Gram negative aerobic bacteria such as Acetic Acid Bacteria, which include Acetobacter and Gluconobacter , have historically caused significant problems to brewers. Although incidences of spoilage have recently reduced as a result of improvements in beer packaging, these bacteria are still a concern in dispense systems in pub breweries, public houses and cask conditioned beers. Gram negative facultative bacteria of the genus Zymomonas can spoil primed cask conditioned beer and cider. There is a wide range of Enterobacteraeceace which are found within brewery environments and they serve as indicator microorganisms for hygiene and sanitation. Gram negative strictly anaerobic bacteria such as Pectinatus and Megasphaera have recently emerged as a significant threat due to the improvement in reduction of oxygen levels in beer and an increase in production of unpasteurised beer. Pectinatus and Megasphaera are sensitive to routine cleaning agents used in breweries, but they can survive and proliferate in biofilms eventually causing spoilage of beer. This review focuses on Gram negative aerobic, facultative anaerobic and strictly anaerobic brewery related spoilage bacteria.
Beer is microbiologically stable due to several intrinsic microbiological properties. Firstly, it has low pH (pH 3.8 - 4.7) and the ethanol content can vary from 0.5% to 10% (w/w). Ethanol causes cellular membrane damage in addition to denaturation of proteins, interfering with metabolism and causing cell lysis of bacteria [
In addition, hurdles for bacterial contaminants are provided by process conditions such as wort boiling, pasteurisation and sterile filtration [
Beer spoilage microorganism can be broadly classified into Gram positive bacteria, Gram negative bacteria and wild yeasts. Gram positive beer spoilage bacteria are regarded as the most hazardous for modern breweries [
Gram negative beer spoilers mainly include anaerobic bacteria belonging to genera Pectinatus, Megasphaera, Selenomonas and Zymophilus. Other significant Gram negative aerobic and facultative anaerobe beer spoilers belong to genera Acetobacter, Zymomonas, Selenomonas, and Obesumbacterium. Certain Enterobacteriaceae such as Rahnella and Hafnia have also been reported in brewing environments [
This review focuses on Gram negative aerobic, facultative anaerobic and strictly anaerobic brewery related spoilage bacteria. Important categories of Gram negative beer spoilage bacteria are comprehensively described in this review. The review deals with current taxonomic status, metabolic aspects, beer spoilage ability and detection methods utilised for these bacteria.
Only a few Gram negative bacteria have been found to be responsible for beer spoilage and these bacteria can be
divided into two categories. The first category includes aerobic and facultative anaerobic bacteria such as Acetic Acid Bacteria, Zymomonas and certain Enterobacteriaceae species. The second category is the anaerobic beer spoilers belong to genera Pectinatus, Megasphaera, Zymophilus, Selenomonas and Propionispora. Pectinatus and Megasphaera are regarded as the most important beer spoilage bacteria, mainly in unpasteurised beer. Spoilage effects of Gram negative bacteria are shown in
Acetic Acid Bacteria (AAB) are industrially important as they can produce organic acid by oxidising sugar to ethanol then to organic acid, principally acetic acid. Gluconobacter are used for production of vinegar commercially. AAB bacteria are also used in various biotechnological applications [
At present AAB taxonomically belongtofamily Acetobacteraceae [
Production of acetic acid from oxidation of ethanol is asignificant characteristic of Acetobacter and Gluconobacter. The process is catalysed by cytoplasmic membrane bound enzymes alcohol dehydrogenase and aldehyde dehydrogenase for production of acetaldehyde from ethanol and acetaldehyde to acetic acid respectively [
AAB occur throughout the brewing process (see
As described, AAB bacteria produce sourness in beer due to formation of acetic acid. Gluconobacter in the beer leads to formation of a pellicle on the surface with cloudiness in beer containing oxygen. Some strains of Gluconobacter produced extran and levan leading to formation of ropiness in the beer with high viscosity [
Zymomonas are short plump rods which occur singly, in pairs and sometimes in chains or rosettes [
Bacteria | Occurrence in brewery environments | Off flavour/aroma and odour | Visual poilage effects | Metabolic products |
---|---|---|---|---|
Acetic acid bacteria [ | ||||
