyclone, less oxygen would be required. While

oxygen transfer is poorer in the shake flasks than in the SCF, less oxygen is required by

the organisrns, and higher concentrations of phospholipid can still be obtaiaed.

Table 5.2.1 Cornparison of extendeci cycle and shake-fiask phospholipid concentrations

Expriment Haidecane Initid Find Percent Lengthof Average Addd P bospholfpid Phqhoiipid Incnue Experiment Productivity

(mL1100mL) Concentration Conceatration (%) @ours)

Extended 0.000 1 -44 39.5 1.5 0.273 Cvc 1e

Extended 0,000 0.99 1.38 39.0 2- 1 O. 186 Cyc Ie

Extended 0-023 1.38 1.56 13.7 1 3 0.138 Cycle

Extwded 0.044 1 .56 1.88 20.0 2.0 0.160 CycIe

Shake Fiask 0.000 1.03 1.88 82.5 24 0.035 Shake Flask 0.000 0.99 1.89 90.9 24 0.038 Shake Flask 0.020 1.18 2.29 942 24 0.046 Shake Fiask 0.020 1.18 2.43 105.5 37 0.034 Shake Fiask 0,040 1.18 2-18 84.6 24 0-042 Shake Fiask 0,040 1.18 2-74 131.9 37 0,042 Shake Flask 0.060 1-18 2.25 90.5 24 0.045 Shake Flask 0,060 1.18 2.92 147.0 37 0,047

Table 52.2 Hydtocarbon use during extended cycles and shake flask expaiments

Experiment Rcs idd CI‘&, Total Cl& Total Cl&, Percent CI-, Addd to CI'& at End of UI«I CI&

Presentat System inSystem Erpcriment ( d l Used s t a r t (mu ( d l (mLl ('W (mL)

Extended 0.043 0.000 0.043 0.027 0.016 372 Cycle

Extwded 0.032 0.000 0.032 0.017 0.0 15 46.9 Cycle

Extended 0.017 0-023 0.040 0.013 0.027 67.5 Cycle

Extended 0.0 13 0,044 0.057 N/A N/A N/A

- - -

Shake Flask 0.06 1 0.000 0.06 1 6.001- - p 0,060 98.4 Shake Fiask 0.064 0,000 0.064 0.005 0.059 92.2 Shake Fiask 0.022 0.020 0.042 0.003 0.039 92.9 Sbake Flask 0.022 0.020 0.042 0.002 0.040 95.2 Shake Fiask 0.022 0,040 0.062 0.005 0.057 911

- - - - - - - - - --

Shake Fiask 0.022 0.040 0.062 0.004 0.058 93.6 Shake Flask 0.022 0.060 0.082 0.005 0.077 93.9 Shake Flask 0.022 0.060 0.082 0.002 0,080 97.6

While the induction of synchrony in the ceii population of the fermentor w u

successfiil in increasing the biosuffàctant concentration, it was not able to eliminate the

presence of residuai hydrocarbon. Regafdless of the cycling scheme employed and the

degree of synchrony of the cells, there seerned to exist a minimum hydrocatbon

concentration within the fennentor below which the organism was no longer capable of

consuming that substrate.

There are two likely explanations for this phenornenon. One involves mass

transfer considerations, where the consumption of hexadecane by the organism was

directly related to the hexadecane concentrations within and outside the cells. The other is

related to the amount o f some other nutrient in the broth. An entire synchronous culture

could stop consuming hydrocarbon due to an unfavourable ratio of certain nutrient

concentrations in the fermentation broth, whereas the effect could be masked in an

asynchronous population. However, h m this study it can only be concluded that the

induction of synchrony alone does not allow for the elimination of residual hydrocarbon

in the fermentation vessel. The direct relation between synchrony of the culture and

p hospholipid production, however, is clear.

6 Conclusions

It was found that the concentration of a phospholipid biosurf'actant p d u c e d by

Coryne&ac?enùm alholyticum could be m e d using fluorescence spectroscopy. The

phospholipid concentration was shown to correlate linearly with fluorescence intensity-

The procedure was rapiâ, required o d y small sample volumes (c 2 ml), and the only

sample preparation required was dilution with distilleci water.

Adding hexadecane to the SCF continuousiy over the course of a fermentation

established a system in which no excess hydrocarbon was present in the reactor at any

tirne. However, biosurfactant production by C. alkanoiytiarn in this modined SCF

system ceased. The resdts do not support the hypothesis that soIely the presence of an

insoluble substrate in a fermentation systern induces biosurfactant production by

microorganisms.

Synchronous cultures of C. a lkan~l~curn were shown to produce much higher

concentrations of phospholipid than asynchronous cultures. During standard SCF cycles,

the organism produced 1.10 g/L of product, compared to 0.75 g/L for asynchronous

cultures in the SCF and 0.69 g/L in previously cited work. Extension of the cell cycle, in

both the SCF reactor and in separate vessels, allowed higher phospholipid concentrations

to be attained. Extendeci cycles in the SCF fermentor gave concentrations as high as 1.88

g/L. Two-stage fermentations resulted in concentration levels as great as 2.92 g/L, 4.2

times as high as the results found in the literature.

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Appendix 1

Fluorescence Emission Spectra

Figure AL1 Fluorescence emission spcctra for uninoailated nutrient bi0t.h. +=283 nm. Peaks shown ocair at )ti;ia=283 nm, =322 nm, and k-. 4 6 4 nm, Note that not ail sarnples used to produce the figures in this appendk wae düutcd by the same factor.

Figure A13 Fluorescence emission spectrr for uninocuhd m i n d dts medium ht.tim=283 nm. P d show occur u A 4 8 3 MI, =3 18 nm, and L -566 nm. Note thaî not aii samples used to promice the figures in this appendà were diluted by the same fiictor.

Figure A1.4 Fluorescence ~mission s p a ~ ~ for a cuiture growing on the mineral salts medium. 4 4 8 5 nm. Peak shown occur at nm, '324 nm, L i r n = 447 nm, and =572 nrn Note that not 111 samples used to producc the figures in this appendk wcm diduted by the suae fktor.

Figure A1.S Fluorescence emUsion speara for the supemrtrint of a centrifiiged $ample fkom a culture growing on the m i n d dts medium. L-u(ioa=283 nm Peaks shown occur at hem;ri-==283 nm, =3 17 NII, A .;- 444 MI, md 4 6 6 MI

Note that not al1 sarnples used to produce the fi- in thU appendix were diluted by the same factor.

Figure A1.6 Fluorescence emissi~n rpactri for the remsupendeci peliet of a centrifûgad sample fkom a culture growing on the minerai dts medium. &-h=î83 nm P& shown ocair at A 4 8 3 nm, =322 nrn, -6 nm, and hr-, 4 6 4 nm. Note that not aU sunples used to produœ the figures h this appendix wcre diluted by the sarne factor.

Figure A1.7 Fluorescence emission spectn. for the orgadc phase of a -pie extractcd using chioroform. ASCiLI1#=283 nm Perks shown occur at k a 8 3 nm A =322 nm, A 4 4 3 nm, and 4 6 4 nm. Note that not ail samples used to produce the figures in this appendk were diiuted by the same &or.