Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6762-3
MINI-REVIEW
Adaptation and tolerance of bacteria against acetic acid Janja Trček 1,2 & Nuno Pereira Mira 3 & Laura R. Jarboe 4,5
Received: 10 March 2015 / Revised: 5 June 2015 / Accepted: 15 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Acetic acid is a weak organic acid exerting a toxic effect to most microorganisms at concentrations as low as 0.5 wt%. This toxic effect results mostly from acetic acid dissociation inside microbial cells, causing a decrease of intracellular pH and metabolic disturbance by the anion, among other deleterious effects. These microbial inhibition mechanisms enable acetic acid to be used as a preservative, although its usefulness is limited by the emergence of highly tolerant spoilage strains. Several biotechnological processes are also inhibited by the accumulation of acetic acid in the growth medium including production of bioethanol from lignocellulosics, wine making, and microbe-based production of acetic acid itself. To design better preservation strategies based on acetic acid and to improve the robustness of industrial biotechnological processes limited by this acid’s toxicity, it is essential to deepen the understanding of the underlying toxicity
* Janja Trček [email protected] * Laura R. Jarboe [email protected]
mechanisms. In this sense, adaptive responses that improve tolerance to acetic acid have been well studied in Escherichia coli and Saccharomyces cerevisiae. Strains highly tolerant to acetic acid, either isolated from natural environments or specifically engineered for this effect, represent a unique reservoir of information that could increase our understanding of acetic acid tolerance and contribute to the design of additional tolerance mechanisms. In this article, the mechanisms underlying the acetic acid tolerance exhibited by several bacterial strains are reviewed, with emphasis on the knowledge gathered in acetic acid bacteria and E. coli. A comparison of how these bacterial adaptive responses to acetic acid stress fit to those described in the yeast Saccharomyces cerevisiae is also performed. A systematic comparison of the similarities and dissimilarities of the ways by which different microbial systems surpass the deleterious effects of acetic acid toxicity has not been performed so far, although such exchange of knowledge can open the door to the design of novel approaches aiming the development of acetic acid-tolerant strains with increased industrial robustness in a synthetic biology perspective. Keywords Acetic acid tolerance . Acetic acid bacteria . Acetic acid/acetate transporters . Intracellular pH . Food preservatives . Vinegar . Biomass fermentation
1
Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroška cesta 160, 2000 Maribor, Slovenia
2
Faculty of Chemistry and Chemical Engineering, University of Maribor, Maribor, Slovenia
Introduction: why is the study of acetic acid tolerance important?
3
Department of Bioengineering, Instituto Superior Técnico, Institute of Bioengineering and Biosciences, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001 Lisbon, Portugal
Food production and spoilage
4
Department of Chemical and Biological Engineering, Iowa State University, Sweeney Hall, Ames, IA 50011, USA
5
Interdepartmental Microbiology Program, Iowa State University, Ames, IA, USA
Humanity has long relied on acetic acid as an antimicrobial agent to enable food storage: acetic acid is the main product of acetic acid bacteria during production of certain foods and drinks, such as vinegar, kombucha beverage, nata de coco,
Appl Microbiol Biotechnol
kefir, and cocoa bean fermentation (Sievers and Swings 2005). Depending on the strain of acetic acid bacterium and the process used (one or two steps), a final concentration of acetic acid up to 20.5 vol% can be achieved today during industrial vinegar production (Heinrich Frings Information). Beside the high productivity, a high acetic acid resistance is still the most appreciated feature of starter cultures to be used in vinegar production (Trček et al. 2006a, b, 2007). However, acetic acid can also play an undesirable role during food production and storage when the ethanol in wine, beer, juice, and fruits is converted to acetic acid, thereby resulting in undesirable alterations of the sensorial properties of the drinks (Sievers and Swings 2005).
