microbial production of poly(glutamic acid)...siegfried, your experience and expertise with reactors...
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Faculty of Bioscience Engineering
Academic year 2013 – 2014
Microbial production of poly(glutamic acid)
Iris Tavernier
Promotors: Prof. dr. ir. Nico Boon and Prof. dr. ir. Siegfried E. Vlaeminck
Tutor: Ing. Joeri Coppens
Master’s dissertation submitted in fulfillment of the requirements for the degree of Master of Science in
Bioscience Engineering in Food and Nutrition Sciences
Faculty of Bioscience Engineering
Academic year 2013 – 2014
Microbial production of poly(glutamic acid)
Iris Tavernier
Promotors: Prof. dr. ir. Nico Boon and Prof. dr. ir. Siegfried E. Vlaeminck
Tutor: Ing. Joeri Coppens
Master’s dissertation submitted in fulfillment of the requirements for the degree of Master of Science in
Bioscience Engineering in Food and Nutrition Sciences
The author and the promoters give permission to use this thesis for consultation and to copy parts of it for
personal use. Every other use is subject to the copyright laws, more specifically the source must be
extensively specified when using results from this thesis.
De auteur en promotoren geven de toelating deze scriptie voor consultatie beschikbaar te stellen en delen
ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het
auteursrecht, in het bijzonder met betrekking tot de verplichting uitdrukkelijk de bron te vermelden bij het
aanhalen van de resultaten uit deze scriptie.
Ghent, 6th of June 2014
The author, The promotors,
Iris Tavernier Prof. dr. ir. Nico Boon Prof. dr. ir. Siegfried Vlaeminck
I
Acknowledgements
“If we knew what it was we were doing, it would not be called research, would it?”
Albert Einstein
This year has undoubtedly been the most intensive year of my five years studying at this Faculty of
Bioscience Engineering. A year consisting out of blissful moments when an experiment finally succeeded,
but also a year of frustrations when I had made a stupid mistake and had to start over or if a technical
failure ruined my experiment. As K32-Tom once said: “ we definitely learned a lot, especially about how
to deal with failure.”
The help of several people has been indispensable for successfully finishing my thesis. First of all, I would
like to thank prof. Nico Boon and prof. Siegfried Vlaeminck. Nico, thank you for the sincere interest in
my research, for your broad knowledge and your unfailing support. Siegfried, your experience and
expertise with reactors and that 14,01 g/mole weighing atom was essential for my experimental work.
Thank you both for thoroughly proofreading my thesis.
Joeri, you have given me a lot of freedom to find my own way. Your door was always open for questions
and suggestions and you have motivated me in times I did not see light at the end of the experimental
tunnel anymore. Thank you for you indestructible confidence in my capacities.
I also want to thank all the people of LabMET for the pleasant atmosphere in the labs and the practical
help. Special thanks go to Sofie, Jessica and Greet for the help with the SEC-HPLC, to Jana for the help
with the microbial work and to Mike for the technical support. Also thank you to the members of the N-
ecology cluster for all the useful advice.
I would like to thank all the thesis students for the pleasant vibe, both in the labs and during the frequent
breaks. Wim and Tom, the K32-team, you made those early Friday morning clean-ups less awful and the
K32 a bit less lonely. Stijn and Ruben, thank you for sometimes making me forget my thesis, for the
relaxing frisbee and swimming breaks and for the (too) late evenings in the Overpoort and Café Koepuur.
Without my parents and family, successfully finishing my studies would not have been possible. I would
like to thank them sincerely for all they have done for me, for all the opportunities I got and for the
confidence and support they have given me.
Robin, it was a heavy year with some ups and downs, but we made it! Thank you for your unfailing
support, your endless love and your enthusiasm. We have almost reached our 1000-days, up for the next
1000?
II
Abstract
More and more it is becoming clear that waste streams have to be seen as resources instead of waste to
allow an evolution towards a more sustainable society. The industrial fixation and subsequent removal of
nitrogen requires enormous amounts of energy and therefore more sustainable recovery alternatives are
researched. In this master thesis, nitrogen recovery by means of production of high-value compounds with
a high nitrogen content was examined. More specifically it is investigated whether production of
poly(amino acids), under the form of poly(γ-glutamic acid) or γ-PGA is possible by means of an open
culture from synthetic waste water and/or by means of a co-culture with in-situ glutamic acid production.
Not only would this result in a possible recovery of nutrients in waste streams, it would also strongly
decrease the production cost of γ-PGA since the necessity to work with pure cultures and the high cost of
the input products are the two cost-determining aspects of this production process. The overall goal of this
master thesis was to innovatively reduce the costs of the production process and hence enable the
industrial use of this biologically produced polymer.
The first set of experiments aimed at the production of γ-PGA by means of an open culture, as this would
avoid the use of pure cultures. Two rotating biological contactors (RBC) were inoculated with a
nitrifying/denitrifying inoculum and fed with synthetic wastewater. To induce γ-PGA production, a feast-
famine regime was applied since different types of bacteria produce γ-PGA when they are in the early
stationary phase. Further, the biofilm was dehydrated since several bacteria produce γ-PGA to protect
their cells from dehydration. The feed had a high C/N ratio to induce the production of compounds with a
high nitrogen content. The influent of the first RBC contained glutamic acid, the influent of the second
RBC did not contain organic nitrogen. In the first RBC on average 55% of the added COD and 66% of
the added nitrogen was removed. In the second RBC on average 3.8% of the added COD and 27% of the
added nitrogen was removed. Since the presence of glutamic acid was the most important difference
between the two RBC, its presence appears to have a significant effect on the growth of the biofilm.
Unfortunately, in neither of the RBC γ-PGA could be detected in the effluent or in the biofilm.
In the second set of experiments, the aim was to develop a co-culture of glutamic acid (Brevibacterium
divericatum) and γ-PGA producing bacteria (Bacillus licheniformis), eliminating the requirement of
glutamic acid as input product. Growth experiments were performed in different media to obtain a
medium that supports growth of both species. Trypton appeared to be essential for the growth of
Brevibacterium divericatum and also stimulated the growth of Bacillus licheniformis. In a next stage of
the research project, the productivity of these bacteria in different media was analysed to assess whether
co-cultivation was possible. Glutamate overproduction in C. glutamicum is induced by biotin limitation,
by specific detergents, by high sugar concentrations and by a sublethal concentration of penicillin. The
highest glutamic acid concentration and productivity was obtained in the medium with a high sugar
concentration, the highest γ-PGA concentration and productivity were obtained in a medium with and
without high sugar concentrations. In the final step, the bacteria were grown together and the γ-PGA
productivity was monitored. The highest γ-PGA concentration and productivity were obtained in the
medium without high sugar concentrations and without TWEEN80. This research shows that production
of γ-PGA with a co-culture is possible. However, further research to optimize this production method is
essential.
III
Samenvatting
Om de evolutie naar een meer duurzame samenleving te bewerkstelligen dient afval niet meer gezien te
worden als onbruikbaar maar als bron van materialen en energie. De industriële fixatie en latere
verwijdering van stikstof vereisen enorme hoeveelheden energie. Daarom moeten meer duurzame
herwinningsalternatieven gezocht worden. In deze masterproef wordt onderzocht of stikstofherwinning
door middel van de productie van hoogwaardige stoffen met een hoog stikstofgehalte mogelijk is. Meer
specifiek wordt onderzocht of de productie van polyaminozuren, onder de vorm van poly(γ-
glutaminezuur) of γ-PGA mogelijk is met een mengcultuur groeiend op synthetische afvalwater en/of met
een co-cultuur met in-situ glutaminezuur productie. Niet alleen zou dit kunnen resulteren in een
herwinning van nutriënten uit afvalstromen, het zou ook sterk de productiekosten van γ-PGA verlagen. De
noodzaak om te werken met zuivere culturen en de hoge kosten van de input producten zijn namelijk de
twee kostprijs bepalende factoren zijn van het γ-PGA productieproces. De algemene doelstelling van deze
masterproef is om innovatief de kosten van het productieproces te verminderen en zo de industriële
productie van γ-PGA aan te zwengelen.
Om het gebruik van pure culturen te vermijden, werd in de eerste reeks experimenten getracht een γ-PGA
producerende open cultuur te verkrijgen. Twee roterende biologische contactoren (RBC) werden
geïnoculeerd met nitrificatie/denitrificatie slib en gevoed met synthetisch afvalwater. Om γ-PGA
productie te induceren werd een “feast-famine” regime toegepast aangezien verschillende types bacteriën
γ-PGA produceren in de vroeg stationaire fase. Verder werd de biofilm ook gedehydrateerd omdat
verschillende bacteriën γ-PGA produceren om hun cellen te beschermen tegen uitdroging. De voeding had
een hoge C/N ratio om microbiële productie van verbindingen met een hoog stikstof gehalte te stimuleren.
De voeding van de eerste RBC bevatte glutaminezuur, de voeding van de tweede RBC bevatte geen
organische stikstof. In de eerste RBC werd gemiddeld 55% van de toegevoegde COD en 66% van de
toegevoegde stikstof verwijderd. In de tweede RBC werd gemiddeld 3.8% van de toegevoegde COD en
27% van de toegevoegde stikstof verwijderd. Aangezien de aanwezigheid van glutaminezuur het
belangrijkste verschil was tussen de twee RBC, blijkt de aanwezigheid van glutaminezuur een significant
effect op de groei van de biofilm hebben. Helaas kon bij geen van beide RBC γ-PGA gedetecteerd
worden in het effluent of in de biofilm.
In de tweede reeks experimenten werd een co-cultuur van glutaminezuur producerende bacteriën
(Brevibacterium divericatum) en γ-PGA producerende bacteriën (Bacillus licheniformis) ontwikkeld.
Glutaminezuur dient hierdoor niet meer toegevoegd te worden in het productiemedium waardoor de
kostprijs van het productieproces daalt. Groei-experimenten werden uitgevoerd in verschillende media om
een medium te ontwikkelen dat de groei van beide soorten ondersteund. Trypton bleek essentieel voor de
groei van Brevibacterium divericatum en stimuleerde ook de groei van Bacillus licheniformis. In de
volgende fase van het onderzoek werd de productiviteit van deze bacteriën in verschillende media
geanalyseerd om te beoordelen of co -cultivatie was mogelijk. Glutamaat overproductie in C. glutamicum
wordt veroorzaakt door biotine limitatie, door specifieke detergenten, door hoge concentraties suiker en
door een subletale concentratie van penicilline. De hoogste concentratie glutaminezuur en maximale
productiviteit werden verkregen in het medium met een hoge suikerconcentratie, de hoogste γ-PGA
concentratie en maximale productiviteit werden verkregen in een medium met of zonder hoge
IV
concentraties suiker. In de finale fase werden de bacteriën tezamen opgekweekt en werd de γ-PGA
productiviteit opgevolgd. De hoogste γ-PGA concentratie en productiviteit werden bekomen in het
medium zonder hoge suiker concentraties en zonder TWEEN80.
Dit onderzoek toont aan dat de productie van γ-PGA met een co-cultuur mogelijk is. Verder onderzoek om
deze productiemethode te optimaliseren is echter essentieel.
