Zymomonas mobilis

WP3: leavening trials in model and real doughs for comparison purposes

Subtask 3.1:

• use of Zymomonas alone
• addition of glucose, fructose or sucrose to model dough formulation
• addition of hydrolytic enzymes (glucoamylase…)
• process parametrization (CO2 production, dough volume increase, HPLC determination of sugars, organic acids and ethanol time course)

Subtask 3.2:

• use of the association Zymomonas- Lactobacillus
• trials in model dough (flour-water) and in real dough formulation
• pinpoint the best combination in terms of cell concentration
• process parametrization (CO2 production, dough volume increase, HPLC determination of sugars, organic acids and ethanol time course)
• evaluation of the new microbial association during dough leavening

Zymomonas mobilis:

a unique microorganism

Doctorate Course in Food Systems

XXXI Cycle

OBJ

last century studies resume about microorganism Zymomonas mobilis and its possible application in the industrial field

Focus on:

history of isolation and identification of microorganism

Z. mobilis phenotypical description

uses of the microorganism in agrifood, biotechnological and energetical fields

Isolation and identification of the genus Zymomonas

At the end I will show the future developments about my PhD project, related to the use of Zymomonas mobilis in the production of leavened baked food

1912:

Barker and Hillier

isolated and purified a bacterial strain which caused cider sickness

1924-1931:

Lindner studied the

aguamiel fermentation to

obtain Pulque, a Mexican

alcoholic beverage (4-6% ethanol)

strong aroma and flavour, gas formation, reduction of sweetness and development of turbidity

discovered the organism involved in this fermentation process

1936:

Kluyver and van Niel created the genus Zymomonas

a bacterium which he called

Termobacterium mobile

Didn’t give a latin taxon name to this new organism

a motile bacterium able to ferment sucrose, fructose and glucose to ethanol and CO2

1937:

Shimwell isolated a strain causing dense turbidity and unpleasant odor from the beer and in particular from the brewery yards

"for polarly flagellated bacteria causing alcoholic fermentation"

[Barker B.T.P. and Hillier V.F. 1912. Cider sickness. J. Agric. Sci. 5:67-85]

[Lindner P. 1931. Termobacterium mobile, ein mexikanisches bakterium als neues Eisauerungsbakterium fur Rubenshnitzel, Z. Ver. Dsch. Zuckerind. 81:25-36]

Zymomonas mobilis

[Kluyver A.J. and van Niel K. 1936. Prospects for a natural classification of bacteria. Zentralb. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2 94:369-403]

In 1950 he called it

Saccharomonas (anaerobia var immobilis or lindneri - Pseudomonas lindneri)

to indicate the physiological resemblance to Saccharomyces and the morphological affinity with Pseudomonas

1951-1956:

Millis demonstrated that the cider sickness organism was Zymomonas: Z. anaerobia var. pomaceae, differed from Z. mobilis in only two features

[Shimwellv J.L. 1937. Study of a new type of beer disease bacterium (Achromobacter anaerobia) producing alcoholic fermentation of glucose. J. Inst. Brew. London 43:507-509]

[Shimwellv J.L. 1950. Saccharomonas, a proposed new genus for bacteria producing a quantitative alcoholic fermentation of glucose. J. Inst. Brew. London 56:179-182]

[Millis N.F. 1956. A study of the cider-sickness bacillus-a new variety of Zymomonas anaerobia. J. Gen. Microbiol. 15:521-528]

1965:

Ault recognized Zymomonas as a serious contaminant in beers

heavy turbidity and unpleasant odor of rotten apple due to acetaldehyde and H2S

[Ault R.G.1965. Spoilage bacteria in brewing-A review. J. Inst. Brew. London 71:376-391]

1977:

Swings and De Ley compared approximately 40 different strains of Zymomonas

all the strains belong to a single species: Zymomonas mobilis with two subspecies:

Zymomonas mobilis subsp. mobilis

Isolated from Pulque or palm wine and used for industrial ethanol production

Zymomonas mobilis subsp. pomaceae

Responsible for beer and cider spoilage

[Swings J. and De Ley J. 1977. The biology of Zymomonas. Bacteriol. Rev. 41:1-46]

Z. mobilis phenotypical description

Morphology and growth

Metabolism/physiology

- Gram-negative rods, 2 to 6 μm long, 1 to 5 wide

- Either motile or nonmotile, motility can be easily lost

- Catalase positive, oxidase negative

- Facultative anaerobe

- High ethanol concentrations tolerance (up to 13% w/v)