Food safety and public health Our reliance on acetic acid and on other weak organic acids that are also used as food preservatives (such as benzoic and sorbic acids (Brul and Coote 1999)) is hindered by the emergence of spoilage bacterial and fungal strains highly tolerant to these acids, this being particularly relevant in the context of pathogenic microbes. For example, enterohemorrhagic Escherichia coli O157:H7 is more acetate-resistant than the non-pathogenic K12 strain (Diez-Gonzalez and Russell 1997) and E. coli isolates associated with outbreaks were also found to be more acetate-resistant than strains simply isolated from fermented foods or other sources (McKellar and Knight 1999). It should be noted that acetic acid is also present in the human gastrointestinal and genitourinary tracts and in the blood stream, often being more than twice as abundant as other short-chain fatty acids (Cummings et al. 1987; Owen and Katz 1999). Thus, increased tolerance to acetic acid could possibly enable increased survival of microbes in these environments. In fact, it has been demonstrated that invasive candidiasis caused by the pathogenic yeast Candida albicans in the mice gut is restrained by the presence of bacteria that produce acetic acid (Yamaguchi et al. 2005).
By-product in microbial processes Acetic acid is also produced as a by-product or overflow metabolite of many biotechnological processes, and therefore, it is desirable for the production organism to be tolerant of acetic acid (Kleman and Strohl 1994). At concentrations as low as 5 g/L, these acidic overflow products already begin to affect cell growth and productivity almost equally in Gram-positive and Gram-negative bacteria (Russell 1991a). Specifically, the average MIC value (mmol/L) is 65 for Gram-positive bacteria and 58 for Gram-negative bacteria (Russell 1991a). Acetic acid is also produced as a by-product during wine fermentations, presumably due to the activity of fermenting yeasts and/ or contaminating bacteria. The final concentration of acetic acid attained at the end of the fermentation has a strong effect in the sensorial properties of the wine produced, and it may reduce fermentation yields by deleteriously affecting the metabolic activity of fermenting yeast cells, particularly in the later stages of the fermentation when other toxic metabolites, such as ethanol, also accumulate in the acidic growth medium (Gibson et al. 2007; Paraggio and Fiore 2004; Romano et al. 2003). Here, we give a short review on acetic acid tolerance mechanisms in bacteria with a focus on the knowledge gathered in acetic acid bacteria, the most well-known bacteria tolerant to acetic acid. Knowledge gathered in other tolerant bacterial strains engineered to cope with increased concentrations of acetic acid will also be described, as well, as the environmental factors that influence tolerance to acetic acid and the toxic effects exerted by acetic acid on physiology of bacterial cells. Furthermore, a comparative analysis of these tolerance mechanisms with those active in Saccharomyces cerevisiae will also be performed, taking advantage of the extensive amount of knowledge that has been gathered on the adaptive responses and tolerance mechanisms to acetic acid stress in this yeast species (reviewed in Mira et al. 2010c; Mollapour et al. 2008; Piper et al. 2001).
Acetic acid-tolerant bacteria Acetic acid bacteria
Biorenewables Acetate is both a hindrance and a substrate in terms of microbial production of fuels and chemicals from biomass. It is a hindrance due to its presence at inhibitory concentrations in biomass hydrolysate (Martinez et al. 2001; Mills et al. 2009; Taylor et al. 2012), though at low concentrations, acetate has been observed to slightly increase product titers (FernándezSandoval et al. 2012). It is used as a substrate in the production of polyhydroxyalkanoates, such as poly-3-hydroxybutyrate (PHB) (Wang and Yu 2000).