V
Table of contents
Acknowledgements ....................................................................................................................................... I
Abstract ....................................................................................................................................................... II
Samenvatting ............................................................................................................................................. III
Table of contents ......................................................................................................................................... V
List of abbreviations .............................................................................................................................. VIII
List of figures ............................................................................................................................................. IX
List of tables ............................................................................................................................................... XI
1 Literature study ....................................................................................................................................1
1.1 Introduction ........................................................................................................................................1
1.2 Poly(amino acids) ...............................................................................................................................2
1.2.1 Cyanophycin ................................................................................................................................................. 2
1.2.2 Poly(ε-lysine) ................................................................................................................................................ 3
1.2.3 Poly(γ-glutamic acid) .................................................................................................................................... 4
1.3 Poly(γ-glutamic acid) .........................................................................................................................4
1.3.1 Definition and structure ................................................................................................................................ 4
1.3.2 Applications .................................................................................................................................................. 5
1.3.3 Microbial synthesis ....................................................................................................................................... 5
1.3.4 Production ..................................................................................................................................................... 8
1.4 Glutamic acid .....................................................................................................................................9
1.4.1 Definition ...................................................................................................................................................... 9
1.4.2 Microbial synthesis ..................................................................................................................................... 10
1.4.3 Production ................................................................................................................................................... 11
1.5 Aims .................................................................................................................................................12
2 Materials and Methods ......................................................................................................................13
2.1 Analytical methods ...........................................................................................................................13
2.1.1 Poly(γ-glutamic acid) determination ........................................................................................................... 13
VI
2.1.2 Glutamic acid determination ...................................................................................................................... 13
2.1.3 Determination of nitrogen species .............................................................................................................. 13
2.1.4 Acidity and dissolved oxygen .................................................................................................................... 14
2.1.5 Chemical oxygen demand .......................................................................................................................... 14
2.2 Experimental set-up for γ-PGA production with open cultures ...................................................... 14
2.2.1 Design ........................................................................................................................................................ 14
2.2.2 Feed ............................................................................................................................................................ 15
2.2.3 Inoculum .................................................................................................................................................... 17
2.2.4 Sampling .................................................................................................................................................... 17
2.3 Experimental set-up for γ-PGA production with pure cultures ....................................................... 17
2.3.1 Bacterial strains and inoculum preparation ................................................................................................ 17
2.3.2 Flow cytometry for quantification of bacterial cells .................................................................................. 17
2.3.3 Growth curves ............................................................................................................................................ 17
2.3.4 Glutamate and γ-PGA production experiments .......................................................................................... 19
3 Results ................................................................................................................................................. 21
3.1 Enrichment of a γ-PGA producing open microbial community in the RBC configuration ............. 21
3.1.1 Rotating biological contactor fed with glutamic acid (RBC 1) .................................................................. 21
3.1.2 Rotating biological contactor fed without glutamic acid (RBC 2) ............................................................. 26
3.2 Development of a γ-PGA producing co-culture .............................................................................. 31
3.2.1 Glutamic acid production by Brevibacterium divericatum ........................................................................ 31
3.2.2 γ-PGA production by Bacillus licheniformis ............................................................................................. 35
3.2.3 Co-culture of Brevibacterium divericatum and Bacillus licheniformis ...................................................... 39
4 Discussion ........................................................................................................................................... 43
4.1 Enrichment of a γ-PGA producing open microbial community in the RBC configuration ............. 43
4.1.1 Biofilm formation and toxicity ................................................................................................................... 43
4.1.2 γ-PGA formation ........................................................................................................................................ 45
4.1.3 Future experiments ..................................................................................................................................... 48
4.2 Development of a γ-PGA producing co-culture .............................................................................. 48
4.2.1 Glutamic acid production by Brevibacterium divericatum ........................................................................ 49
4.2.2 γ-PGA production by Bacillus licheniformis ............................................................................................. 50
4.2.3 Co-culture of Brevibacterium divericatum and Bacillus licheniformis ...................................................... 51
4.2.4 Future experiments ..................................................................................................................................... 52
4.3 Final conclusion ............................................................................................................................... 53
References .................................................................................................................................................. 54
Appendices ................................................................................................................................................. 60
VII
Appendix 1: Standard curves for SEC-HPLC ..............................................................................................60
Standard curve for poly-L-glutamic acid (Mw between 50 000 and 100 000 Da) .................................................... 60
Standard curve for poly-L-glutamic acid (Mw > 1 000 000 Da) ............................................................................... 60
Molecular weight ladder .......................................................................................................................................... 61
Appendix 2: Chromatograms of the open culture experiment RBC 1 .........................................................62
Chromatogram of the influent on 01/11/2013 of RBC 1 .......................................................................................... 62
Chromatogram of the effluent on 01/11/2013 of RBC 1 .......................................................................................... 63
Chromatogram of the supernatans of the biofilm on 01/11/2013 of RBC 1 ............................................................ 64
Appendix 3: Chromatograms of the open culture experiment RBC 2 .........................................................65
Chromatogram of the influent on 01/11/2013 of RBC 2 .......................................................................................... 65
Chromatogram of the effluent on 01/11/2013 of RBC 2 .......................................................................................... 66
Chromatogram of the supernatans of the biofilm on 01/11/2013 of RBC 2 ............................................................ 67
VIII
List of abbreviations
AOB Ammonium oxidizing bacteria
C/N Carbon over nitrogen ratio
COD Chemical oxygen demand
DO Dissolved oxygen
EPS Extracellular polymeric substances
HRT Hydraulic retention time
MSG Monosodium glutamate
NOB Nitrite oxidizing bacteria
OD Optical density
Org N Organic nitrogen
PHA Polyhydroxyalkanoate
RBC Rotating biological contactor
SCP Single cell protein
TAN Total ammoniacal nitrogen
TKN Total Kjeldahl nitrogen
VER Volume exchange ratio
γ-PGA Poly(γ- glutamic acid)
ε-PL Poly(ε-lysine)
IX
List of figures
Figure 1-1: Anthropogenic fixation of N in terrestrial ecosystems over time in comparison with the natural
biological N fixation on land ..........................................................................................................................1
Figure 1-2: γ -amide linkages in poly(γ-glutamic acid) .................................................................................2
Figure 1-3: Chemical structure of cyanophycin .............................................................................................3
Figure 1-4: Chemical structure of poly(ε-lysine) ...........................................................................................3
Figure 1-5: Conformation of poly-γ-D-glutamate, a levorotary helix stabilized by intramolecular hydrogen
bonds ..............................................................................................................................................................5
Figure 1-6: Bacillus anthracis with capsule, visualized by India ink staining ...............................................6
Figure 1-7: Genetic elements required for γ-PGA synthesis ..........................................................................6
Figure 1-8: Biochemical pathway of the glutamate synthesis by Bacillus subtilis IFO 3335 ........................7
Figure 1-9: Glutamate racemase reactions .....................................................................................................7
Figure 1-10: Course of γ-PGA production by B. licheniformis ATCC 9945A. .............................................8
Figure 1-11: Model of induction of L-glutamate production in C. glutamicum...........................................10
Figure 1-12: Time course production of glutamate and cell mass (g/L) in dextrin (12%) and ammonium
sulphate (2.0%) in L-6 medium at 30°C for Brevibacterium divericatum ...................................................11
Figure 2-1: Experimental set-up rotating biological contactor.....................................................................15
Figure 3-1: Organic nitrogen and TAN influent and effluent concentrations RBC 1 ..................................22
Figure 3-2: Removal and production rates of nitrogen compounds and COD in RBC 1 .............................23
Figure 3-3: Percentage COD and nitrogen removal in RBC 1 .....................................................................24
Figure 3-4: Evolution of the effluent concentrations of the nitrogen components in one cycle of RBC 1 ..25
Figure 3-5: The evolution of the effluent COD concentration and the DO in RBC 1..................................25
Figure 3-6: The biofilm growing on the rotator in the RBC fed with glutamic acid ....................................26
Figure 3-7: Organic nitrogen and TAN influent and effluent concentrations RBC 2 ..................................27
Figure 3-8: Nitrogen and COD loading and removal rates in RBC 2 ..........................................................28
Figure 3-9: Percentage COD and nitrogen removal in RBC 2 .....................................................................29
X
Figure 3-10: Evolution of the concentration of the effluent nitrogen components in one cycle of RBC 2 . 30
Figure 3-11: The evolution of the COD concentration and the DO in RBC 1 ............................................ 30
Figure 3-12: The biofilm growing on the rotator in the RBC fed without glutamic acid ............................ 31
Figure 3-13: Growth experiment 1 Brevibacterium divericatum ................................................................ 32
Figure 3-14: Growth experiment 2 Brevibacterium divericatum ................................................................ 32
Figure 3-15: Glutamic acid production experiment – Brevibacterium licheniformis in optimal γ-PGA
production medium with trypton (without glutamic acid) ........................................................................... 33
Figure 3-16: Glutamic acid production experiment – Brevibacterium divericatum in optimal γ-PGA
production medium with trypton and 100 g/L glucose (without glutamic acid) ......................................... 34
Figure 3-17: Glutamic acid production experiment – Brevibacterium divericatum in optimal PGA
production medium with trypton and 1 mL/L TWEEN80 (without glutamic acid) .................................... 35
Figure 3-18: Growth experiment 1 Bacillus licheniformis .......................................................................... 36
Figure 3-19: Growth experiment 2 Bacillus licheniformis .......................................................................... 36
Figure 3-20: γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA production medium
with trypton ................................................................................................................................................. 37
Figure 3-21: γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA production medium
with trypton and 100 g/L glucose ................................................................................................................ 38
Figure 3-22: γ-PGA production experiment – Bacillus licheniformis in optimal PGA production medium
with trypton and 1 mL/L TWEEN 80 .......................................................................................................... 39
Figure 3-23: γ-PGA production experiment – Bacillus licheniformis and Brevibacterium divericatum in
optimal γ-PGA production medium with trypton (without glutamic acid) ................................................. 40
Figure 3-24: γ-PGA production experiment – Bacillus licheniformis and Brevibacterium divericatum in
optimal γ-PGA production medium with trypton and 100 g/L glucose (without glutamic acid) ................ 41
Figure 3-25: γ-PGA production experiment – Bacillus licheniformis and Brevibacterium divericatum in
optimal γ-PGA production medium with trypton and 1 mL/L TWEEN80 (without glutamic acid) ........... 41
Figure 4-1: The deamination mechanism of glutamic acid ........................................................................ 45
XI
List of tables
Table 1-1: Comparison of different γ-PGA producing bacilli ........................................................................9
Table 1-2: Kinetic parameters for glutamate production .............................................................................11
Table 2-1: Course of the cycle RBC ............................................................................................................14
Table 2-2: Overview of the influent concentrations RBC ............................................................................15
Table 2-3: Overview of the operational parameters of the RBC ..................................................................16
Table 2-4: Composition trace element solution A........................................................................................16
Table 2-5: Composition trace element solution B ........................................................................................16
Table 2-6: Growth media Bacillus licheniformis .........................................................................................18
Table 2-7: Optimal glutamic acid production medium and L6 medium for growth of Brevibacterium
divericatum ...................................................................................................................................................19
Table 2-8: Types of media used to support growth of and production by Bacillus licheniformis and
Brevibacterium divericatum .........................................................................................................................19
Table 3-1: Cell count of the glutamic acid production experiment – Brevibacterium divericatum in optimal
γ-PGA production medium with trypton (without glutamic acid) ...............................................................33
Table 3-2: Overview of glutamic acid production parameters .....................................................................34
Table 3-3: Cell count of the γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA
production medium with trypton ..................................................................................................................37
Table 3-4: Cell count of the γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA
production medium with trypton and 100 g/L glucose ................................................................................38
Table 3-5: Overview of γ-PGA production parameters for production experiments with Bacillus
licheniformis .................................................................................................................................................39
Table 3-6: Cell count of the γ-PGA production experiment – Bacillus licheniformis and Brevibacterium
divericatum in optimal γ-PGA production medium with trypton ................................................................40
Table 3-7: Overview of γ-PGA production parameters for production experiments with co-culture of
Bacillus licheniformis and Brevibacterium divericatum ..............................................................................42
Table 4-1: COD and nitrogen concentration entering and leaving RBC 1 during 56 operation days ..........43
Table 4-2: COD and nitrogen concentration entering and leaving RBC 2 during 56 operation days ..........44
XII
Table 4-3: Comparison of the influent concentration for PHA production in feast-famine regime and γ-
PGA production in feast-famine regime ...................................................................................................... 46
Table 4-4: Comparison of the maximal glutamic acid concentration between a pure culture of
Brevibacterium divericatum and a co-culture ............................................................................................. 52
1
1 Literature study
1.1 Introduction
Nitrogen is an essential element for humans as it is part of proteins which are the building blocks of our
body. The consumption of animal and plant proteins is necessary for our survival and the use of synthetic
nitrogen fertilizers in agriculture to produce sufficient amounts of these proteins has become
indispensable. NH3 chemically produced in the Haber-Bosch process is used in the preparation of mineral
fertilizers (80%) and for other industrial purposes (20%), such as the production of nylon, plastics, glues,
… (Figure 1-1) (Galloway, Townsend et al. 2008, Gu, Chang et al. 2013). However, since the Haber-
Bosch process requires high temperatures, high pressures and thus enormous amounts of energy (12 000
kWh /ton NH3-N), it is a non-sustainable production method for ammonia (Appl 1997, Maurer, Schwegler
et al. 2003). The recovery of nitrogen present in waste streams can be an alternative for the sequential
removal and recapturing of nitrogen. However, the resource efficiency and environmental friendliness of
both pathways have to be evaluated to allow a fair comparison in terms of sustainability. The products of
nitrogen recovery can be used in various industries: algae, duckweed, struvite and stripped ammonia can
be applied as fertilizers and single cell proteins can be used as animal feed.
Figure 1-1: Anthropogenic fixation of N in terrestrial ecosystems over time in comparison with the natural biological N fixation
on land (Gu, Chang et al. 2013).
The crystallization of struvite from waste streams (MgNH4PO4.6H2O) allows the simultaneous recovery of
phosphate and ammonium, but requires high concentrations of ammonia and phosphate in waste streams.
The composition of struvite, containing both phosphorus and nitrogen, makes it a potentially marketable
product for the fertilizer industry. Ammonia stripping results in the removal of ammonia from waste
streams. By increasing the pH to 10 or more and increasing the temperature to 70°C, the NH3/NH4+
equilibrium shifts to NH3. The ammonia can then be recovered under the form of ammonium sulphate or
other depending on the capturing acid, which can also be applied as a fertilizer.
Single cell protein (SCP) is the protein extracted from cultivated microbial biomass. It can be used for
protein supplementation of a staple diet. Currently SCP is produced from many species of microorganisms
2
(algae, fungi and bacteria) which grow on agricultural and industrial wastes (Anupama and Ravindra
2000).
In this masterthesis, nitrogen recovery by means of production of high-value compounds with a high
nitrogen content is researched. More specifically it is investigated whether production of poly(amino
acids), under the form of poly(γ- glutamic acid) or γ-PGA, is possible by means of an open culture from
synthetic waste water and/or by means of a co-culture with in situ glutamic acid production.
1.2 Poly(amino acids)
Unlike proteins, poly(amino acids) typically exist out of only one type of amino acid, at least in the
polymer backbone and they don’t have a fixed length. The amino acids residues are linked together by
amide bonds, but not with α-amide linkages as is the case for proteins (Figure 1-2). The amide bonds
involve other side chain functions. Poly(amino acids) are synthesized by relatively simple enzymes, in a
ribosome-independent manner (Oppermann-Sanio and Steinbuchel 2002). Three different poly(amino
acids) can be synthesized by microorganisms: poly(γ-glutamic acid), poly(ε-lysine) and cyanophicin.
Figure 1-2: γ -amide linkages in poly(γ-glutamic acid)
1.2.1 Cyanophycin
Cyanophycin or multi-L-arginyl-poly(L-aspartic acid) (Figure 1-3) consists out of equimolar amounts of
aspartic acid and arginine arranged as a poly-aspartic acid backbone to which arginine residues are linked
to the β-carboxyl group of each aspartate by its α-amino group (Mooibroek, Oosterhuis et al. 2007).
Cyanophycin has different possible applications: it can be used as water softener, as a biodegradable poly-
acrylate substitute after removal of the arginyl residues or as a dispersant. In nature, cyanophycin is
produced by most cyanobacteria (Anabaena sp., Synechocystis sp., Synechococcus sp., …). In non-
heterocyst-forming bacteria, the cyanophycin granules are distributed in the protoplast. In heterocyst-
forming bacteria, cyanophycin granules are present in these heterocysts (Oppermann-Sanio and
Steinbuchel 2002). At cellular pH and physiological ionic strength, cyanophycin is insoluble (Mooibroek,
Oosterhuis et al. 2007). Cyanophycin accumulates during the transition of the cells from the exponential to
the stationary phase as a temporary nitrogen reserve. In the typical aquatic habitat of cyanobacteria, the
possibility to accumulate nitrogen before extracellular ammonia is exhausted gives a competitive
advantage over other organisms (Mackerras, de Chazal et al. 1990).
3
Figure 1-3: Chemical structure of cyanophycin
Unfortunately, many problems are related to the microbial production of cyanophycin by cyanobacteria. It
is produced intracellular, cyanobacteria are slow growers and only limited amounts of cyanophycin are
produced. Therefore cyanobacteria are unsuitable for large-scale production, with respect to cost
effectiveness (Oppermann-Sanio and Steinbuchel 2002). To solve this problem, the cphA genes, coding
for cyanophicin synthetase (CphA), have been introduced in various other bacteria, such as E. coli, and
even in plants (Frey, Oppermann-Sanio et al. 2002, Mooibroek, Oosterhuis et al. 2007). So far, in contrary
to poly(ε-lysine) and poly(γ-glutamic acid), cyanophycin has not yet been commercialized.
1.2.2 Poly(ε-lysine)
Poly(ε-lysine) or ε-PL is a cationic, water soluble, biodegradable, edible and non-toxic homopolyamide
and consists out of 20 to 30 L-lysine residues, having amide linkage between the ε-amino and α-carboxyl
group (Figure 1-4). ε-PL can be used to forms hydrogels with a high capacity for absorption of water.
Crosslinked ε-PL can also be used as a cationic adsorbent in pharmaceutical applications. Poly(ε-lysine) is
industrially produced as an antimicrobial compound for food applications (JNC America 2014).
It is produced as an extracellular material by Streptomyces albulus strain 346, a mutant of this strain and
ergot fungi, a group of fungi of the genus Claviceps (Nishikawa and Ogawa 2002).
Figure 1-4: Chemical structure of poly(ε-lysine)
4
ε-PL molecules, being surface active and cationic, have been shown to have a wide antimicrobial activity
against Gram-positive bacteria, Gram-negative bacteria and even against yeasts and fungi (Shima,
Matsuoka et al. 1984). The inhibitory effect of ε-PL on microbial growth can be explained by the fact that
ε-PL molecules are electrostatically absorbed to the cell surface, followed by the stripping of the outer
membrane and abnormal distribution of the cytoplasm. This ultimately leads to physiological damage of
the treated cells (Shima, Matsuoka et al. 1984).