Acetobacter | Wort, beer dispenses and cask condition ales and barrel aged ales, brewery biofilm. | Sour, vinegar | Haze, Ropiness | Acetic acid |
Glucanobacter | Wort, beer dispense and cask condition ales | Sour, vinegary | Haze | Acetic acid, acetate |
Zymomonas | Primed beers (not found in lagers) | Fruity, rotten apple, rotten egg, sulphudic | Haze Ropiness | Acetaldehyde and H2S |
Enterobacteriaceae [ | ||||
Obesumbacterium | Pitching yeast and fermenting wort | Parsnip, sulphury | Haze | Dimethyl sulphides (DMS), diacetyl, higher alcohols and N-nitrosamines, acetoin |
Citrobacter | Brewing liquor , fermenting wort | Parsnip, sulphury | Dimethyl sulphides (DMS), diacetyl, lactic acid, acetaldehyde | |
Rahnella | Pitching yeast, Early stages of fermentation (wort) | Fruity, sulphury, | - | Dimethyl sulphides (DMS), diacetyl, methyl acetate, ethyl acetate |
Klebseilla | Fermenting wort, biofilm | Unpleasant odour | - | 4-vinylguaicol, Dimethylsulphides (DMS), diacetyl. |
Obligatory anaerobes [ | ||||
Pectinatus | Low alcohol unpasteurised beer, beer filling area, biofilm | Rotten egg, unpleasant odour | Turbidity | Acetic acid, propionic acid, lactic acid, succinic acid, H2S, acetoin, methyl mercaptan and other sulphur compounds |
Megasphaera | Low alcohol, unpasteurised beer, beer filling area, biofilm | Unpleasant odour | Turbidity | H2S, butyric acid, isobutyric acid, caprioc acid, valeric acid, isovaleric acid. |
Selenomonas | Pitching yeast | Unpleasant odour | Turbidity | Acetic, lactic, and propionic acids. |
Zymophilus | pitching yeast or brewery waste | Unpleasant odour | Turbidity | Acetic acid and propionic acid |
References: [
These bacteria are found on the glucose rich sugarcane juice, agavesapand palm trees as a naturally occurring fauna [
Spoilage due to Zymomonas is quite a common problem in ciders; a motile rod shape bacterium responsible for sick cider has been well studied. The original source of contamination by Zymomonas species in the brewery and cider house is still unknown. Soil is suggested to be the possible source of contamination in beer [
The contamination incidents due to Zymomonas are limited to ales supplemented with primed sugar and spoilage problems due to these bacteria have never been encountered in lager beers [
The Enterobacteriaceae [
Obesumbacterium proteus belongs to the family Enterobacteriaceae and is an extensively studied beer spoilage microorganism. O. proteus shows negative reaction to Gram staining and is aerobic or facultatively anaerobic. Morphologically it is a short rod but it has also been reported to show pleomorphic rod morphology in the presence of yeast in fermenting wort [
The genus Obesumbacterium contains only one species, O. proteus, and it was isolated as a contaminantwithin the brewing yeast culture. Formerly O. proteus was classified as Flavobacterium proteus [
O. proteus is found in pitching yeast and fermenting wort, and it has never been reported in beer due to its inability to grow below pH 3.9. O. proteusis encountered in initial stages of fermentation where it competes with yeast for nutrients resulting inaslower rate of fermentation. O. proteus also produces metabolites such as dimethyl sulfoxide (DMS), acetoin, lactic acid, propanol, isobutanol and 2, 3-butandiaol. DMS imparts parsnip flavour to contaminated beer [
Brewery related Enterobacteriaceae serve as hygiene indicator microorganisms and are not normally able to grow in finished beer. They may, however, grow during the initial stages of the brewing process, causing unwanted off-flavours in the final product [
Due to implementation of effective cleaning and sanitation procedures in modern breweries and effective removal of oxygen from post fermentation processes, spoilage due to aerobic Gram negative bacteria such as Acetobacter and Glucanobacter has been significantly reduced [
Pectinatus was reported as a new genus of Gram negative, catalase negative, motile, obligate beer spoilage bacteria in the 1970s when it was first isolated from a brewery in the United States in unpasteurized beer stored at 30˚C [
Pectinatusfrisingnesis can fermentcellobiose, inositol and N-acetyl glucosamine but it cannot utilise xylose and melibiose which can be utilised by P. cervisiiphilus [
The genus Pectinatus currently comprises three brewery related species: P. cerevisiiphilus [
Characteristics | Bacterial species | |||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | |
Inhabit | spoiled beer | brewery bottling hall | spoiled beer | spoiled beer | spoiled beer | spoiled beer |
