In his excellent review on beer spoilage micro-organisms, Rainbow (1981) noted that 'beer is resistant to microbial spoilage because of its relatively low nutritional status, its content of products of yeast metabolism, its adverse values of pH and redox potential and its content of hop bitter substances'. This, in a nutshell, explains the relative robustness of beer to microbial spoilage. Although the 'low nutritional status' is self-evident, it is worth reiterating that yeast removes the vast majority of assimilable nutrients from wort, leaving behind generally high-molecular-weight material of limited appeal to the vast majority of micro-organisms. Whether this is a major factor in providing innate protection is debatable as the nutrient status of beer is boosted by release of nutrients through yeast autolysis and subsequent priming with fermentable sugars. More significant factors in limiting susceptibility to spoilage are 'the products of yeast metabolism', specifically the twin guns of ethanol (> 3.5% v/v) and pH (« 4). Telling insights in their contribution to minimising the risk of spoilage comes from experiences with low/no alcohol beers (LAB/NABs) and products with pHs above those of beer. Both are innately more susceptible to spoilage by the usual spectrum of spoilage organisms as well as others that are typically suppressed by these parameters. Additionally, other beer components (hop iso-a-acids, carbon dioxide, etc.) bolster resistance to spoilage. Of course, the robustness of the product to microbial spoilage is best viewed as being an intrinsic 'benefit' and should in no way detract from a commitment to minimising risk through hygienic operations and practices.
The few systematic investigations into the 'spoilability' of beer have focused on the resistance to spoilage by the Gram-positive organisms, Lactobacillus and Pediococcus (Dolezil & Kirsop, 1980; Fernandez & Simpson, 1995; Hammond et al., 1999). Given the unpredictability of brewing microbiology, it is no surprise to find a somewhat confused picture with little by way of generic insights. As ever, methodology may be at the root of the confusion. The earlier work of Dolezil and Kirsop (1980) was unable to correlate resistance to spoilage with any beer parameters. However, as noted by Fernandez and Simpson (1995), the bacteria used by Dolezil and Kirsop (1980) had not been 'trained' to grow in beer prior to inoculation. Fernandez and Simpson (1995), using 14 strains of hop-resistant lactic acid bacteria and 17 different lagers, were able to correlate resistance to spoilage to a number of beer parameters. These were generally in accord with expectation, such that spoilage was related to beer pH, various nutrients (free amino nitrogen, maltotriose etc.) and the undissociated forms of hop bitter acids and sulphur dioxide. Intriguingly, beer colour correlated strongly with resistance to spoilage. These parameters, together with carbon dioxide (Hammond et al., 1999), may be viewed as providing beer with innate protection to spoilage by lactic acid bacteria. Inevitably though, there are differences between the response of different species of Lactobacillus, strains of Lactobacillus species and sensitivity of different beers to spoilage. Indeed, it is tempting to conclude that the only predictable thing about microbiology is its lack of predictability!
Despite the ever increasing sophistication of methods to 'fingerprint' bacteria (see Section 126.96.36.199), the typical 'first stab' at identification of an unknown bacterium involves a handful of simple questions and methods. As many brewery bacteria are fastidious in their nutrient requirements and environment, much can be gleaned from simply identifying where the contaminant was isolated. Overlaid on this are considerations such as the growth medium the organism was recovered from and, importantly, the size and shape of the cells under the microscope. Some bacteria are typically spherical (cocci) and others more elongated rods (bacilli). Further diagnostic information can be obtained from whether the cells are single or organised into groups (pairs, tetrads, clusters or chains).
In the majority of cases, depending on need and available resources, the above criteria provide sufficient information for the routine cursory identification of a bacterial contaminant. However, two laboratory methods - the catalase test and the Gram test - are frequently used to describe bacteria and can be used to 'add value' in routine microbiological trouble shooting. Of the two, the catalase test is much the simpler but less revealing diagnostically. The method probes for the presence of catalase, one of a number of enzymes that protect microbial cells from toxic oxygen species. Quite simply, an aliquot of hydrogen peroxide (3% v/v) is added to the microbial colony or culture and the presence or absence of gas (oxygen) evolution scored as, respectively, catalase positive or negative. Typically, anaerobic organisms are catalase negative. Although more convoluted, the Gram test is worthy of greater comment, particularly as it remains the basis of the division of bacteria into two classes, 'Gram positive' and 'Gram negative'.
The Gram test was devised in 1884 by a Danish bacteriologist, Hans Christian Gram, who, at the time was working in the morgue of the city hospital in Berlin (for an historical review see Scherrer, 1984). The method involves the stepwise staining and counterstaining of a thin film of bacteria on a microscope slide and results in Gram positive bacteria being stained purple and Gram negative cells being stained red. Typically, the result is assessed visually without recourse to microscopy. Although long a routine method in bacteriology, it has its limitations, notably being both tedious and time consuming. Further, the outcome is sensitive to factors such as staining technique and reagent stability together with the physiology and age of bacteria under test.