1.2.3 Poly(γ-glutamic acid)
Both the production of cyanophycin and of poly(ε-lysine) have disadvantages that the production of
poly(γ-glutamic acid) does not have. Cyanophycin is produced intracellularly which makes it more
difficult to harvest. Poly(ε-lysine) is only produced by a limited amount of species, which would make
production in an open culture more challenging. Furthermore, ε-PL has a wide antimicrobial activity.
Poly(γ-glutamic acid) is produced by several Bacilli sp. and is excreted extracellulary (vide infra).
1.3 Poly(γ-glutamic acid)
1.3.1 Definition and structure
Poly(γ-glutamic acid) or γ-PGA is a water soluble, anionic, biodegradable and non-toxic homo-polyamino
acid. γ-PGA and its derivatives are therefore interesting for a broad range of industrial fields, such as food,
cosmetics, medicine and water treatments. γ-PGA has a high relative molecular mass (Mr= 100 000 –
1 000 000) and its stereochemical structure can be divided into three types: a homopolymer of D-glutamic
acid (γ-D-PGA), a homopolymer of L-glutamic acid (γ-L-PGA), and copolymer of both D- and L-
glutamic acid (γ-DL-PGA) (Ashiuchi, Nakamura et al. 2003). The amide bond between these units is
formed between the α-amine group and the γ-carboxyl group (Figure 1-2). This is in contrast to proteins
where the amide bond is formed between the α-amine group and the α-carboxyl group. γ-PGA is therefore
resistant against breakdown by proteases, which only recognize the α-amide bond. An additional
difference with proteins is that γ-PGA is not formed by the sequential processes of transcription and
translation, but by a membrane-bound γ-PGA-synthase complex catalyzing the polymerization of L-
glutamic acid to γ-PGA in a ribosome independent manner (Birrer, Cromwick et al. 1994, Kunioka 1997).
Based on the theoretical models of Zanuy and Aleman (2001) the structure of a γ-PGA molecule in
aqueous solution was modeled as a levorotary helix stabilized by hydrogen bonds (Figure 1-5). γ-PGA
originating from Bacillus licheniformis has a flexible conformation which is dependent on both the pH and
the γ-PGA concentration. Below pH 7.0, the structure of γ-PGA mainly exists out of α-helices. Above pH
7.0, β-sheets are formed, as negative charges are exempted more efficiently in a β-sheet conformation
(Candela and Fouet 2006).
5
Figure 1-5: Conformation of poly-γ-D-glutamate, a levorotary helix stabilized by intramolecular hydrogen bonds
1.3.2 Applications
The possible field of application of γ-PGA is very diverse. In medicine γ-PGA can for example be used as
a drug carrier as it is biocompatible with human tissue and also biodegradable. Another possible medicinal
application is the use of γ-PGA as biological adhesive for tissue adhesion and closing air and liquid leaks
during surgery. Furthermore, γ-PGA has reactive carboxyl groups that can serve as point of attachment for
pharmaceutical agents. By regulating the biodegradability of γ-PGA, the therapeutic agent can be released
more rapidly or more slowly in the body (Tansey, Ke et al. 2004).
In the cosmetics industry, the hydrating properties of γ-PGA can be used in various applications such as
facial creams. γ-PGA can also be used in the food sector as a stabilizer or a texture enhancer. Addition of
γ-PGA increases the bioavailability of calcium by improving the solubility and the intestinal absorption.
Thus, γ-PGA may be an interesting therapeutic tool in the treatment of osteoporosis. In wastewater
treatment γ-PGA can be used as a bio-sorbent for the removal of heavy metals such as Ni2+, Cu2+, Mn2+ en
Al3+. It can also be used as a flocculant. Other applications of γ-PGA include the use as a contrast agent,
cryoprotectant or biodegradable plastic (Bajaj and Singhal 2011).
Today, γ-PGA is commercially produced by Ajinomoto as a ‘debittering’ agent in salt substitute products
containing potassium chloride (Yamaguchi 2007). Meiji Seika Kaisha also produces γ-PGA for food
applications (Kubota 1992).
1.3.3 Microbial synthesis
γ-PGA is produced mainly by Gram positive bacteria (e.g. Bacilli spp.), although also a species of
Archaea and a eukaryotic organism are described to produce γ-PGA (Candela and Fouet 2006). The
synthesis of γ-PGA has different physiological functions. For pathogenic strains such as Bacillus antracis
or Staphylococcus epidermis, the synthesis of surface associated γ-PGA is essential for virulence because
the formation of a γ-PGA capsule prevents the access of antibodies to the bacterium (Figure 1-6). Bacilli
living in the soil, such as B. licheniformis and B. subtilis, produce the anionic γ-PGA extracellular as a
viscous slimy layer to immobilize toxic metal ions. γ-PGA produced by bacteria in the early stationary
growth phase, can be used as carbon – and nitrogen source during the late stationary phase. γ-PGA is then
degraded with a depolymerase or hydrolase. Halophilic archaea secrete the highly hygroscopic γ-PGA to
increase the availability of water in the salty micro-environment of the cell (Oppermann-Sanio and
Steinbuchel 2002, Kimura, Tran et al. 2004, Bajaj and Singhal 2011).
6
Figure 1-6: Bacillus anthracis with capsule, visualized by India ink staining (Department of Health 2012)
To understand how γ-PGA production is regulated, the genetic organization of the genes involved in γ-
PGA production is of importance. When a γ-PGA capsule is formed, the involved genes are called cap
genes. When γ-PGA is released in the environment, the genes are called pgs (poly glutamate synthetase)
genes (Bajaj and Singhal 2011).
The genes related to the synthesis of the γ-PGA can be found on plasmids, for example in the case of
Bacillus anthracis, or in the genomic DNA, as is the case for Bacillus subtilis. The pgsBCA genes of
Bacillus subtilis IFO 3336 are highly homogenous with capBCA genes of Bacillus anthracis (Shih and
Van 2001, Candela, Mock et al. 2005) (Figure 1-7).
Figure 1-7: Genetic elements required for γ-PGA synthesis (Candela and Fouet 2006)
Currently, some research focuses on metabolic and genetic engineering of microorganisms producing γ-
PGA. The pgsBCA gene sequence has successfully been expressed in Escherichia coli which could result
in a more abundant production at moderate prices ((Ashiuchi, Shimanouchi et al. 2004, Bajaj and Singhal
2011). Furthermore site-directed mutagenesis can be used to modify the γ-PGA synthesizing enzymatic
complex for controlled γ-PGA production.
The γ-PGA-synthesis complex is embedded in the cell membrane and requires the presence of glutamic
acid, ATP and Mg2+ as a cofactor (Ashiuchi, Shimanouchi et al. 2004). Some γ-PGA producing bacteria
can form the necessary glutamic acid themselves, but these generally have a low γ-PGA productivity.
Other more productive bacteria require glutamic acid from the extracellular environment to produce γ-
PGA. The biosynthesis of γ-PGA proceeds in 3 steps: first, the intracellular synthesis of L- and D-
7
glutamic acid for glutamic acid independent γ-PGA producers, second, the combination of these glutamic
acid units and third, the transport of the γ-PGA through the membrane (Bajaj and Singhal 2011).
A possible biochemical pathway of the glutamate synthesis by Bacillus subtilis IFO 3335 can be found in
figure 1-8. L-glutamic acid can be formed out of α-keto glutarate by an aminotransferase. This enzyme
transfers one of the amino groups of glutamine to α-keto glutarate, resulting in L-glutamic acid.
Figure 1-8: Biochemical pathway of the glutamate synthesis by Bacillus subtilis IFO 3335 (1. glutamine:2 oxoglutarate
aminotransferase; 2. glutamine synthetase; 3. L-glutamic acid:pyruvic acid aminotransferase; 4. alanine racemase; 5. D-
glutamic acid:pyruvic acid aminotransferase; 6. γ-PGA polymerase) (Kunioka 1997)
L-glutamic acid can also be transformed into D-glutamic acid by the action of a glutamate racemase
(Figure 1-9). D-glutamic acid and L-glutamic acid can both be used by γ-PGA-polymerase to form γ-
PGA. There is however still a lot of uncertainty concerning the exact biochemical pathway for γ-PGA
synthesis (Kunioka 1997).
Figure 1-9: Glutamate racemase reactions (Cava, Lam et al. 2011)
8
1.3.4 Production
The bacteria that secrete γ-PGA extracellularly are interesting for γ-PGA production processes since the
required purification is easier and the bacteria producing a γ-PGA capsule are pathogenic. Production
conditions have to be optimized for every individual bacterial strain to attain maximal γ-PGA production.
As stated previously, two different groups can be distinguished: glutamic acid independent γ-PGA
producing bacteria and glutamic acid dependent γ-PGA producing bacteria. The γ-PGA producers with the
highest productivity are usually glutamic acid dependent. However, there has to be a trade-off between
this higher productivity and the high cost of glutamic acid (Cao, Geng et al. 2011).
Since glutamate is formed from α-ketoglutarate, an intermediate of the Krebs cycle, glutamic acid
production can be the result of conversion of for example glucose, glycerol or other intermediates of the
Krebs cycle (Bajaj and Singhal 2011). Glycerol cannot only be used as carbon source, but also stimulates
polyglutamyl synthetase, the enzyme that catalyses the polymerization of glutamate to γ-PGA.
Furthermore, glycerol is in the production medium also responsible for a reduction of the chain length of
γ-PGA and thus for a reduction of the viscosity in the production broth (Wu, Xu et al. 2010). Glycerol also
facilitates the intake of extracellular substrates and enhances the permeability of the cell membrane for
intracellular produced γ-PGA (Wu, Xu et al. 2008).
As a nitrogen source, inorganic nitrogen present as NH4+ is preferably used. Next to carbon and nitrogen,
also salts play an important role. Addition of CaCl2 reduces the viscosity of the medium and promotes the
activity of different important enzymes in the γ-PGA pathway which causes an increase in the
consumption of extracellular glutamate and therefore also the γ-PGA yield.
As can be seen in figure 1-10, the cells of B. licheniformis ATCC 9945A started to grow within 5 hours
after inoculation. The concentration of citric acid and L-glutamic acid rapidly decreased and the
concentration of γ-PGA simultaneously increased, reaching a maximum at the start of the stationary phase
(Yoon, Hwan Do et al. 2000).
Figure 1-10: Course of γ-PGA production by B. licheniformis ATCC 9945A. Time profiles: (□) dry cell weight, (■) γ-PGA, (●)
citric acid, (○) L-gutamic acid, (▲) glycerol concentrations (Yoon, Hwan Do et al. 2000)
In the table below (Table 1-1), different γ-PGA producing organisms are compared in terms of the
concentration of the produced γ-PGA, productivity and conversion rate which is the ratio of γ-PGA to the
amount of L-glutamic acid added to the medium (Xu, Jiang et al. 2005).
9
Table 1-1: Comparison of different γ-PGA producing bacilli (Xu, Jiang et al. 2005)
Strain Main nutrients Culture
time (h)
γ-PGA
(g/l)
Productivity
(g/l/h)
Conversion
rate to
glutamic
acid (%)
Reference
B.
licheniformis
ATCC 9945a
Glutamic acid
(20 g/l), glycerol
(80 g/l), citric
acid (12 g/l)
96 17–23 0.18–0.24 85–115
(Birrer,
Cromwick et
al. 1994)
B. subtilis
IFO3335
Glutamic acid
(30 g/l), citric
acid (20 g/l)
48 10–20 0.2–0.4 33–66
(Kunioka and
Goto 1994)
B. subtilis
TAM-4
Fructose
(75 g/l), NH4Cl
(18 g/l)
96 22 0.23 –
(Ito, Tanaka et
al. 1996)
B. subtilis
F-2-01
Glutamic acid
(70 g/l), glucose
(1 g/l)
96 48 0.5 68
(Kubota,
Matsunobu et
al. 1993)
B. subtilis
NX-2
Glutamic acid
(30 g/l), glucose
(30 g/l)
24 30.2 1.26 101
(Xu et al.
2005)
Currently, large scale production is strongly limited by the cost of the input products and the necessity to
work with pure cultures, which results in a very expensive production process. Furthermore, γ-PGA is a
strongly viscous polymer which results in limitations in nutrient – and oxygen transfer when submerged
production processes are used (Cromwick, Birrer et al. 1996). Therefore extensive research is being done
to obtain bacteria which have a sufficiently high productivity, can use an inexpensive starting product
such as glycerol (Zhang, Zhu et al. 2012) or which can perform the production on solid state materials
such as soy meal (Bajaj, Lele et al. 2008), dairy manure (Chen, Chen et al. 2005) or pig manure (Chen,
Chen et al. 2005).
1.4 Glutamic acid
1.4.1 Definition
Monosodium glutamate or MSG is a non-essential amino acid which is applied as a flavor enhancer and is
especially used in east Asian dishes (Ault 2004). Glutamic acid was first isolated as a pure substance in
1866 and has since then become the basis of a trillion-dollar worldwide industry. It can be produced by
extraction from protein hydrolysates of wheat gluten, by chemical synthesis or by microbial processes.
However, the hydrolysis process results in a variety of environmental problems due to the use of hydrogen
chloride gas and chemical synthesis results the production of a racemic mixture of the enantiomeric forms
of glutamic acid. Therefore, mainly the microbial production processes are used today (Sano 2009).
The discovery of Corynebacterium glutamicum, which is capable of producing L-glutamic acid with high
productivity from sugars, paved the way for the success of the fermentation technique in amino acid
production (Kinoshita, Udaka et al. 1957). The worldwide annual production of glutamate by
Corynebacterium glutamicum is over 1.5 million tons (Shimizu and Hirasawa 2007) and the annual
market growth for most amino acids is 10% and higher (Hermann 2003).
10
1.4.2 Microbial synthesis
Several bacterial species have been proven to produce glutamic acid, for example some lactic acid bacteria
(Zareian, Ebrahimpour et al. 2012), but all bacteria useful for industrial glutamate production (eg.
Corynebacterium glutamicum) are coryneform bacteria, which are gram-positive, non-spore-forming, non-
motile and require biotin for growth (Sano 2009).
The principal pathway for glutamate production is via the enzyme glutamate dehydrogenase from α-
ketoglutarate when the ammonium concentration is sufficiently high. α-Ketoglutarate is a member of the
tricarboxylic acid cycle and thus of the central carbon metabolism (Shimizu and Hirasawa 2007).
Growth of C. glutamicum requires the presence of biotin in its growing medium since biotin is the
cofactor of the enzyme acetyl-CoA carboxylase, which is indispensable for biosynthesis of fatty acids.
Extracellular glutamate accumulation only occurs under biotin-limiting conditions. C. glutamicum can
produce significant amounts of glutamate in the presence of excess biotin, if detergent compounds such as
Tween 40 of Tween 60 are added. Also the addition of penicillin enhances the overproduction of
glutamate by C. glutamicum (Shimizu and Hirasawa 2007). So far, the mechanism of excessive glutamate
production by coryneform bacteria is not fully understood. A recent study showed that the activity of 2-
oxoglutarate dehydrogenase complex (ODHC) is greatly reduced under all the aforementioned conditions
which leads to an increase in the carbon flow towards the synthesis of glutamate at the ODHC branch
point (Shimizu and Hirasawa 2007). The current hypothesis explaining the excretion of glutamic acid is
that the membrane tension alters under conditions in which glutamate accumulates in the medium, such as
if biotin is limited, because the fatty acid biosynthesis is inhibited. This alteration triggers a change in
conformation of the NCgl1221 protein which causes the export of glutamic acid (Figure 1-11) (Nakamura,
Hirano et al. 2007).