Acetic acid bacteria are the most prominent acetic acidtolerant microorganisms. They are Gram-negative aerobic rods of the α-Proteobacteria that are presently classified into 19 genera. The taxonomy of acetic acid bacteria has been updated many times over the past few years (Cleenwerck and De Vos 2008; Trček and Barja 2015). The species with the highest acetic acid tolerance, such as Gluconacetobacter e u ro p a e u s, G l u c o na c e t o b a ct e r o b ed i e n s, a n d Gluconacetobacter intermedius (Trček et al. 2000, 2006a), have been recently reclassified from genus
Appl Microbiol Biotechnol
Gluconacetobacter to Komagataeibacter (Yamada et al. 2012). These species are responsible for industrial production of vinegar in submerged bioreactors with final concentration of acetic acid above 10 % (Boesch et al. 1998; Sievers et al. 1992; Trček et al. 1997). These submerged bioreactors differ from bioreactors with static cultures in having the bacteria dispersed throughout the whole bioreactor and continually aerated with fine bubbles. Also, the species of genus Acetobacter, such as Acetobacter aceti, Acetobacter pasteurianus, and Acetobacter pomorum, are used for vinegar production but mainly for the production of vinegars with final concentration of acetic acid not exceeding 6 %, such as in cider, malt, and rice vinegar (González et al. 2004; Gullo et al. 2006; Nanda et al. 2001; Slapšak et al. 2013; Sokollek et al. 1998; Wu et al. 2010). These bioprocesses are usually performed with immobilized or surface cultures (Ghommidh et al. 1982; Osuga et al. 1984). Acetic acid bacteria oxidize ethanol to acetic acid in two sequential reactions catalyzed by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), both of these enzymes being located on the periplasmic side of the cytoplasmic membrane and linked to the respiratory chain (Matsushita et al. 1994). Durine et al. (1979) suggested that the ADH of acetic acid bacteria was possibly a quinoprotein. Further on, it was confirmed by Ameyama et al. (1981) that ADH and ALDH of acetic acid bacteria had the pyrroloquinoline quinone (PQQ) prosthetic group. Later, it was revoked that PQQ is a cofactor of ALDH (Takemura et al. 1994). Thurner et al. (1997) proposed that the putative cofactor of ALDH complex of Komagataeibacter europaeus is the molybdopterin. Besides oxidizing ethanol to acetic acid, some genera of acetic acid bacteria, such as Gluconacetobacter, Acetobacter, and Komagataeibacter, can also oxidize acetic acid to CO2 and H2O (Sievers and Swings 2005). This phenomenon is called overoxidation and results in diauxic growth of acetic acid bacteria. The overoxidation of acetate is enabled by enzymes of the tricarboxylic acid (TCA) cycle and acetyl-CoA synthetase. Between these two exponential phases, a stationary phase appears whose length differs among strains of acetic acid bacteria. During the overoxidation process, the glyoxylate cycle could play an important role by supplying oxaloacetate to the TCA cycle (Saeki et al. 1999; Sakurai et al. 2013). Other bacteria Acetic acid can also be produced anaerobically from H2 and CO2 by acetogenic bacteria. Among this group of bacteria, the most well-known species are Clostridium aceticum, Acetobacterium woodii, and Moorella thermoacetica (Diekert 1992). They utilize the acetyl-CoA WoodLjungdahl pathway for CO2 reduction with electrons coming from H2. Most acetogens have pH optima at pH >6 and are
sensitive to acetate and acetic acid. Novel species, such as Clostridium drakei, that produces acetate at moderately low pH might have also higher tolerance to acetic acid (Drake et al. 2008). An example of highly acetate-resistant Gram-positive bacterium is Staphylococcus capitis (Lasko et al. 2000), a close relative to Staphylococcus epidermidis and Staphylococcus aureus, whose natural ecological niche is the skin (Naber 2009). Evolution as a means to improve acetic acid tolerance In addition to bacteria that naturally have a high acetic acid tolerance, other bacteria have been evolved or engineered for increased tolerance. Classical evolution methods involving strain enrichment under selective pressure have been used with the goal of improving acetate tolerance in order to improve acetate production by Clostridium thermoaceticum (Reed et al. 1987) and A. aceti (Steiner and Sauer 2003a). This technique has also been used to increase acetate tolerance of Zymomonas mobilis (Joachimsthal et al. 1998) and E. coli (Steiner and Sauer 2003b; Fernández-Sandoval et al. 2012). Improving acetate tolerance in E. coli has also been addressed through the use of global transcription machinery engineering (gTME) (Chong et al. 2013) and scalar analysis of library enrichments (SCALES) (Sandoval et al. 2011). Insights into the mechanisms for increased tolerance by these strains are discussed below in the BMechanisms of tolerance to acetic acid stress^ section.