The steps in Gram's staining are detailed in Fig. 8.1. Although subject to debate, it is generally accepted that the two-stage staining with crystal violet and then Gram's iodine (iodine and potassium iodide) leads to the formation of a water insoluble complex. Treatment with ethanol differentiates between bacteria by washing out the purple stain from the Gram-negative cells, which are then counterstained red with safranin. Seemingly, the crystal violet-iodine complex is retained within the Grampositive cells but lost from the Gram-negative cells. Retention or loss of the complex is explained by the cell wall structure of the two classes of bacteria. Although broadly similar (see Fig. 8.2), the peptidoglycan matrix layer in Gram-positive cells is many times thicker than the Gram-negative cells. Thus, the physical mass and thickness of the cell wall is believed to explain the differing responses of bacteria to the Gram test. It is noteworthy that compared to Gram-negative cells, Gram-positive bacteria are generally more sensitive to growth inhibition by dyes and many antibiotics but are more resistant to enzymic digestion. Unlike other eukaryotic cells, yeast is strongly Gram-positive.
A rapid alternative to the Gram's test has been described (Lin, 1980), which exploits the differing viscosity of pure bacterial colonies when treated with 3% (w/v) potassium hydroxide solution. Gram-negative bacteria form a viscous mass whereas Gram-positive colonies fail to exhibit viscosity. This is thought to reflect the
Air dry, heat fix
Fig. 8.1 The Gram stain.
• peptidoglycan ■ plasma membrane
Fig. 8.2 Differences between the cell walls of Gram-positive and Gram-negative bacteria.
Fig. 8.2 Differences between the cell walls of Gram-positive and Gram-negative bacteria.
extraction of DNA from the Gram-negative bacteria. The correlation between the 'KOH' method and the Gram test is best described as 'directional'. In a survey of 466 bacteria, 91.4% of the Gram negative and 88% of the Gram positive correlated with KOH lysis (Moaledj, 1986).
Inevitably, the differentiation of bacteria is more complex and extensive than indicated here. Like yeast (Section 188.8.131.52), API test strips have found application in the differentiation and identification of brewery bacteria (see for example Ingledew et al., 1980). However, Priest (1996) has reported reservations in accuracy and repeatability of diagnostic strips. From a taxonomic perspective, the definitive reference work for bacteria is Bergey's Manual of Determinative Bacteriology (Holt et al., 1994), which is now in its ninth edition. As with yeast, bacterial taxonomy is increasingly influenced by the application, and consequent insights, of new and evolving molecular methods. A user-friendly overview of the classification of brewing bacteria can be found in Priest (1981).
184.108.40.206 Gram-negative bacteria. The major Gram-negative bacteria found in breweries are summarised in Table 8.1. For an authoritative review, see Van Vuuren (1996) in Brewing Microbiology. For a wider appreciation of Gram-negative bacteria see Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). Specific brewing reviews can be found on Zymomonas (Dadds & Martin, 1973), Entero-bacteriaceae (Priest et al., 1974) and the obligate anaerobes (Chelack & Ingledew, 1987).
Arguably, in comparison to the Gram-positive bacteria, the threat of the various Gram-negative bacteria is under reasonable control. With changes in process, raw materials and market demands some - the acetic acid bacteria (Fig. 8.3) and Zymomonas (Fig. 8.4) - have had their day and are now more of a niche concern. O. proteus (Fig. 8.5) remains a concern for its role in the formation of ATNCs (Section 220.127.116.11) and ease of recycling via yeast re-pitching. Day-to-day management of this microbiological threat is achieved through regular acid washing (see Section 7.3.3 and
Table 8.1 Gram-negative bacteria.
Family Genus Major species
Acetic acid bacteria Acetobacter A. aceti
Acetic acid bacteria Gluconobacter G. oxydans
Obesumbacterium (syn. Hafnia)
Enterobacter (syn. Rahnella)
E. agglomerans (R. aquatilis) E. cloacae
Enterobacteriaceae Citrobacter ('coliforms')
Pleomorphic, 0.6-0.8 ^m x 1-4 ^m, catalase positive, strict aerobes, oxidises ethanol to carbon dioxide and water
Pleomorphic, some motile with flagella, 0.6-0.8 x 1-4 catalase positive, strict aerobes, oxidises ethanol to acetic acid
Typically rods, 0.3-1 x 1-6 ^m, facultatively anaerobic, diversity of fermentation products (organic acids, butanediol, phenolics), generally sensitive to pH (< 4.4) and ethanol (>2%)
Catalase positive, specific to brewery environments, 'short fat rod' (0.8-1.2 x 1.5-4 |jm), ethanol tolerant (< 6%), found in yeast heads and slurries and consequent danger of recycling contamination
Catalase positive, straight rod (1 x 2-6 |^m)
Specific to aerobic or microaerophilic environments, i.e. dispense and draught (unpasteurised) products but not keg or smallpack, forms a haze and surface film
Specific to aerobic or microaerophillic environments, i.e. dispense and draught products but not keg or smallpack, forms a haze, surface film and viscous 'ropiness'
Generally (but not exclusively) limited to wort and early fermentation, most common is O. proteus - which (within the Enterobacteriaceae) best survives fermentation
Grows in wort and during early fermentation, produces sulphur off-products (DMS), inhibits fermentation rate and results in high beer pH, reduces nitrate to nitrite leading to the formation of ATNCs
Occasional contaminant of pitched wort, can accelerate fermentation, produces organic acids and DMS, does not survive fermentation
Similar to O. proteus in associating with yeast and in surviving fermentation conditions, forms diacetyl and DMS, increases initial fermentation rate, final pH higher
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