Figure 1-11: Model of induction of L-glutamate production in C. glutamicum (Nakamura, Hirano et al. 2007)
11
1.4.3 Production
The industrial production process for L-glutamic acid is done using sterile aerobic batch fermentation
process with cells in suspension. The L-glutamic acid released by the microorganism into the fermentation
solution is then obtained by crystallization (Leuchtenberger, Huthmacher et al. 2005). Currently, extensive
research is done to genetically engineer C. glutamicum and develop the strain further to obtain higher
production yields and efficiencies (Hermann 2003).
The coryneform bacteria used for this research was Brevibacterium divericatum. According to Nadeem,
Niaz et al. (2011) a medium containing 12% dextrin as a carbon source and 2% ammonium sulphate as a
nitrogen source resulted in maximal volumetric productivity (2 g/(L.h)). Apart from a suitable nitrogen
and carbon source, also biotin is essential for glutamate production as stated previously.
The production of glutamate is simultaneous to the growth for Brevibacterium divericatum (Figure 1-12).
Figure 1-12: Time course production of glutamate and cell mass (g/L) in dextrin (12%) and ammonium sulphate (2.0%) in L-6
medium at 30°C for Brevibacterium divericatum (Nadeem, Niaz et al. 2011)
Several kinetic parameters for glutamic acid production were determined for Brevibacterium divericatum
by (Nadeem, Niaz et al. 2011) and for Corynebacterium glutamicum by Khan et al., 2005 and Bona and
Moser, 1997 (Table 1-2).
Table 1-2: Kinetic parameters for glutamate production
Strain Main nutrients Glutamate
(g/l)
YP/S
(g glutamate/g
substrate)
YP/X
(g glutamate/
g biomass)
Reference
Brevibacterium
divericatum
NIAB SS-67
120 g/L dextrin,
20 g/L (NH4)2SO4 60.9 0.55 5.2
(Nadeem, Niaz
et al. 2011)
Corynebacterium
glutamicum
MTCC 2745
50 g/L glucose,
5 g/L urea 25.1 0.48 3.216
(Khan, Mishra
et al. 2005)
Corynebacterium
glutamicum
ATCC 13869
70 g/L
glucose.H2O,
0.24 g/L (NH4)2SO4
32.74 0.356 3.539
(Bona and
Moser 1997)
12
1.5 Aims
Poly(γ-glutamic acid) is an interesting poly(amino acid) of which the potential applications are manifold.
However, the use of γ-PGA is currently constrained by the expensive production process. Currently,
poly(γ-glutamic acid) is produced in an aerobic batch process using pure cultures of glutamic acid-
dependent γ-PGA producing strains. The necessity to work with pure cultures and the high cost of the
input products are the two cost-determining aspects of this production process.
The aim of this master thesis is to innovatively decrease the costs of the production process and hence
enable the industrial use of this biologically produced polymer.
In the first part of this research project the development of an open γ-PGA producing culture is
investigated, which would eliminate the necessity of a pure culture production process. This research part
focusses on the enrichment of an open microbial culture for γ-PGA production by applying a feast-famine
and dehydrating regime on the biofilm growing in an RBC configuration.
The goal of the second stage of the project, is to develop a co-culture of glutamic acid and γ-PGA
producing bacteria, eliminating the requirement of glutamic acid as input product. By evaluating which
compounds of both optimal production media are indispensable and which are not, a medium supporting
growth of and production by both bacteria is developed.
13
2 Materials and Methods
2.1 Analytical methods
2.1.1 Poly(γ-glutamic acid) determination
For the analysis of poly-γ-glutamic acid Size Exclusion Chromatography (SEC) was used. The Dionex
U3000RS HPLC (Dionex, USA), was fitted with a PL aquagel-OH MIXED-H permeation column, pore
size: 8μm, 300 x 7.5 mm (Agilent Technologies, Belgium). Samples (20 µL) were eluted with a 0.1 mM
sodium chloride at a flow rate 1 mL/min and detected at 220 nm.
Standard curves were prepared for poly-L-glutamic acid sodium salt with a molar weight between 50 000
and 100 000 and a molar weight higher then 1 000 000 (Sigma – Aldrich, USA). The retention times for
molecules with different molecular weights were determined using a Gel Filtration Markers Kit for protein
molecular weights between 12 000-200 000 Da (Sigma – Aldrich, USA). The standard curves and the
molecular weight ladder can be found in appendix 1.
The presence of γ-PGA was analyzed in both the effluent of the RBC and in the biomass. To analyze the
presence of γ-PGA in the biomass, 1 gram of biomass was scraped of the surface of the rotator. This
sample was centrifuged during for 30 min at 10 000 rpm and 4 °C, after which the supernatant was
analyzed on SEC-HPLC.
2.1.2 Glutamic acid determination
For the quantitative determination of L-glutamic acid the K-Glut 07/12 assay (Megazyme, Ireland) was
used. The absorbance was measured at 492 nm in a 96-well plate in the Tecan Infinite M200 Pro, with i-
Control software (Tecan Group Ltd., Germany).
2.1.3 Determination of nitrogen species
Nitrite and nitrate
For the quantitative determination of nitrite and nitrate, anion exchange chromatography (IC) was used.
The ion chromatography device used for this research was the Metrohm 761 Compact IC (Metrohm,
Switzerland) with the anion column Metrosep A Supp 5 – 150. The anion column was protected with a
guard column (Metrosep A Supp 4/5 Guard). As eluent a mixture of 1.0 mM NaHCO3, 3.2 mM Na2CO3
and 5% vol. acetone at a flow rate of 0.7 mL min-1 was used. Detection was done based on electric
conductivity.
Total ammonia nitrogen
The total ammonia nitrogen (TAN) was determined by steam distillation. A maximum of 20 mL of the
sample is added in the distillation tube. The nitrogen content has to be between 0.1 and 6 mg NH4+-N/L.
Also minimum 3 blanks and a control sample were analyzed. To the sample tubes 0.4 g of MgO was
added to ensure a strong alkaline environment. A Gerhardt Vapodest 30 (C. Gerhardt GmbH & Co. KG,
Germany) ammonia distillation apparatus was used. The ammonia which is captured in this acid solution
as (NH4)3BO3 was tritrimetrically determined with 0.02 N HCl using a Metrohm 719 S (Titrino;,
Switzerland) for the automatic titration.
14
Kjeldahl nitrogen
The Kjeldahl nitrogen (TKN) includes both organic nitrogen and TAN. The organic nitrogen can be
determined by the substraction of the TAN from the Kjeldahl nitrogen. The organic nitrogen present in the
sample is transformed into (NH4)2SO4 by means of destruction at 400°C in a destruction block (Foss
Tecator 2020 digestor, The Netherlands) with sulphuric acid (H2SO4) (98%) and potassium and copper
sulphate (K2SO4,CuSO4) as catalysts. The ammonia coming from the organic nitrogen and the already
present ammonia were then determined by the method used to determine TAN.
2.1.4 Acidity and dissolved oxygen
The pH of the influent and effluent samples of the RBC were measured with a Consort C532 multi-
parameter analyzer pH probe (Consort, Belgium). The dissolved oxygen concentration was measured
every two days using luminescence with a Hach HQ30d with LDO101 HQ30d DO-probe (Hach, Lang
GmbH, Germany).
2.1.5 Chemical oxygen demand
The chemical oxygen demand or COD was measured with NANOCOLOR COD 1500 and NANOCOLOR
COD 160 test kits (Machery – Nagel, Germany). The COD is determined by a silver-catalyzed oxidation
with potassium dichromate in the presence of sulfuric acid. The COD concentration is determined with the
NANOCOLOR 500D digital photometer (Machery-Nagel, Germany).
2.2 Experimental set-up for γ-PGA production with open cultures
2.2.1 Design
Two rotating biological contactors of the type Venta airwasher LW 14 (Airsain, Belgium) were used. The
set-up can be found in figure 2-1.
The Venta airwasher LW 14 contains a rotor existing out of 40 disks with 3 mm space between these
disks. The disk diameter is 7.5 cm. The total surface of the rotor is 1.32 m². The rotation speed was 1.5
rpm. The rotor was for 50% submerged, up to a volume of 3.62 liter.
In a 4 hour lasting cycle, the automatic valve first opens to release 1 liter effluent. Subsequently, 1 liter of
influent is pumped in the system with a peristaltic pump during 15 minutes. The final volume of the RBC
is then 3.62 liter. The Volume Exchange Ratio (%VER) was 27.8% . The rotor of the RBC stopped
rotating during 15 minutes every hour to dehydrate the biofilm. The cycle was regulated by means of
timers (Chacon – TH 858C, Chacon N.V, Belgium) (Table 2-1).
Table 2-1: Course of the cycle RBC
Time point
0:00 – 0:15 Effluent removal
0:15 – 0:30 Influent dosage
0:30 – 4:00 Reaction time
4:00 - 4:15 Effluent removal
4:15 - 4:30 Influent dosage
15
Figure 2-1: Experimental set-up rotating biological contactor (P: pump, M: motor)
2.2.2 Feed
The feed of the RBC was refreshed every two days. One RBC was fed with influent containing glutamic
acid, the other RBC was fed without glutamic acid in the influent. The influent concentrations for both
RBC are given in table 2-2. The operational parameters can be found in table 2-3. The N loading rates for
both RBC are the same. However, this results in a slightly higher COD loading rate for RBC 1 compared
to RBC 2.
Table 2-2: Overview of the influent concentrations RBC
Influent concentrations RBC 1 fed with
glutamic acid
RBC 2 fed without
glutamic acid
Glycerol (g/L) 0.195 0.390
Glucose (g/L) 0.223 0.445
Glutamate (g/L) 1.002 0
NH4Cl (g/L) 0.286 0.572
KH2PO4 (g/L) 1.5 1.5
Trace element solution A (mL/L) 1 1
Trace element solution B (mL/L) 1 1
16
Table 2-3: Overview of the operational parameters of the RBC
Operational parameters RBC 1 fed with
glutamic acid
RBC 2 fed without
glutamic acid
COD-loading rate (mg COD/(m².d)) 6401 4320
N-loading rate (mg N/(m².d)) 680 680
Reactor volume (L) 3.62 3.62
Volume exchange ratio (%VER) 27 27
HRT (hrs) 14.5 14.5
Q (L/d) 6 6
Biofilm surface (m²) 1.32 1.32
pH 7.26 ± 0.34 6.93 ± 0.34
Temperature (°C) 23.69 ± 0.96 22.28 ± 1.32
The phosphate buffer (1.5 g/L KH2PO4) was added to maintain the pH at a value of 7. Trace elements
solution A and B (Table 2-4 and Table 2-5) were added to at a concentration of 1 mL/L.
Table 2-4: Composition trace element solution A
Salt Concentration (g/L)
FeSO4.7H2O 10
Na2H2EDTA.2H2O 13.4
Table 2-5: Composition trace element solution B
Salt Concentration (g/L)
ZnSO4.7H2O 4.4
CoCl2.6H2O 3.2
MnSO4.H2O 10.4
CuSO4 2
(NH4)6Mo7O24.4H2O 2.2
NiCl2.6H2O 0.203
NaSeO3.5H2O 0.152
H3BO3 0.0115
Na2H2EDTA.2H2O 43.2
17
2.2.3 Inoculum
As inoculum highly activate nitrification – denitrification sludge or HANDS (Avecom nv, Belgium) was
used.
2.2.4 Sampling
Sampling was done every 2 days. The effluent was measured at the end of a 4 hour lasting cycle. The
samples were filtered (0.45 µm) and preserved at 4°C until the moment of analysis.
2.3 Experimental set-up for γ-PGA production with pure cultures
2.3.1 Bacterial strains and inoculum preparation
For the production of poly(γ-glutamic acid) Bacillus licheniformis NCIM 2324 obtained from the National
Collection of Industrial Micro-organisms (India) was used. The growth and maintenance medium
contained 5 g/L peptone, 1.5 g/L yeast extract, 1.5 g/L meat extract and 5 g/L sodium chloride (pH 7).
Bacterial cells were incubated in this growth and maintenance broth at 37 °C during 48 h and then used as
inoculum.
For the production of glutamic acid Brevibacterium divericatum NCIM 2634 obtained from the National
Collection of Industrial Micro-organisms (India) was used. As maintenance and growth medium glucose
yeast extract was used, containing 5 g/L glucose and 3 g/L yeast extract (pH 7). Bacterial cells were
incubated in this broth at 28 °C during 48 h and then used as inoculum.
2.3.2 Flow cytometry for quantification of bacterial cells
Quantification of bacterial cells was performed using flow cytometry and Sybr Green I (SG, Invitrogen,
USA) staining. The stain was prepared by diluting the SYBR Green stock solution 100 times in 0.22 µm
filtered dimethyl sulphoxide (DMSO). 196 µL of the diluted sample was coloured with 2 µL of SYBR
Green stain followed by the addition of 2 µL 0.5 M EDTA solution. The coloured sample was incubated
in the dark at 37°C during 13 minutes.
Flow cytometry measurements were done with an Accuri C6 with autosampler. 25 µL of sample was
measured at high flow rate and with the threshold on FL1 (green fluorescence). The results were processed
in de BD CSampler software and read on a scatter plot of the green (533 ± 15 nm) against the red
fluorescence (> 670 nm).
2.3.3 Growth curves
Microbial growth was followed up by measuring the optical density (OD) at 620 nm using the
spectrophotometer Tecan Infinite M200 Pro with i-Control software (Tecan Group LTD., Germany) and
the Tecan Sunrise with Magellan software (Tecan Group LTD., Germany).
Bacillus licheniformis was grown in the optimal γ-PGA production medium (Bajaj, Lele et al. 2009)
(Table 2-6), in optimal γ-PGA production medium without citric acid, in optimal γ-PGA production
medium with trypton (7.5 g/L) and in optimal γ- PGA production medium with trypton (7.5 g/L) and high
dextrose concentration (120 g/L). The pH of every medium was adjusted to 7.
18
Table 2-6: Growth media Bacillus licheniformis
Optimal γ- PGA
production
medium
Optimal γ- PGA
production
medium without
citric acid
Optimal γ- PGA
production
medium with
trypton
Optimal γ- PGA
production
medium with
trypton and high
dextrin
concentration
Component Concentration
(g/L)
Concentration
(g/L)
Concentration
(g/L)
Concentration
(g/L)
Glucose 25 25 25 0
Dextrin 0 0 0 120
Citric acid 10 0 10 10
Glutamic acid 20 20 20 20
NH4Cl 6 6 6 6
Trypton 0 0 7.5 7.5
Na2HPO4.2H2O 1.1936 1.1936 1.1936 1.1936
NaH2PO4.2H2O 0.7697 0.7697 0.7697 0.7697
MgSO4.7H2O 0.5 0.5 0.5 0.5
CaCl2.6H20 0.298 0.298 0.298 0.298
FeCl3.6H20 0.0281 0.0281 0.0281 0.0281
MnSO4.H2O 0.0305 0.0305 0.0305 0.0305
Brevibacterium divericatum was grown in the optimal glutamic acid production medium as determined by
Nadeem, Niaz et al. (2011) with the salts of the optimal γ- PGA production medium and the L6 medium
with the salts of the optimal γ- PGA production medium both with and without 7.5 g/L trypton (Table
2-7). It was also grown in the optimal γ- PGA production medium, the optimal γ- PGA production
medium with 7.5 g/L trypton and the optimal γ- PGA production medium with 7.5 g/L trypton and 120
g/L dextrine (Table 2-6). The pH of every medium was adjusted to 7.