- High substrate concentrations tolerance (up to 30% glucose)

CARBOHYDRATE TRANSPORT

Z. mobilis catabolizes only three carbon sources:

GLUCOSE and FRUCTOSE

SUCROSE

Enter via the common uniport system (GLF-glucose/fructose transporter) or are converted into sorbitol and gluconolactone

Three sucrose-splitting enzymes:

- an extracellular levansucrase (SacB): forming glucose and levan, a fuctose oligosaccharide

- an extracellular invertase (SacC): forming glucose and fructose

- an invertase (SacA), whose role is unclear

Z. mobilis is unique among prokaryotes for its facilitated diffusion system to trasport glucose and fructose without consumption of metabolic energy

Metabolism of Zymomonas

Zymomonas grows best at 25 - 30 °C; above 30 °C some strains don’t grow and growth at 40 °C is rarely observed. Growth is slow at 15 °C and absent at 4°C

Most strains are able to grow between pH 3.8 and 7.5 and show good acid tolerance

subject of several studies in the last four decades

ENTNER-DOUDOROFF PATHWAY

Zymomonas mobilis produces ethanol and CO2 from few sugars via the ENTNER-DOUDOROFF pathway in conjunction with the enzymes pyruvate decarboxylase and alcohol dehydrogenase

It was the first example of an anaerobic organism using a pathway occurring mainly in strictly aerobic bacteria

The Krebs cycle is knocked off and serves only for biosynthetic purposes

Up to 1 µmol of glucose is converted to ethanol and carbon dioxide per min per mg of cell protein, with the production of only one net mol of ATP per mol of glucose consumed

Embden-Meyerhof-Parnas:

1 GLUCOSE 2 ATP, 2 Pyruvate

Entner-Doudoroff

1 GLUCOSE 1 ATP, 2 CO2, 2 Ethanol

Rate-limiting steps: conversion of

glucose-6-phosphate to 6-phosphogluconate and

of 3-phosphoglycerate to 2-phosphoglycerate

Pyruvate decarboxylase generally present in yeasts

Present in Z. mobilis

Catalyzes the non-oxidative decarboxylation of pyruvate to produce acetaldheyde and carbon dioxide

Phosphofructokinase and hexokinase (key regulatory enzymes in glycolysis) are absent

Two alcohol dehydrogenase isozymes catalyze the reduction of acetaldehyde to ethanol with the oxidation of NADH+

Adh I: zinc dependent

Adh II: contains iron

Future perspectives – PhD project

Proposed research:

• evaluates the possibility of creating a new area of leavened bakery products avoiding the addition of baker’s yeast (Saccharomyces cerevisiae)

Commercialization of yeast-free baked goods will successfully respond to the increasing incidence of adverse effects due to yeast ingestion in Western population

Ethanol production

S. cerevisiae is used all over the world as the major ethanol-producing microorganism

WP1: Zymomonas mobilis biomass production

• screening among different strains for biomass growth yield and determination of CO2 production
• formulation of a food-grade yeast-free culture medium
• process parametrization to identify the best biotechnological conditions to optimize cell production and fermentation performance

Main goals of the project:

Levan formation

WP2: Lactobacillus sanfranciscensis biomass production

• screening among different strains
• formulation of a food-grade yeast-free culture medium
• set-up of the biotechnological condition to produce suitable amount of biomass
• process parametrization to identify the best biotechnological conditions to optimize cell production and fermentation performance

Disadvantages:

Set up of an economically feasible biotechnological process to obtain cells of Z. mobilis and L. sanfranciscensis in high yield

Investigation on the physiological and technological performance of Zymomonas alone or in association with L. sanfranciscensis

Levan:

• extracellular polysaccharide composed solely by fructose units,
• produced by Z. mobilis from the transfructolization reaction catalyzed by the levansucrase enzyme that uses sucrose as substrate

- high aeration cost to produce biomass

- higher biomass conversion rate Yx/s respect to ethanol conversion rate Yp/s

- low tolerance to temperature and ethanol

Development of leavened baked products

Baking, sensorial tests and technological analysis will be performed to complete the research and the description of the products

