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