19
Table 2-7: Optimal glutamic acid production medium and L6 medium for growth of Brevibacterium divericatum
Optimal glutamic acid
production medium L6 medium
Component Concentration (g/L) Concentration (g/L)
Dextrin 120 0
Glucose 0 100
(NH4)2SO4 20 30
Trypton 7.5 7.5
Biotin 0.00006 0.00006
Thiamin HCl 0.005 0.005
Na2HPO4.2H2O 1.1936 1.1936
NaH2PO4.2H2O 0.7697 0.7697
MgSO4.7H2O 0.5 0.5
CaCl2.6H20 0.298 0.298
FeCl3.6H20 0.0281 0.0281
MnSO4.H2O 0.0305 0.0305
2.3.4 Glutamate and γ-PGA production experiments
Glutamate and γ-PGA production was determined in batch experiments. Each 250 mL Erlenmeyer flask
containined 100 mL of sterile, autoclaved production medium. To obtain a medium suitable to support
growth of both Brevibacterium divericatum and Bacillus licheniformis different types of media were used
(Table 2-8).
The medium was inoculated with 1% (v/v) of 48 h old B. licheniformis and/or B. divericatum culture. The
flasks were incubated on a rotary shaker at 32°C and 110 rpm. All the experiments were carried out at
least in triplicate.
20
Table 2-8: Types of media used to support growth of and production by Bacillus licheniformis and Brevibacterium divericatum
Optimal γ- PGA
production medium
Optimal γ- PGA
production medium
with trypton
Optimal γ- PGA production
medium with trypton and
high glucose concentration
Optimal γ- PGA production
medium with TWEEN 80
Component Concentration (g/L) Concentration (g/L) Concentration (g/L) Concentration (g/L)
Glucose 25 25 100 25
Citric acid 10 10 10 10
Glutamic acid (not for
B. divericatum) 20 20 20 20
NH4Cl 6 6 6 6
Trypton 0 7.5 7.5 7.5
Na2HPO4.2H2O 1.1936 1.1936 1.1936 1.1936
NaH2PO4.2H2O 0.7697 0.7697 0.7697 0.7697
MgSO4.7H2O 0.5 0.5 0.5 0.5
CaCl2.6H20 0.298 0.298 0.298 0.298
FeCl3.6H20 0.0281 0.0281 0.0281 0.0281
MnSO4.H2O 0.0305 0.0305 0.0305 0.0305
Tween 80 0 0 0 1 mL/L
21
3 Results
3.1 Enrichment of a γ-PGA producing open microbial community in the RBC
configuration
Both RBC were evaluated in terms of γ-PGA production and nitrogen and COD removal. Nitrite and
nitrate production rates were followed up to assess the activity of competing processes such as nitrification
by ammonium and nitrite oxidizing bacteria (AOB and NOB) in the open community. Total ammoniacal-
nitrogen (TAN) removal rates, organic nitrogen and COD removal rates were followed up to assess the
microbial removal and/or production and to what extent there was a feast-famine regime present in the
reactor system. The removal rate of organic nitrogen was followed up to see whether glutamic acid was
removed and potentially incorporated into poly(γ-glutamic acid).
3.1.1 Rotating biological contactor fed with glutamic acid (RBC 1)
Reactor performance in terms of nitrogen removal
In figure 3-1 the influent and effluent concentrations of TAN, organic nitrogen, nitrate, nitrite and COD
are visualized. During the start-up period no organic nitrogen measurements could be performed due to
technical difficulties with the Kjeldahl analysis apparatus. It stands out that the TAN influent
concentration measured is on average 165% times bigger than what was actually added according to the
medium and that the organic nitrogen influent concentration is on average 35% times smaller then what
was added (Table 2-2). The total nitrogen, the sum of organic nitrogen and TAN, is constant since 65% of
the organic nitrogen was hydrolyzed to ammonia in the influent vessel. Only a limited amount of organic
nitrogen actually reached the reactor, which makes it difficult to make conclusions concerning the organic
nitrogen removal.
The first days ammonia is produced due to the partial degradation (lysis) of the activated sludge which
was used to inoculate the reactor. Proteins are degraded which results in a release of ammonia. After a
short start-up period, an average TAN reduction of 89 ± 5% was reached. 11.1 ± 1.3% of the TAN influent
was converted to nitrite and 5.6 ± 1.6% of the TAN influent was converted to nitrate, which results in an
effective TAN removal of 74 ± 4%. The average pH in RBC 1 was 7.26 ± 0.34, the average temperature
23.7 ± 1.0 °C and the average TAN effluent concentration was 9 ± 3 mg TAN/L (Table 2-3). According to
Anthonisen, Loehr et al. (1976) the free ammonia concentration at this pH, temperature and TAN level is
0.09 mg NH3-N/L. This free ammonia level can result in inhibition of NOB (inhibition between 0.08 and
0.82 mg NH3-N/L), but not in AOB inhibition (inhibition between 8 and 120 mg NH3-N/L).
The average nitrite influent concentration was very low (0.10 ± 0.05 mg NO2- -N/L), the average nitrite
effluent concentration was 13.8 ± 1.8 mg NO2- -N/L, which means on average 13.7 ± 1.8 mg NO2
- -N/L
was produced. According to Anthonisen, Loehr et al. (1976) the free nitrous acid concentration at this pH,
temperature and total nitrite effluent level is 0.0018 mg HNO2-N/L, which does not result in inhibition of
NOB (inhibition between 0.06 and 0.83 mg HN02-N/L), nor in AOB inhibition (inhibition between 0.2
and 2.8 mg HNO2-N/L). The average nitrate influent concentration was 0.72 ± 0.16 mg NO3- -N/L, the
average nitrate effluent concentration was 6.60 ± 1.6 mg NO3- -N/L, which means on average 5.88 ± 1.6
mg NO3- -N/L was produced. The COD influent concentration shows a downward trend, the COD effluent
concentrations remains constant. On average 55 ± 3% of the COD is removed.
22
Figure 3-1: Organic nitrogen and TAN influent and effluent concentrations RBC 1
23
In figure 3-2 the total nitrogen removal rate and the removal and production rates of the different nitrogen
compounds can be found. The total nitrogen removal rate only takes the organic removal in account
starting from operation day 21. On average 504 ± 44 mg NH4+-N/(m².day) was removed with a maximum
of 782 mg NH4+-N/(m².day). The removal of organic nitrogen, in this case glutamic acid, is more variable.
This is due to the fact that the influent concentration of the added organic nitrogen was not constant as can
be concluded from figure 3-1.
The nitrite production rate is 132 ± 8 mg NO2--N/(m².day) and the nitrate production rate is 68 ± 7 mg
NO3--N/(m².day).
Reactor performance in terms of COD removal
In figure 3-3 the percentage of the COD and nitrogen removal is shown. The removal percentages are the
ratio of the effluent concentration and the influent concentration. On average 55 ± 3% of the COD and 66
± 5% of the nitrogen is removed. Since all the COD (glucose, glycerol, glutamic acid) given to the RBC is
easily degradable, it can be concluded that the feast-famine regime is not reached. To obtain more
conclusive results concerning the feast-famine regime, one cycle was followed up.
Figure 3-2: Removal and production rates of nitrogen compounds and COD in RBC 1
24
Figure 3-3: Percentage COD and nitrogen removal in RBC 1
Follow-up of one cycle
On operation day 56, one cycle was followed-up to obtain more conclusive results concerning the removal
and production of nitrogen compounds and COD removal.
In figure 3-4 it can be seen that all the ammonia is removed in one cycle. However, there is still 4.88 mg
organic N/L left. It can be concluded that there is no feast-famine regime. The nitrate concentration clearly
increases towards the end of the cycle, up to a concentration of 7.3 mg NO3- -N/L. The nitrite
concentration remains low throughout the cycle with a maximum of 1.2 mg NO2--N/L. The AOB and
NOB are both active since only nitrate is detected. After half an hour, the total nitrogen concentration
remains approximately constant as a result of the activity of AOB and NOB.
From figure 3-5 it can be derived that only 76% of the COD and 81% of the nitrogen is removed at the
end of a 4 hour lasting cycle. The highest activity can be seen in the beginning of the cycle: the dissolved
oxygen is lowest in the beginning of the cycle. It can be concluded that there is no feast-famine regime for
COD or nitrogen.
Production of γ-PGA
Samples taken after a dehydration period of 15 minutes of the effluent and of the supernatans of the
biomass were analyzed for the presence of poly(γ-glutamic acid). No γ-PGA was detected in both cases.
The chromatogram of an influent sample, an effluent sample and a supernatant sample can be found in
appendix 2.
25
Figure 3-4: Evolution of the effluent concentrations of the nitrogen components in one cycle of RBC 1
Figure 3-5: The evolution of the effluent COD concentration and the DO in RBC 1
26
Figure 3-6: The biofilm growing on the rotator in the RBC fed with glutamic acid
3.1.2 Rotating biological contactor fed without glutamic acid (RBC 2)
Reactor performance in terms of nitrogen removal
In figure 3-7 the influent and effluent concentrations of TAN and organic nitrogen for RBC 2 is
visualized. RBC 2 was not fed with glutamic acid; therefore no organic nitrogen was present in the
influent. In the start-up period no organic nitrogen measurements could be performed due to technical
difficulties with the Kjeldahl analysis apparatus.
The first 7 days there is production of ammonia due to the partial degradation (lysis) of the activated
sludge which was used to inoculate the reactor. An average TAN reduction of 31 ± 6% was reached. 4.7 ±
1.8% of the TAN influent was converted to nitrite and 39 ± 15% of the TAN influent was converted to
nitrate, which results in an effective TAN removal of 18 ± 4% . The difference between the COD influent
and effluent concentration is very limited: on average only 4 ± 8% of the COD is removed.
The average pH in RBC 2 was 6.93 ± 0.34, the average temperature 22.3 ± 1.3 °C and the average TAN
effluent concentration 98 ± 7 mg TAN/L (Table 2-3). According to Anthonisen, Loehr et al. (1976) the
free ammonia concentration at this pH, temperature and TAN level is 0.39 mg NH3-N/L. This free
ammonia level inhibits the NOB activity (inhibition between 0.08 and 0.82 mg NH3-N/L), but not the
AOB acitivity (inhibition between 8 and 120 mg NH3-N/L).
The average nitrite influent concentration was very low (0.04 ± 0.04 mg NO2- -N/L), the average nitrite
effluent concentration was 1.97 ± 0.56 mg NO2- -N/L, which means on average 1.90 ± 0.57 mg NO2
- -N/L
was produced. According to Anthonisen, Loehr et al. (1976) the free nitrous acid concentration at this pH,
temperature and total nitrite effluent level is 0.0006 mg HNO2-N/L, which does not result in inhibition of
NOB (inhibition between 0.06 and 0.83 mg HN02-N/L), nor in AOB inhibition (inhibition between 0.2
and 2.8 mg HNO2-N/L). The average nitrate influent concentration was 2.3 ± 1.6 mg NO3- -N/L, the
average nitrate effluent concentration was 20.5 ± 2.8 mg NO3- -N/L, which means on average 18.2 ± 2.4
mg NO3- -N/L was produced.
27
Figure 3-7: Organic nitrogen and TAN influent and effluent concentrations RBC 2
28
In figure 3-8 the total nitrogen removal rate and the removal and production rates of the different nitrogen
compounds can be found. The total nitrogen removal rate only takes the organic removal in account
starting from operation day 21. On average 143 ± 28 mg NH4+-N/(m².day) was removed with a maximum
of 338 mg NH4+-N/(m².day).
The nitrite production rate is 7 ± 3 mg NO2--N/(m².day) and the nitrate production rate is 83 ± 11 mg NO3
-
-N/(m².day).
Figure 3-8: Nitrogen and COD loading and removal rates in RBC 2
Reactor performance in terms of COD removal
In figure 3-9 the percentage of the COD and nitrogen removal is shown. The removal percentages are the
ratio of the effluent concentration and the influent concentration. On average 10% ± 6% COD and 5 ± 5%
of nitrogen is removed. Very little of the added COD is degraded, which means that the biofilm is not
properly growing. The negative COD removal indicates excretion or cell lysis. To obtain more conclusive
results concerning the feast-famine regime, one cycle was followed up.
29
Figure 3-9: Percentage COD and nitrogen removal in RBC 2
Follow-up of one cycle
On operation day 56, one cycle was followed-up to obtain more conclusive results concerning the removal
and production of nitrogen compounds and COD removal.
As can be seen in figure 3-10, only 6.4 mg NH4+-N/L is removed during the 4 hour lasting cycle. There is
some initial production of nitrite, which is nitrified to nitrate later in the cycle. The final nitrate
concentration is 30.2 mg NO3--N/L, the finale nitrite concentration is 0 mg NO2
--N/L. The total nitrogen
concentration remains constant throughout the 4 hour lasting cycle.
In figure 3-11 it is shown that the highest COD removal is achieved when the slope of DO rapidly
increases. 40% of the COD is removed.
It can be concluded that the activity in the RBC fed without glutamic acid is much lower than the activity
in the RBC fed with glutamic acid (Figure 3-12).
Production of γ-PGA
Samples taken after a dehydration period of 15 minutes of the effluent and of the supernatans of the
biomass were analyzed by means of SEC-HPLC for the presence of poly(γ-glutamic acid). No γ-PGA was
detected in both cases. The chromatogram of an influent sample, an effluent sample and a supernatans
sample can be found in appendix 3.
30
Figure 3-10: Evolution of the concentration of the effluent nitrogen components in one cycle of RBC 2
Figure 3-11: The evolution of the COD concentration and the DO in RBC 1
31
Figure 3-12: The biofilm growing on the rotator in the RBC fed without glutamic acid
3.2 Development of a γ-PGA producing co-culture
The goal of these experiments was to develop a co-culture of glutamic acid and γ-PGA producing bacteria,
eliminating the requirement of glutamic acid as an input product for γ-PGA production. First, the bacteria
were grown in different types of media to assess which media are capable of supporting the growth of
both Brevibacterium divericatum and Bacillus licheniformis. In the following experimental stage, these
media were tested for their capability to support production of both glutamic acid by Brevibacterium
divericatum and γ-PGA by Bacillus licheniformis. To conclude the experiments, both strains were
cultivated together without the addition of glutamic acid, to check for γ-PGA production.