WP4: dough leavened production

• production of one bread-like and one sweet (i.e. croissant) leavened product
• evaluation of the influence of the other ingredients (NaCl, fats..) on fermentation performance
• process parametrization
• determination of the rheological properties employing technological tests such as rheofermentometer as well as image analysis and NIR spectroscopy
• shelf-life tests, taking into account both microbial and technological deterioration of the products during storage time
• evaluation of the possibility of developing a starter culture ready to use by the consumer: identification of the best production and storage conditions of the obtained biomass

Zymomonas mobilis has emerged as a potential alternative to the yeast

Used in:

- food industry as: fructose source, thickening, gelling and suspending agent, color and flavor vehicle;

- medicine as: hypo-cholesterol, immune modulator and anti-inflammatory agent

Advantages:

WP5: process economics

• operating costs (chemicals, use of steam, water and electricity) estimation using process simulators (BioPro and SuperPro Designer®) initially for a pilot plant bioreactor and up to a 1 m3 production plant to produce Zymomonas biomass with good leavening ability
• work out if the production cost of a Zymomonas only or Zymomonas-Lactobacillus leavened good will lead to a competitive sale price, taking as benchmark a gluten-free baked product

It can be an economically valuable sub-product in alcohol production

Literature shows lots of papers concerning ethanol production from Z. mobilis

- high specific sugar uptake rate and ethanol yield

- high ethanol tolerance

- low biomass conversion rate Yx/s

- does not require controlled addition of oxygen

- amenability to genetic improvement

[de Oliveira et al. 2007. Biochem. Eng. J. 37:177-183]

The first report on the great potentialities of Z. mobilis for ethanol production was published by an Australian group of scientists in 1980

Assessed the effects of the carbohydrates sources, sugar concentration, fermentation time and the influence of the medium in levan and biomass production

[Rogers P.L. Lee K.J. and Tribe D.E. 1980. High productivity ethanol fermentations with Zymomonas mobilis. Process. Biochem. 15:7-11]

Biomass: 0.857 g/L

Levan: 21 g/L

Different substrates

Solutions example

Problems in application

Low-cost substrates

Ethanol yield

In this minireview, Sprenger analyzed Zymomonas carbohydrate uptake systems up to all carbon flux to ethanol, CO2 and by-products

Reported the possibility to circumvent the problems about ethanol production with:

Authors focused on different substrates industrially available to produce ethanol and on strategies for strain improvement through genetic techniques

[Sprenger G.A. 1996. FEMS Microbiol. Lett. 145:301-307]

Authors focused on development of fermentation technology employing hydrolysed waste starch (HWS) stream and involved a comparison of batch culture ethanol production by Z. mobilis and S. cerevisiae

- genes transfer to other organisms

- genetic manipulation of Z. mobilis

In this review, Bai et al., focused on ethanol fermentation technologies from sugar and starch feedstock, critically reviewing some technical and economic key challenges

Z. mobilis produces less biomass than S. cerevisiae and more carbon is funneled to ethanol fermentation

R-plasmid was transfered from E. coli or Pseudomonas by conjugation

An artificial operon was constructed by combining pdc and adhII genes and introduced in E. coli generally with good yields

Other substrates

Glucose

HWS: obtained after separation of the high-value gluten and the main starch fraction

it can provide a low-cost substrate for fuel ethanol production

Cell converts 98% of glucose in ethanol and CO2

Other microorganisms were modified as: Lb. casei, K. planticola, B. subtilis

Fructose

Different renewable resources were tested for ethanol production: maize, potatoes, wheat, milo, cassava…

Efficiency of Z.m. decreases: to overcome that, mutagenic techniques are used especially to broaden substrate range

Highlighted the gap between academic research and industry: many proposed technical developments are not appliable to large scale ethanol production

Ethanol yield is lower (90%) than glucose

formation of byproducts (mannitol, glycerol, sorbitol, acetaldehyde, lactic and acetic acid…)

responsible for the reduction in ethanol yield

Ethanol yield: 97% of the theoretical yield while S. cerevisiae only 90-93%

Both microorganisms were found to be successful in fermenting the HWS supplemented media

1. Effects of sucrose concentration

2. Use of sugar cane syrups (cheaper substrate)

(sucrose concentration: 250 g/L, time: 24h)

Recombinant strains used:

• Z. mobilis CP4 encoding xylose assimilation and pentose phosfate pathway enzymes yielded 86% of theoretical yield
• Z. mobilis ATCC39676 engeneered with plasmid pZB206 grew on arabinose and produced ethanol at 98% of theoretical yield
• Z. mobilis with plasmid pZY507 is useful for production of ethanol from mannitol

Sucrose

Ethanol fermentation with Z. mobilis

Sugarcane and beet molasses

Two different problems:

- the cleavage of the sucrose molecule with the formation of free fructose, oligosaccharides and higher polymers of fructose and levan

- the direct oxidation of glucose linked to reduction of fructose to form sorbitol, polimerization of fructose to polyfructooligosaccharides

cheapest feedstock for ethanol production

Some components can cause growth and fermentation inhibition

Membrane technology can be applied to improve the use for ethanol production

Disadvantages: specific substrate spectrum (only three sugar); growth on sucrose is accompanied by the formation of levan and sorbitol with decrease in ethanol yield

Ethanol fermentation industry often uses heterogeneous raw materials rather than pure glucose

Z.m. had greater productivity and higher yields than industrial strain of S. c.

36 g/L in 11 h

39 g/L in 9 h

Final ethanol

concentration

98% - 90% of theoretical maximum

Levan : 15 g/L, Biomass: 2.366 g/L

Optimum condition: Sucrose: 250g/L,

Ferm time: 24h

Chiara Mapelli

Dough-leavening agent

Z. mobilis provides an equimolar mixture of ethanol and CO2 and theoretically, as for Saccharomyces cerevisiae, the gas evolved can be used to leaven a dough

Could the CO2 produced by Z. mobilis leaven a dough ?

Yes! But....

In dough with no added sugar the principal fermentable sugar is maltose liberated from the starch by amylases

Thanks for your attention!

Limitation in use of Zymomonas mobilis as dough leavening agent is represented by its inability to use starch and maltose as carbon source

ALTERNATIVES?

Chiara Mapelli

ADDITION, into dough formulation, of GLUCOSE, FRUCTOSE OR SUCROSE

The use in bread-making has rarely reported

ADDITION, into dough formulation of ENZYMATIC PREPARATIONS that can liberate glucose from starch

SET-UP of an UNCONVENTIONAL MICROBIAL ASSOCIATION between Zymomonas mobilis and Lactobacillus sanfranciscensis

Lactobacillus sanfranciscensis

About..Lb. sanfranciscensis

Journal Club, Academic Year 2015-2016

Heterofermentative Lactic Acid Bacterium (LAB)

It is the most frequently Lactobacillus isolated from the sourdough

Cleavage of maltose via a maltose phosphorylase

More over its metabolic activities imparts positive features like production of aromatic compounds and extended shelf life of leavened products

Zymomonas mobilis

Biotechnological innovation

L. sanfranciscensis hydrolyzes maltose, than keeps one mole of glucose at intracellular level and releases the other one in the medium that could be used by Zymomonas mobilis

Up to now no investigations have been carried out or reported in the literature about use of an unconventional microbial association between Zymomonas and Lactobacillus sanfranciscensis

There is only an oral presentation at the 6th “Sourdough symposium” given by Dr. Musatti A. about promising initial results of experimental tests

PhD

Assessed the applicability of using Z. mobilis as alternative agent to S. cerevisiae in baking

Increase the variety of bakery goods

optimizing Z. mobilis biomass growth yield

Evaluated the growing performance of two different strains in a culture medium lacking yeast extract

Investigated the use of Z. mobilis in dough as alternative to the commercial S. cerevisiae

Z.m.

S.c.

• Z. mobilis and S. cerevisiae produce the same volume of CO2 from a given level of glucose

• Leavening ability of Z. m. per cell weight, related to speed of CO2 production, was double than yeast

• Ethanol productivity of Z. m. and S. c. were 2.53 and 0.87 (g EtOH/g cell mass as dry basis/h)

creating a new area of leavened bakery products avoiding the addition of baker’s yeast in response to the increasing incidence of adverse effects due to yeast ingestion

S.c.

Evaluated effects of NaCl and sucrose on leavening ability

Z.m.

Z. mobilis, when glucose is present, is able to rise a dough with similar or higher gas production rates and total amount of gas evolved to S. cerevisiae

The addition of NaCl to the dough was inhibitory and reduced the leavening ability in both, particularly in Zymomonas

Application

Journal Club, Academic Year 2015-2016