G + C Content (%) | 38.6 | 39.1 | 38.4 | 42.4 - 4.8 | 40.5 | 43.1 |
Width x Length (µm) | 0.7-1.0-30 | 0.6 - 0.8 × 3 - 50 | 0.7 - 0.9 × 3 - 50 | 1.5 - 2.1 | 1.2 - 1.9 × 1 - 1.4 | 1 - 1.4 × 0.8 - 1.2 |
Temperature (˚C) | ||||||
Range | 10 - 45 | 15 - 30 | 15 - 37 | 10 - 37 | 10 - 30 | 10 - 30 |
Optimum | 30 | 30 | 30 | 30 | 30 | 30 |
pH | ||||||
Range | 3.5 - 8.5 | 4.0 - 8.0 | 3.5 - 8.0 | - | - | - |
Optimum | 6.5 | 7 | 6.5 | - | - | - |
catalase activity | - | + | - | - | - | - |
Spoilage ability | absolute beer spoiler | potential beer spoiler | absolute beer spoiler | potential beer spoiler | potential beer spoiler | potential beer spoiler |
1) Pectinatus cerevisiiphilus; 2) Pectinatus frisingensis; 3) Pectinatus haikarae; 4) Megasphaera cerevisiae; 5) Megasphaera paucivorans; 6) Megasphaera sueceinsis; References: [
Pectinatus was considered to be brewery specific. Recently two new species of Pectinatus have been recovered from salty pickle waste water, namely P. brassicae [
Previously, Gram negative anaerobic bacteria belonging to the genus Pectinatus were affiliated to sub branch sporomusa in the family Acidamincocaceae of class Clostridia [
Most Pectinatus species have been isolated from beer and brewery environments but their natural environment and source of contamination are not well understood [
Pectinatus have also been reported in pitching yeast and CO2 recovery systems [
Brewery related Pectinatus species are non-spore forming, motile rods with flagella attached laterally to one side of the cells. Young cells show an X shaped pattern formation during movement and old cells show slow snake like movement [
Genus Megasphaera, originally described by Rogosa [
Brewery related Megasphaera species share common ecological niches with Pectinatus but are less wide- spread [
Growth occurs in the temperature range 15˚C - 37˚C and optimum growth is reported to be at 28˚C [
Other Gram negative, anaerobic beer spoilers phylogenetically related to Pectinatus and Megasphaera belong to genera Zymophilus, Selenomonas and Propionispira. Selenomonaslacticiflex and Propionispira are non-spore forming, motile rods and may lose mobility on repeated culturing Selenomonaslacticiflex and Propionispira are more sensitive to acidic environments than Pectinatus and Megasphaera and has been isolated from pitching yeast in Germany and Finland [
Conventional methods for detection of spoilage microorganisms in beer and other beverages generally involves pre-enrichment of the sample with a non-selective medium, followed by enrichment on selective or differential agar [
For AAB bacteria Frateur’s differential medium, AE medium, Reinforced AE medium and YPM medium have been described in the literature [
Detection of Zymomonas in the brewery using MYPG (malt yeast extract glucose and peptone) agar supplemented with 50 ppm actidione and 3% ethanol or beer with 100 ppm actidione has been reported [
For detection of Enterobacteriaceae in wort and yeast slurries the use of MacConkey agar supplemented with actidione (10 ppm) for suppression of yeasts is recommended by the European Brewing Convention [
For Gram negative strictly anaerobic bacteria such as Pectinatus and Megasphaera, SMMP (Selective Medium for Megasphaera and Pectinatus), NBB medium, and MRS medium with several modification have been described [
There is a small range of non-pathogenic beer spoilage bacteria which can survive, grow and spoil beer. Very few cases of beer spoilage have been reported in recent years due to high standards of hygiene and technological improvements within the brewing industry. However, due to food and beverage safety concerns, strict regulations regarding food and beverage production and maintaining high quality of products, beer spoilage microorganisms are of severe concern to breweries worldwide. Acetic Acid Bacteria such as Acetobacter and Gluconobacter were important beer spoilers. Due to implementation of effective cleaning and sanitation procedures in modern breweries and effective removal of oxygen from post fermentation processes, these bacteria are of minor importance in commercial brewing. However these bacteria are concern to dispense systems in pub breweries, public houses and cask conditioned beers [
Pectinatus and Megasphaera have been postulated to emerge due to high levels of hygiene and significant reduction in oxygen levels in beer and increased production of unpasteurised beer [
Paradh Ashtavinayak,Hill Annie Elizabeth, (2016) Review: Gram Negative Bacteria in Brewing. Advances in Microbiology,06,195-209. doi: 10.4236/aim.2016.63020