3.2.1 Glutamic acid production by Brevibacterium divericatum
Growth experiments
In figure 3-13 the first growth experiment is visualized. In the optimal glutamic acid production medium,
the carbon source exists out of 120 g/L of dextrin and the nitrogen source is 20 g/L (NH4)2SO4. The L6
medium contains 100 g/L glucose and 30 g/L (NH4)2SO4. The optimal γ-PGA production medium
contains 25 g/L glucose and 6 g/L NH4Cl, but no trypton. For the optimal glutamic acid production
medium the stationary phase is reached after 32 hours. The maximal optical density (OD) is 0.77 ± 0.06.
In the L6 medium with trypton, the stationary phase is reached after 80 hours. The maximal optical
density (OD) is 1.65 ± 0.10. Comparing the growth in the L6 medium with trypton to the growth in the L6
medium without trypton (ODmax 0.15 ± 0.01), we can conclude that trypton is an essential component for
the growth of Brevibacterium divericatum. The absence of trypton in the optimal γ-PGA production
medium could be the reason why no growth is detected. The maximal optical density in the optimal γ-
PGA production medium is 0.20 ± 0.04.
To further optimize the growth medium of Brevibacterium divericatum a second growth experiment was
performed. The growth was compared in a medium with and without a high (120 g/L) dextrin
concentration and the importance of the presence of trypton was tested. In figure 3-14 the second growth
experiment is visualized. In the optimal glutamic acid production medium an ODmax of 0.67 ± 0.02 was
reached. In the optimal γ-PGA production medium with trypton a maximal OD of 1.30 ± 0.01 was
reached. In the medium with high dextrin concentration growth inhibition is visible, the maximal OD is
1.03 ± 0.13. Based on this experiment it was concluded to continue with the optimal γ-PGA production
medium with trypton both with and without high sugar concentrations.
32
Figure 3-13: Growth experiment 1 Brevibacterium divericatum
Figure 3-14: Growth experiment 2 Brevibacterium divericatum
33
Glutamic acid production experiments
In figure 3-15 the glutamic acid production by Brevibacterium divericatum in the optimal γ-PGA medium
with trypton is visualized. The glutamic acid production starts after 23 hours when the stationary growth
phase is reached. A maximal concentration of 67 ± 7 mg/L of glutamic acid was obtained. A maximal
productivity of 151 ± 11 mg/(L.day) glutamic acid was reached after 72 hours. From table 3-1 can be
concluded that the cell count remained constant after 23 hours. A cell count of 2.109 cells per mL is
assumed as final cell concentration since this cell count is reached after 23 hours and does not further
change.
Figure 3-15: Glutamic acid production experiment – Brevibacterium licheniformis in optimal γ-PGA production medium with
trypton (without glutamic acid)
Table 3-1: Cell count of the glutamic acid production experiment – Brevibacterium divericatum in optimal γ-PGA production
medium with trypton (without glutamic acid)
Hours events/mL Standard error
0 7.00.106 1.00.106
17 1.25.109 0.04.109
23 1.97.109 0.15.109
42 1.95.109 0.06.109
48 1.63.109 0.08.109
65 1.98.109 0.02.109
34
In figure 3-16 the glutamic acid production by Brevibacterium divericatum in the optimal γ-PGA medium
with trypton and 100 g/L of glucose is visualized. The glutamic acid production starts after 48 hours. A
maximal concentration of 439 ± 15 mg/L of glutamic acid was obtained after 164 hours or approximately
7 days. A maximal productivity of 120 ± 57 mg/(L.day) glutamic acid was reached after 164 hours.
Figure 3-16: Glutamic acid production experiment – Brevibacterium divericatum in optimal γ-PGA production medium with
trypton and 100 g/L glucose (without glutamic acid)
In figure 3-17 the glutamic acid production by Brevibacterium divericatum in the optimal γ-PGA medium
with trypton and 1 mL/L TWEEN80 is visualized. The glutamic acid production starts after 141 hours. A
maximal concentration of 272 ± 52 mg/L of glutamic acid was obtained after 236 hours. A maximal
productivity of 124 ± 67 mg/(L.day) glutamic acid was reached after 236 hours.
Overview of glutamic acid production parameters
Table 3-2: Overview of glutamic acid production parameters
Maximal glutamate
concentration (mg/L)
Maximal productivity
(mg glu/(L.day))
Optimal γ-PGA medium
with trypton 67 ± 7 151 ± 11
Optimal γ-PGA medium
with trypton and 100
g/L glucose
439 ± 15 120 ± 57
Optimal γ-PGA medium
with trypton and 1
mL/L TWEEN80
272 ± 52 124 ± 67
35
Figure 3-17: Glutamic acid production experiment – Brevibacterium divericatum in optimal PGA production medium with
trypton and 1 mL/L TWEEN80 (without glutamic acid)
3.2.2 γ-PGA production by Bacillus licheniformis
Growth experiments
In figure 3-18 the growth curves of Bacillus licheniformis in optimal γ-PGA production medium with and
without citric acid can be found. This medium also contains 25 g/L glucose, 20 g/L glutamic acid as
carbon sources and 20 g/L glutamic acid and 6 g/L NH4Cl as nitrogen sources. The growth with citric acid
reaches an ODmax of 0.28 ± 0.05 while the ODmax without citric acid is 0.16 ± 0.01. It can therefore be
concluded that citric acid is important for the growth of Bacillus licheniformis.
In figure 3-19 the growth curves of Bacillus licheniformis in optimal γ-PGA production medium with and
without trypton and dextrin can be found. The growth in optimal γ-PGA production medium without
trypton reaches an ODmax of 0.21 ± 0.02, the ODmax with trypton is 1.49 ± 0.01 and the ODmax with trypton
and high dextrin concentration is 0.910 ± 0.001.
The addition of trypton has a positive influence on the growth of Bacillus licheniformis. Growth in a
medium with a high sugar concentration results in growth inhibition.
36
Figure 3-18: Growth experiment 1 Bacillus licheniformis
Figure 3-19: Growth experiment 2 Bacillus licheniformis
37
γ-PGA production experiments
γ-PGA-production was assessed in the optimal γ-PGA production medium with trypton, with trypton and
100 g/L glucose and with trypton and 1mL/L TWEEN80.
In figure 3-15 the γ-PGA production by Bacillus licheniformis in the optimal γ-PGA medium with trypton
is visualized. The γ-PGA production started after 18 hours. A maximal concentration of 1150 ± 159 mg/L
of γ-PGA was obtained after 212 hours. A maximal productivity of 753 ± 25 mg/(L.day) of γ-PGA was
reached after 28 hours. From table 3-3 can be concluded that the cell count reached its maximum after 43
hours: 5,97.109 ± 0,20.109 cells/mL which is assumed as final cell concentration. TAN concentrations in
the medium were followed up, but no TAN removal was detected. 45 ± 3% of the added glutamic acid
was removed after 212 hours and used for γ-PGA production and growth. The yield or the amount of γ-
PGA produced after 212 hours per amount of glutamic acid removed was 0.13 ± 0.02 mg γ-PGA produced
/mg of glutamic acid removed.
Figure 3-20: γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA production medium with trypton
Table 3-3: Cell count of the γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA production medium with
trypton
Hours Events/mL Standard error
0 3,00.107
43 5,97.109 0,20.109
91 2,80.109 0,19.109
38
In figure 3-21 the γ-PGA production by Bacillus licheniformis in the optimal γ-PGA medium with trypton
and 100 g/L glucose is visualized. The γ-PGA production started after 18 hours. A maximal concentration
of 1084 ± 184 mg/L of γ-PGA was obtained after 91 hours. A maximal productivity of 786 ± 124 mg/(L.
day) of γ-PGA was reached after 43 hours. From table 3-3 can be concluded that the cell count reached its
maximum after 43 hours: 5.89.109 ± 0.20.109 cells/mL. TAN and glutamic acid were followed up, 26 ± 2%
TAN was removed after 212 hours and 23 ± 2% of glutamic acid was removed and used for γ-PGA
production and growth. The yield or the amount of γ-PGA produced after 212 hours per amount of
glutamic acid removed was 0.20 ± 0.01 mg γ-PGA produced / mg of glutamic acid removed.
Figure 3-21: γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA production medium with trypton and 100
g/L glucose
Table 3-4: Cell count of the γ-PGA production experiment – Bacillus licheniformis in optimal γ-PGA production medium with
trypton and 100 g/L glucose
Hours Events/mL Standard error
0 3,00.107
43 5,89.109 0,20.109
91 0,98.109 0,07.109
In figure 3-22 the γ-PGA production by Bacillus licheniformis in the optimal γ-PGA medium with trypton
and 1 mL/L TWEEN80 is visualized. The γ-PGA production starts after 141 hours. A maximal
concentration of 61 ± 32 mg/L of γ-PGA was obtained after 212 hours. A maximal productivity of 13 ± 10
mg/(L.day) of γ-PGA was reached. 33 ± 7% of the added glutamic acid was removed and used for γ-PGA
production and growth. The yield or the amount of γ-PGA produced after 212 hours per amount of
glutamic acid removed was 0.010 ± 0.005 mg γ-PGA produced / mg of glutamic acid removed.
39
Figure 3-22: γ-PGA production experiment – Bacillus licheniformis in optimal PGA production medium with trypton and 1 mL/L
TWEEN 80
Overview of γ-PGA production parameters
Table 3-5: Overview of γ-PGA production parameters for production experiments with Bacillus licheniformis
Maximal
concentration
(mg γ-PGA/L)
Yield (mg γ-PGA
produced / mg of
glutamic acid removed)
Maximal
productivity
(mg γ-PGA/(L. day))
Optimal γ-PGA
medium with trypton 1150 ± 159 0.13 ± 0.02 753 ± 25
Optimal γ-PGA
medium with trypton
and 100 g/L glucose
1084 ± 184 0.20 ± 0.01 786 ± 124
Optimal γ-PGA
medium with trypton
and 1 mL/L
TWEEN80
61 ± 32 0.010 ± 0.005 13 ± 10
3.2.3 Co-culture of Brevibacterium divericatum and Bacillus licheniformis
γ-PGA production experiments
γ-PGA production was assessed using a co-culture of Brevibacterium divericatum and Bacillus
licheniformis in the optimal γ-PGA production medium with trypton, with trypton and 100 g/L glucose
and with trypton and 1mL/L TWEEN80.
In figure 3-23 the γ-PGA production of the co-culture of Brevibacterium divericatum and Bacillus
licheniformis in the optimal γ-PGA medium with trypton is visualized. The γ-PGA production started after
42 hours. A maximal concentration of 390 ± 3 mg/L of γ-PGA was obtained after 74 hours. A maximal
productivity of 286 ± 41 mg/(L.day) of γ-PGA was reached after 50 hours. From table 3-6 can be
40
concluded that the cell count reached its maximum after 66 hours: 1,45.109 ± 0,04.109 cells/mL which is
assumed as final cell concentration. The maximal glutamic acid concentration was 647 ± 69 mg/L.
Figure 3-23: γ-PGA production experiment – Bacillus licheniformis and Brevibacterium divericatum in optimal γ-PGA
production medium with trypton (without glutamic acid)
Table 3-6: Cell count of the γ-PGA production experiment – Bacillus licheniformis and Brevibacterium divericatum in optimal γ-
PGA production medium with trypton
Hours Events/mL Standard error
0 1,00.104
19 1,07.109 0,09.109
42 1,19.109 0,03.109
50 1,41.109 0,03.109
66 1,45.109 0,04.109
In figure 3-24 the γ-PGA production of the co-culture of Brevibacterium divericatum and Bacillus
licheniformis in the optimal γ-PGA medium with trypton and 100 g/L glucose is visualized. The γ-PGA
production started after 69 hours. A maximal concentration of 222 ± 27 mg/L of γ-PGA was obtained after
141 hours. A maximal productivity of 151 ± 60 mg/(L.day) of γ-PGA was reached after 69 hours. The
maximal glutamic acid concentration was 946 ± 211 mg/L.
In figure 3-24 the γ-PGA production of the co-culture of Brevibacterium divericatum and Bacillus
licheniformis in the optimal γ-PGA medium with trypton and 1 mL/L TWEEN80 is visualized. The γ-
PGA production started after 48 hours. A maximal concentration of 302 ± 28 mg/L of γ-PGA was
obtained after 69 hours. A maximal productivity of 169 ± 115 mg/(L.day) of γ-PGA was reached after 69
hours. The maximal glutamic acid concentration was 157 ± 32 mg/L.
41
Figure 3-24: γ-PGA production experiment – Bacillus licheniformis and Brevibacterium divericatum in optimal γ-PGA
production medium with trypton and 100 g/L glucose (without glutamic acid)
Figure 3-25: γ-PGA production experiment – Bacillus licheniformis and Brevibacterium divericatum in optimal γ-PGA
production medium with trypton and 1 mL/L TWEEN80 (without glutamic acid)
42
Overview of γ-PGA production parameters for co-culture
Table 3-7: Overview of γ-PGA production parameters for production experiments with co-culture of Bacillus licheniformis and
Brevibacterium divericatum
Maximal
concentration (mg/L)
Productivity
(mg/(L.day)
Optimal γ-PGA medium
with trypton 390 ± 3 286 ± 41
Optimal γ-PGA medium
with trypton and 100 g/L
glucose
222 ± 27 151 ± 60
Optimal γ-PGA medium
with trypton and 1 mL/L
TWEEN80
302 ± 28 169 ± 115
43
4 Discussion
4.1 Enrichment of a γ-PGA producing open microbial community in the RBC
configuration
The goal of this experiment was the production of γ-PGA by means of an open culture, as this would
avoid the use of pure cultures and therefore decrease the production costs of γ-PGA. One RBC was fed
with glutamic acid and one was fed without glutamic acid to select for glutamic acid independent γ-PGA
producing bacteria or for a co-culture of glutamic acid producers and glutamic acid dependent γ-PGA
producing bacteria. Glutamic acid independent γ-PGA producers or a co-culture are of great interest
because glutamic acid is a rather costly substrate ($2000/ton in bulk, (Xian Lyphar Biotech Co. 2014)).
Furthermore, problems such as limitation in volumetric oxygen mass transfer caused by the increase in
viscosity due to polymer accumulation are eliminated using RBC.
It is described in literature that different types of bacteria produce γ-PGA when they are in the early
stationary phase (Oppermann-Sanio and Steinbuchel 2002). To mimic this type of stress, the biofilm in the
RBC was subjected to a feast-famine regime. Furthermore, it is also known that several bacteria produce
γ-PGA to protect their cells from dehydration (Kimura, Tran et al. 2004, Bajaj and Singhal 2011). To
impose this type of stress and dehydrate the biofilm, the rotators were stopped for 15 minutes every hour.
Other stress factors inducing γ-PGA production are high salt concentrations and the presence of toxic
metal ions.
4.1.1 Biofilm formation and toxicity
RBC fed with glutamic acid
On average 55% of the added COD and 66% of the added nitrogen was removed. For every gram of
microbial biomass formed, 0.05 g N is required for the synthesis of proteins and nucleic acids and 2.5 g
of substrate COD is required for cell growth.
In table 4-1 can be seen that in total 12.23 g/L of COD was removed during the 56 operation days,
which would result in 4.89 g/L of biomass. From this calculation can be derived that only 0.24 g/L of
the removed nitrogen was assimilated, the remaining 2.28 g/L of nitrogen would then be removed via
other pathways. If the experiment were to be repeated the weight of the should be followed-up regularly to
follow-up the growth of the biofilm.
Table 4-1: COD and nitrogen concentration entering and leaving RBC 1 during 56 operation days
in out removed
COD (g/L) 21.17 8.94 12.23
N (g/L) 2.99 0.47 2.52
COD/N 7.08 19.0 4.85
C/N 3.25
44
The anoxic denitrification is the conversion of NO3− and NO2
− to gaseous nitrogen (N2). In a medium
containing a bulk DO level of 2 mg/L or more, a DO concentration of 0.1 mg/L or less in the center of the
biofilm is possible. Hence, conditions of limited oxygen supply can exist and therefore denitrification is a
possibility.
The free ammonia concentration was 0.09 mg NH3-N/L, which could result in a limited inhibition of the
NOB activity, but not of the AOB activity (Anthonisen, Loehr et al. 1976). For glutamic acid and γ-PGA
producers, this concentration of free ammonia should not be a problem since high concentrations of
ammonium are added to the optimal media. The existing free nitrous acid concentration (0.0018 mg
HNO2-N/L) does not result in inhibition of NOB or AOB activity.
RBC fed without glutamic acid
On average 3.8% of the added COD and 27% of the added nitrogen was removed. These limited removal
efficiencies and the bad biofilm formation, which can be seen in figure 3-12, show the bad growth of the
biomass in the RBC fed without glutamic acid. The only difference with RBC 1, is the absence of
glutamic acid in the influent of RBC 2 and a slightly lower C/N ratio. The C/N ratio of the influent of
RBC 1 is 3.25 and of the influent of RBC 2, C/N is 2.21. The presence of glutamic acid appears to have a
significant effect on the biofilm formation.
The composition of the influent of RBC 2 is comparable with the influent of the senior OLAND reactor, in
which oxygen-limited autotrophic nitrification/denitrification takes places. The only nitrogen source is
NH4+-N as (NH4)2SO4. As a carbon source NaHCO3 and no COD is added. In the master thesis of
De Wilde (2011-2012), the COD/N ratio was increased from 0 to 2 and also here the nitrogen and
COD removal efficiencies decreased.
In table 4-2 can be seen that in total 2.02 g/L of COD was removed during the 56 operation days,
which would result in 0.808 g/L of biomass. From this calculation can be derived that only 0.04 g/L of
the removed nitrogen was assimilated, the remaining 0,55 g/L of nitrogen would then be removed via
other pathways. Also here, conditions of limited oxygen supply can exist and denitrification is a
possibility.
Table 4-2: COD and nitrogen concentration entering and leaving RBC 2 during 56 operation days
in out removed
COD (g/L) 17.73 15.71 2.02
N (g/L) 2.88 2.28 0.59
COD/N 6.16 6.89 3.42
C/N 2.21
To assess possible ammonia toxicity, the level of free ammonia was calculated. The free ammonia
concentration was 0.39 mg NH3-N/L, which inhibits the NOB activity, but not the AOB activity
(Anthonisen, Loehr et al. 1976). This is visible in figure 3-10: the NOB activity increases if the TAN
concentration decreases. A possible alteration to the feed could be a decrease of the TAN concentration.
45
The existing free nitrous acid concentration (0.0006 mg HNO2-N/L) does not result in inhibition of NOB
or AOB activity.
4.1.2 γ-PGA formation
No γ-PGA was formed in the biofilm or in the effluent of both RBC. This could be due to several reasons
which are discussed in detail in the next paragraphs.
RBC fed with glutamic acid
a. Composition of the influent
From the experimental data can be deduced that very little of the glutamic acid in the influent actually
reached the reactor. From the TAN measurements can be concluded that 65% of the nitrogen originating
from glutamic acid was converted to ammoniacal nitrogen in the influent tank. This is probably due to the
non-sterile conditions in the influent tank. Bacteria growing in the influent tank could effectuate the
deamination of glutamic acid, resulting in ammonium production
Figure 4-1: The deamination mechanism of glutamic acid (1: glutamic acid, GDH: glutamate dehydrogenase, 2: α-keto glutarate)
To avoid this reaction, the influent was regularly refreshed and the influent tank was cleaned thoroughly.
This was nonetheless insufficient to prevent glutamic acid degradation. Also cooling of the influent could
help to minimize the bacterial activity. Autoclavation of the influent was not possible in practice due to
the large influent volume.
As glutamic acid is the monomer for the production of the polymer γ-PGA, the low glutamic acid
concentrations in the influent can explain why no γ-PGA production was measured. In figure 4-1 is
visualized that deamination results in α-keto glutarate. This compound alone cannot be used for γ-PGA
production (Figure 1-8). However, according to Bajaj and Singhal (2009) the addition of α-keto glutarate
in a glutamic acid containing medium can significantly increase the γ-PGA production.
The C/N ratio in the RBC fed with glutamic acid equaled 3.2. As this C/N ratio is lower than 5, the
production of proteins and polyaminoacids was favored over microbial sugar production. Nevertheless it
seemed that this ratio was insufficient to stimulate γ-PGA production. Therefore it could be a possibility to
use the same C/N ratio used in the optimal γ-PGA production media.
If the media used for growth of γ-PGA producing bacteria, such as Bacillus subtilis, are compared with the
feed that was used for supporting growth of the biofilm in the RBC, it is clear that the concentrations of
the carbon and nitrogen sources required for γ-PGA production by for example Bacillus subtilis are much
higher. The growing of a known γ-PGA producing bacterium, Bacillus licheniformis, in the RBC medium
did not result in γ-PGA production. In the optimal γ-PGA production medium the C/N ratio is 8 with as
carbon sources glucose, glutamic acid and citric acid and as nitrogen sources glutamic acid and NH4Cl
(Table 2-6). In literature it is described that citric acid is important for γ-PGA production but it was not
added because the main focus was on the use of cheap carbon sources (Yoon, Hwan Do et al. 2000).
46
Pure γ-PGA producing cultures produce γ-PGA to approximately a concentration of 20 g/L (Table 1-1). If
the influent concentrations would be 100 times lower compared to the optimal medium, one can expect a
γ-PGA concentration of 0,20 g/L. However, γ-PGA would be concentrated within the biofilm and
therefore the continuous harvest of the biofilm on the RBC would be easier compared to the conventional
aerobic batch processes applied. In this research, it was opted to use low influent concentrations to
develop a γ-PGA production method from nitrogen containing waste streams. Furthermore, these low
influent concentrations stimulate growth of γ-PGA producing bacteria with low nutrient requirements and
this would further reduce the cost of the production process.
b. Feast-famine regime
From the experimental data it can be concluded that no real feast-famine regime is obtained and therefore
probably no nitrogen/carbon storage occurred. In the 4 hour lasting cycle all the ammonia is removed, but
this is only the case during 15 minutes. Furthermore, there is still 4.88 mg organic N/L left and only 48%
of the easily degradable COD is removed.
Also for the open culture production of polyhydroxyalkanoates (PHA) the feast-famine strategy is applied.
Various micro-organisms use PHA as carbon/energy or reducing power storage material. Accumulation of
PHA by open cultures occurs under transient conditions mainly caused by intermittent feeding (Ma, Chua
et al. 2000). During the feast periods when there is an excess of external substrate, storage polymers are
formed. When all the external substrate is consumed, the stored polymer can be used as a carbon and
energy source. However, if external substrate is continuously present, growth becomes more important
over storage and the bacteria without the ability to produce storage molecules obtain a competitive
advantage over the bacteria with this ability (Salehizadeh and Van Loosdrecht 2004).
Johnson, Jiang et al. (2009) describes a culture enrichment for PHA using open sequencing batch reactors
based on a 12 hour batch cycle. In table 4-3 the influent characteristics for PHA and γ-PGA production in
feast-famine regime are compared. For PHA production a higher COD concentration is applied, but a
lower N concentration, which is normal since PHA is a means for bacteria to store carbon and energy,
while γ-PGA is a means to store nitrogen and carbon. However, the order of magnitude is the same. A
possible alteration could be applying a longer lasting batch cycle or decreasing the influent concentrations
to obtain real famine.
Table 4-3: Comparison of the influent concentration for PHA production in feast-famine regime and γ-PGA production in feast-
famine regime
Concentrations used for γ-
PGA production in RBC 1
Concentration used for PHA production
SBR (Johnson, Jiang et al. 2009)
8,52 g COD/d 34,56 g COD/d
0,9 g N/d 0,52 g N/d
c. Stress from dehydration
Another possible explanation for the absence of γ-PGA production is a limited dehydration stress.
However, the biofilm did look slimy which suggests the presence hydrophilic molecules, binding the
water to the biofilm to prevent dehydration (Figure 3-6). The presence of these extracellular polymeric
substances (EPS) suggests that the applied dehydration stress was sufficient.
Extracellular polymeric substances (EPS) exist out of a mixture of proteins, lipids, nucleic acids,
polysaccharides and other polymers excreted by micro-organisms (Sheng, Yu et al. 2010). Cells produce
47
the EPS layer to protect them against dewatering and harmful toxic substances (Sutherland 2001), but
cells can also use the EPS layer as an extracellular source of carbon, nitrogen or energy in conditions of
nutrient shortage (Sutherland 2001, Zhang and Bishop 2003).
Nutrient levels have a significant effect on EPS production and composition. EPS production and the
carbohydrate content of this EPS is increased when phosphorus is depleted (Hoa, Nair et al. 2003, Liu, Liu
et al. 2006). The C/N ratio influences the proteins to carbohydrates ratio of the EPS layer. At a C/N ratio
lower of 5, the EPS layer is high in proteins but low in carbohydrates. At a C/N ratio of more than 40, the
amount of protein decreases sharply (Bura, Cheung et al. 1998, Durmaz and Sanin 2001, Liu, Liu et al.
2006, Ye, Ye et al. 2011). The EPS which is detected are thus mainly proteins, but unfortunately not γ-
PGA.
d. Degradation of the formed γ-PGA by other bacteria
Another possibility is that the formed EPS and/or γ-PGA is degraded by other bacteria in the open culture
as a source of nitrogen and/or carbon. According to Akagi, Higashi et al. (2006) several enzymes such as
lipases and proteases are capable of degradation at a temperature of 37°C. It is possible that bacteria which
are located more deeply in the biofilm and are nutrient-limited excrete these enzymes and use the EPS and
potentially the γ-PGA for their maintenance.
RBC fed without glutamic acid
The limited removal efficiencies and bad biofilm formation was an indication that no γ-PGA would be
formed. Since glutamic acid was not present in the influent of RBC 2, there was a γ-PGA production
inducing factor less compared to the RBC 1. Other possible explanations for the absence of γ-PGA are
discussed in the next paragraphs.
a. Composition of the influent
No glutamic acid or other type of organic nitrogen was added to the feed of RBC 2 to enrich for glutamic
acid independent γ-PGA producing bacteria or for a co-culture of glutamic acid producers and glutamic
acid dependent γ-PGA producing bacteria.
If the media used for growth of glutamic acid independent γ-PGA producing bacteria, such as Bacillus
amyloliquefaciens, are compared with the feed that was used to support growth of the biofilm in the RBC,
it is clear that the concentrations of the carbon and nitrogen components required for γ-PGA production by
this type of bacteria are much higher and the medium is more complex (Cao, Geng et al. 2011, Zhang, Zhu
et al. 2012). The C/N ratio is higher than for the glutamic acid dependent strains: 17 in the case of Zhang,
Zhu et al. (2012) and 50 in the case of Cao, Geng et al. (2011). The C/N ratio in the feed of RBC 2 was 2,
which means that too much nitrogen was added compared to the amount of carbon. However, one would
expect that the COD would be degraded, and the nitrogen remained in the reactor. This was not the case:
neither the COD nor the TAN was completely degraded.
If the influent composition would be altered to have the same types of components and ratio between the
components but at lower absolute concentrations, it could start γ-PGA production. Also here, the addition
of citric acid has been proven to positively influence on the γ-PGA production (Zhang, Zhu et al. 2012).
Glutamic acid independent γ-PGA producing bacteria generally have a lower productivity than the
glutamic acid dependent strains. Bacillus amyloliquefaciens LL3 for example has a productivity of 0.70
g/L which is much lower than 20 g/L for glutamic acid dependent strains (Cao, Geng et al. 2011). If the
48
influent concentrations would be 100 times lower compared to the optimal medium, one can expect a γ-
PGA concentration of 0,007 g/L. Glutamic acid independent strains with a high production capacity have
also been isolated. Bacillus subtilis TAM-4 can produce 22.1 g/L γ-PGA in a medium containing fructose
as carbon source and ammonium chloride as nitrogen source (Shih and Van 2001). Optimization of the
growth conditions for this strain would therefore also result in a decrease of the production cost of γ-PGA.
b. Feast-famine regime
From the experimental data can be concluded that no feast-famine regime is obtained since the only 21%
of the COD is removed at the end of the cycle. It is remarkable that the COD concentration increases
again after 2 hours, since after 2 hours the 45% of the COD is removed. It appears that some cell lysis is
occurring after 2 hours.
c. Effect of technical errors
From the results and figure 3-12 can be concluded that the biofilm was not growing, even though the
reactor was re-inoculated after a technical error which caused complete dehydration of the biofilm.
4.1.3 Future experiments
Several alterations could be made to the parameters of the experiment to obtain production of γ-PGA. First
of all, changes to the influent could be made. The C/N ratio and the influent components should be based
on the C/N ratio and components of the optimal γ-PGA production media. Citric acid has to be added to
the influent to stimulate γ-PGA production. Also, a feast-famine regime has to be obtained. This can be
done by lowering the influent concentrations and maintaining the duration of the cycle at 4 hours or by
extending the cycle time to for example 6 hours and maintaining the influent concentrations. To stress the
bacteria, the reactor could be spiked with high salt concentrations since salt stress has been described to
also induce γ-PGA production. However, one has to be careful not to obtain a salt tolerant or halophilic
open culture in the RBC.
The initial goal to produce γ-PGA from nitrogen containing waste streams will be difficult since it is clear
that γ-PGA producing bacteria require specific growth circumstances and medium components, such as
citric acid and glycerol. It should however definitely be possible to obtain an enrichment of proteins in the
EPS layer. A possible application of this technique could be the production of single cell proteins in a
continuous mode.
4.2 Development of a γ-PGA producing co-culture
To establish a synthetic co-culture of a glutamic acid producing bacterium and a γ-PGA producing
bacterium, growth experiments were performed in different media, to obtain a final medium that would
support growth of both species. In a next stage of the research project, the productivity of these bacteria in
different media was analysed to assess whether co-cultivation was possible. In the final step, the bacteria
were grown together and the γ-PGA productivity was followed-up.
Co-cultivation would eliminate the necessity to use glutamic acid for γ-PGA production and thereby
reduce the production costs of γ-PGA, which would allow a broader application of this interesting
biopolymer.
49
4.2.1 Glutamic acid production by Brevibacterium divericatum
Growth experiments
From the first growth experiment can be concluded that the addition of trypton is necessary for the growth
of Brevibacterium divericatum (Figure 3-13). Trypton is an enzymatic digest of casein used as a nitrogen
source in culture media. Casein is the main protein of milk and a rich source of amino-acid nitrogen
especially tryptophane.
In the second growth experiment it is shown that Brevibacterium divericatum shows good growth in the
optimal γ-PGA production medium with trypton (Figure 3-14). The high sugar concentration results in
growth inhibition due to osmotic stress. However, according to Nadeem, Niaz et al. (2011), high sugar
concentration also results in high glutamic acid production.
Glutamic acid production
Based on the results of the growth experiments, it was decided to test the glutamic acid productivity in
three media: the optimal γ-PGA production medium with trypton, in the optimal γ-PGA production
medium with trypton and high sugar concentration and in the optimal γ-PGA production medium with
trypton and 1 mL/L TWEEN80. TWEEN80 was chosen based on the research of Jyothi, Sasikiran et al.
(2005) in which it was added to cassava starch residues on which Brevibacterium divericatum showed
significant glutamic acid production.
From table 3-2 can be concluded that the maximal concentration was highest in the case of the optimal γ-
PGA medium with trypton and 100 g/L glucose (439 ± 15 mg/) and the lowest in the case of the optimal γ-
PGA medium with trypton (67 ± 7 mg/L). A potential function of L-glutamate excretion is carbohydrate
storage during periods when sugar is in excess and cell growth is inhibited by the absence of biotin. The
excreted L-glutamate might then be used as a carbon source when biotin becomes available once more
(Nakamura, Hirano et al. 2007). It has also been described that overproduction of glutamic acid occurs
when the membrane tension alters under influence of detergents. However, according to Kataoka,
Hashimoto et al. (2006) TWEEN80 does not appear to induce overproduction of glutamic acid, while
TWEEN40 and TWEEN60 do appear to have this effect. These findings are in contrast with the research
done by Jyothi, Sasikiran et al. (2005). A future experiment could be to use of TWEEN40 or TWEEN60
instead of TWEEN80 to assess the glutamic acid production. The limited glutamic acid production in the
optimal γ-PGA medium with trypton is probably due to the absence of a factor inducing glutamic acid
overproduction and the absence of a factor altering the membrane tension, which could explain why no
glutamic acid is excreted extracellular.
Comparison with previous experiments
If the obtained maximal glutamic acid concentration and maximal productivity are compared with results
in literature (Table 1-2), it stand out that the results obtained in literature are 60 or more times higher for
Corynebacterium glutamicum. Khan, Mishra et al. (2005) obtained final glutamate concentrations of 25,1
g/L in a medium containing 50 g/L glucose and 5 g/L urea as main nutrients. In this research however, pH,
foaming and dissolved oxygen were controlled in a batch fermentor which would explain why higher
glutamic acid production is obtained. No previous research has been done with the strain used in this
research (Brevibacterium divericatum NCIM 2634). From the obtained results can be concluded that this
strain is not interesting for industrial production since the produced glutamic acid concentrations are too
low.
50
4.2.2 γ-PGA production by Bacillus licheniformis
Growth experiments
In the first growth experiment Bacillus licheniformis was grown in optimal γ-PGA production medium
with and without citric acid (Figure 3-18). Citric acid is a rather expensive component ($900/ton,
(Weifang Tenor Chemical Co. 2014)) which is why its necessity for growth of Bacillus licheniformis was
tested. From this experiment was concluded that citric acid is important. Yoon, Hwan Do et al. (2000) also
stresses the importance of citric acid for the production of γ-PGA, since it acts as one of the precursors for
polymer production.
Since the goal was the development of a medium suitable to support growth of both Brevibacterium
divericatum and Bacillus licheniformis, the growth of Bacillus licheniformis was tested in the same media
as Brevibacterium divericatum in the second experiment and similar results were obtained (Figure 3-19).
Also here, trypton definitely has a positive impact on the growth of Bacillus, but its presence is not
essential for growth. The high sugar concentration resulted in growth inhibition of B. licheniformis due to
the osmotic stress high sugar concentrations induce.
Production experiments
Based on the results of the growth experiments, it was decided to test the γ-PGA productivity in three
media: the optimal γ-PGA production medium with trypton, in the optimal γ-PGA production medium
with trypton and high sugar concentration and in the optimal γ-PGA production medium with trypton and
1 mL/L TWEEN80. These media are the same ones used to test for glutamic acid production by
Brevibacterium divericatum.
From table 3-5 can be concluded that both the optimal γ-PGA medium with trypton (maximal
concentration 1150 mg γ-PGA/L) and the optimal γ-PGA medium with trypton and 100 g/L glucose
(maximal concentration 1084 mg γ-PGA/L) support production of γ-PGA. The productivity in the optimal
γ-PGA medium with trypton and 100 g/L glucose is slightly higher than in the optimal γ-PGA medium
with trypton: 786 ± 124 mg γ-PGA/(L.day) versus 753 ± 25 mg γ-PGA/(L.day).
The effect of TWEEN80 could have been two-fold. According to Ashiuchi, Shimanouchi et al. (2004) the
γ-PGA production activity of Bacillus subtilis subsp. chungkookjang was completely lost after the
addition of TWEEN80 due to the localization of the γ-PGA synthesis complex: TWEEN80 resulted in the
solubilisation of the membranous enzymatic system. This suggested that the γ-PGA synthetic complex had
to be associated with the membrane to remain in an active form. According to Wu, Xu et al. (2008)
however, the γ-PGA production by Bacillus subtilis CGMCC 0833 was stimulated due to an increase in
cell permeability which facilitated the uptake of extracellular substrates and the secretion of γ-PGA.
In the experiment with the TWEEN80 containing medium, only very low concentrations of γ-PGA were
produced: 61 ± 32 mg γ-PGA/L, which suggests that the activity of the γ-PGA synthetic membrane
complex was lost after addition of TWEEN80. Furthermore the concentration of γ-PGA dropped to zero at
the end of the experiment which would also support this hypothesis.
Comparison with previous experiments
Bajaj and Singhal (2009) conducted research with the same strain and a similar medium. Concentrations
of 25 g/L and more were obtained. A first difference is the use of glycerol as primary carbon source
instead of glucose, a second difference is the isolation method of γ-PGA. Bajaj and Singhal (2009) first
51
centrifuged the diluted culture broth, poured the γ-PGA containing supernatant in 4 vol of methanol and
kept it for 12 hours at 4°C and centrifuged again. This isolation method will result in a higher recuperation
of the produced γ-PGA, compared to a simple 0.22 µm filtration as was done in this research. In table 1-1
an overview of the kinetic parameters of some γ-PGA producing bacteria are shown. The obtained γ-PGA
concentrations reached are 10 to 20 times higher than in this research, while the bacteria were also grown
in batch without any parameter control.
It was also remarkable that the production of γ-PGA was unreliable and irregular. The starting point of the
production was different and there was a very large variation in the amount of γ-PGA produced. Bacillus
licheniformis NCIM 2324 is therefore not a good strain to use for industrial production.
4.2.3 Co-culture of Brevibacterium divericatum and Bacillus licheniformis
A co-culture of Brevibacterium divericatum and Bacillus licheniformis was grown in the optimal γ-PGA
production medium with trypton, in the optimal γ-PGA production medium with trypton and high sugar
concentration and in the optimal γ-PGA production medium with trypton and 1 mL/L TWEEN80. None of
these media contained glutamic acid.
From table 3-7 can be concluded that there is γ-PGA in all three media which means that Bacillus
licheniformis can use the glutamic acid produced by Brevibacterium divericatum for γ-PGA production.
This type of collaboration between these bacteria had not yet been described in literature.
From the production experiments with Brevibacterium divericatum and Bacillus licheniformis, one can
expect the highest γ-PGA production in the optimal γ-PGA production medium with trypton and high
sugar concentration for the co-culture, since there is both glutamic acid production and γ-PGA production
in this medium. From table 3-7 however can be concluded that both the highest γ-PGA concentration and
productivity is reached in the optimal γ-PGA production medium with trypton but without 100 g/L
glucose or 1 mL/L TWEEN80 (resp. 390 ± 3 mg γ-PGA/L and 286 ± 41 mg γ-PGA/(L.day)). The
obtained concentrations of γ-PGA are lower in co-culture, compared to production by pure culture of
Bacillus licheniformis, where the maximal obtained concentration in the optimal γ-PGA medium with
trypton is 1150 mg γ-PGA/L and the maximal productivity is 753 ± 25 mg γ-PGA/(L. day).
This could be due to glutamic acid shortage: at the moment where Bacillus licheniformis enters the
stationary phase, the glutamic acid concentration drops to zero. However, there is residual glutamic acid at
the end of the growth experiment with the co-culture experiment. Furthermore it stands out that the
maximal glutamic acid concentration obtained in the co-culture is higher than the pure culture of
Brevibacterium divericatum. It could increase γ-PGA production if Bacillus licheniformis were added to
the culture when Brevibacterium divericatum has reached maximal productivity for glutamic acid.
It is also remarkable that the γ-PGA concentration drops to zero in the optimal γ-PGA production medium
with trypton and 1mL/L TWEEN80 after 141 hours. This is again a conformation of the hypothesis that
the membrane-bound γ-PGA synthetic complex is deactivated by the addition of this detergent.
52
Table 4-4: Comparison of the maximal glutamic acid concentration between a pure culture of Brevibacterium divericatum and a
co-culture
Maximal glutamate concentration
in pure culture of Brevibacterium
divercatum (mg glutamic acid/L)
Maximal glutamate concentration
in co-culture (mg glutamic acid/L)
Optimal γ-PGA medium
with trypton 67 ± 7 647 ± 69
Optimal γ-PGA medium
with trypton and 100
g/L glucose
439 ± 15 946 ± 211
Optimal γ-PGA medium
with trypton and 1
mL/L TWEEN80
272 ± 52 157 ± 32
4.2.4 Future experiments
In these series of experiments a proof-of-concept is given: production of γ-PGA is possible with a co-
culture. The most interesting medium is the optimal γ-PGA production medium with trypton, since here
the glutamic acid is replaced with the addition of Brevibacterium divericatum without the addition of for
example higher glucose concentrations or TWEEN80. However, in a pure culture of only Brevibacterium
divericatum the glutamic acid production in the optimal γ-PGA medium with trypton was very limited
probably due to the absence of a factor inducing glutamic acid overproduction and the absence of a factor
altering the membrane tension, which could explain why no glutamic acid is excreted extracellular. A
possible alternative research line could be to use TWEEN40 or TWEEN60.
The use of other strains could result in higher production rates. For the glutamic acid production,
Coynebacterium glutamicum is a more promising option because this type of bacterium is already used in
industry for glutamic acid production. For the γ-PGA production, several strains of Bacillus subtilis are
described in literature to produce higher concentrations of γ-PGA, compared to Bacillus licheniformis.
Genetic engineering could also possibly increase productivity and decrease the production costs of γ-PGA.
Ashiuchi, Nawa et al. (2001) cloned the pgsBCA gene in E. coli which enzymatically synthesized
elongated γ-PGA in the presence of ATP and D-glutamate. Also coryneform bacteria have been
genetically modified to produce γ-PGA. These bacteria have some important advantages over for example
E. coli. First of all, they are generally recognized as safe. Coryneform bacteria also have the ability to
produce high levels of glutamic acid, which can be used for γ-PGA production. Furthermore, the
fermentation characteristics are well-known and finally, coryneform bacteria are also gram-positive, like
Bacillus, so the rigid structure of their cell walls seems more suitable for the display of PgsBCA for γ-
PGA production (Sung, Park et al. 2005).
Further research will also have to focus on the improvement of the kinetic parameters for γ-PGA
production with co-culture by determining the optimal pH, optimal DO level and optimal temperature and
maintaining these parameters throughout the production process. First, the conditions of batch processes
have to be optimized and controlled. In a next step, a continuous system, such as a chemostat, can be
developed.
53
4.3 Final conclusion
The first approach for γ-PGA production, using an open culture in RBC configuration and low influent
concentrations, appears to be an unpromising technique. It should be possible to obtain an enrichment of
proteins in the EPS layer and possibly also γ-PGA. However, γ-PGA will probably only be present in low
concentrations and in a complex mixture which would require rather complicated downstream processing.
A possible application could be the production of single cell proteins in a continuous mode.
The use of a co-culture appears to be a more realistic approach. However, other bacterial strains or
genetically engineered strains have to be used. For the glutamic acid production, Coynebacterium
glutamicum is a more promising option than Brevibacterium divericatum and for the γ-PGA production,
several strains of Bacillus subtilis are described in literature to produce higher concentrations of γ-PGA,
compared to Bacillus licheniformis.
If the same concentrations and same productivity can be obtained with the co-culture compared to the pure
culture, which is not unrealistic since the production parameters still have to be further optimized,
glutamic acid can be removed from the feed. Glutamic acid costs $2000 per ton and for the production of
1 g/L γ-PGA about 1 g/L glutamic acid is required. The production cost of one ton of γ-PGA
($500 000/ton) would therefore decrease with $2000 dollar/ton.
To conclude, the co-culture and the use of genetically engineered coryneform bacteria appear to be the
two most promising techniques to decrease the production costs of γ-PGA.
54
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Appendices
Appendix 1: Standard curves for SEC-HPLC
Standard curve for poly-L-glutamic acid (Mw between 50 000 and 100 000 Da)
The retention time of low molecular weight poly-L-glutamic acid was 6,554 ± 0,013 minutes. A standard
curve was prepared for concentrations between 5 mg/L poly-L-glutamic acid and 200 mg/L poly-L-
glutamic acid.
Area [mAU*min] = 0,3602. concentration [mg/L low Mw poly-L-glutamic acid] + 0,1018 (R² = 0,9986)
Standard curve for poly-L-glutamic acid (Mw > 1 000 000 Da)
The retention time of high molecular weight poly-L-glutamic acid was 6,13 ± 0,075 minutes. A standard
curve was prepared for concentrations between 5 mg/L poly-L-glutamic acid and 500 mg/L poly-L-
glutamic acid.
Area [mAU*min] = 0,3642.concentration [mg/L high Mw poly-L-glutamic acid] + 0,6661 (R² = 0,9994)
61
Molecular weight ladder
The Gel Filtration Markers Kit for Protein Molecular Weights between 12 000-200 000 Da resulted in a
linear relationship for molecular weights between 150 kDa (alcohol dehydrogenase) and 2 000 kDa (blue
dextran).
Molecular weight [kDa] = 22672,6 . retention time [min] + 18 715 (R² = 0.9981)
Blue Dextran
Thyroglobulin
ApoferritinAlcohol
dehydrogenase0
500
1000
1500
2000
2500
6,2 6,4 6,6 6,8 7
Mo
lecu
lar w
eig
ht
(kD
a)
Retention time (min)
62
Appendix 2: Chromatograms of the open culture experiment RBC 1
Chromatogram of the influent on 01/11/2013 of RBC 1
63
Chromatogram of the effluent on 01/11/2013 of RBC 1
64
Chromatogram of the supernatans of the biofilm on 01/11/2013 of RBC 1
65
Appendix 3: Chromatograms of the open culture experiment RBC 2
Chromatogram of the influent on 01/11/2013 of RBC 2
66
Chromatogram of the effluent on 01/11/2013 of RBC 2
67
Chromatogram of the supernatans of the biofilm on 01/11/2013 of RBC 2