Things that acetate does Acetate leads to cross protection to other stresses As with many inhibitors, adaptation to acetate primes tolerance to further acetate challenge; this has been reported for E. coli O157:H7 (Brudzinski and Harrison 1998) and Acetobacter senegalensis (Shafiei et al. 2013). However, short-term adaptation to acetate can also prime tolerance to a variety of other stressors. In the case of Salmonella typhimurium, 1 h of adaptation to 100 mM acetate at pH 7.0 increased survival of this bacterium during challenge with HCl at pH 3.0 (Kwon and Ricke 1998). Similar experiments performed with E. coli K12 and E. coli O157:H7 showed that pre-exposure to acetic acid stress increased survival during challenge with citric acid at pH 3.0 (Arnold et al. 2001). E. coli’s adaptation to acetate challenge also enabled increased tolerance to oxidative stress and thermal challenge (Arnold et al. 2001). It was also found that adaptation of Pseudomonas putida S12 to acetate challenge increased tolerance to several organic solvents, including decane, styrene, and toluene (Weber et al. 1993).
Appl Microbiol Biotechnol
This activation of a variety of stress response systems is consistent with the observed increase in expression of stress response pathways in E. coli following acetate exposure and adaptation (Arnold et al. 2001; Kirkpatrick et al. 2001; Polen et al. 2003). Specifically, the activated pathways in response to acetic acid were found to be regulated by the alternative sigma factor RpoS. Thus, it is not surprising that deletion of rpoS prevents priming of acid tolerance during acetate challenge (Arnold et al. 2001). Diffusion of acetic acid through the cell membrane Acetic acid is a weak one carbon-chain carboxylic acid with a moderate pKa value of 4.75. The acid dissociation constant directly connects the amount of protonated and ionic forms of acetic acid as a function of the pH value of the cultivation medium. This function is mathematically described by the Henderson-Hasselbalch equation (Hasselbalch 1917): pH ¼ pKa þ log
½A‐ ½HA
According to the Henderson-Hasselbalch equation, 50 % of the acid is protonated at the pH of pKa value. Thus, at a pH below 4.75, most of the acetic acid is in its undissociated form (CH3COOH), 90 % of it being protonated at a pH of 3.75 and 99 % protonated when the pH drops to 2.75. Due to their lipophilicity, the undissociated acetic acid molecules can permeate the cell membrane simply by passive diffusion, thereby dissociating directly in the cytosol. This is a marked difference from what occurs with strong acids like HCl, which acidify the growth medium but are not efficient in the acidification of microbial cytosol as protons diffuse poorly through the cell envelope. The accumulation of protons in the cytosol causes a decrease of internal pH to values that may not be tolerable to the cell, thereby leading to a strong reduction of metabolic activity and, eventually, to cell death (Axe and Bailey 1995; Russell 1991a). As described below, another consequence of cytosol acidification is dissipation of the electrochemical gradient maintained across the cell membrane, this being an essential demand for secondary transport. Various effects of anion accumulation As described above, one of the most dramatic effects of acetic acid entry into the cell is the acidification resulting from the protons released upon chemical dissociation of the acid. However, the acetate that is released upon dissociation of acetic acid also accumulates at high values inside microbial cells, this event being impactful on cell physiology. When held at a constant external pH, E. coli K12 accumulates
intracellular acetate in a roughly linear response to increasing acetate dosage (Diez-Gonzalez and Russell 1997). Intracellular pools of 500 mM were measured in the presence of 80 mM acetate at pH 5.9 (Diez-Gonzalez and Russell 1997) and greater than 3 M for 340 mM external acetate at pH 6.5 or pH 5.5 (Steiner and Sauer 2003a). A detailed characterization during challenge with 8 mM acetate at pH 6.0 showed that the internal acetate pool can reach 170 mM within just 2 min (Roe et al. 1998). While E. coli O157:H7 pool sizes were similar to E. coli K12 at low external acetate concentrations, the intracellular concentration leveled off at approximately 300 mM when the external concentration surpassed 40 mM (DiezGonzalez and Russell 1997). Not surprisingly, this acetate anion accumulation results in increased membrane potential; a dose-dependent response was observed during E. coli K12 challenge with increasing amounts of acetate at pH 6.8 (Bakker and Mangerich 1983). The accumulation of acetate is accompanied by perturbations of other anion pools. Most notably, glutamate is depleted in E. coli K12 cells under acetic acid stress (Roe et al. 1998). In specific, E. coli cells challenged with 8 mM acetate at pH 6.0 exhibited internal glutamate pools of 10 mM, comparing to a 60 mM pool of glutamate registered in unstressed cells (Roe et al. 1998). The drop in glutamate internal concentration occurred concomitantly with the increase in internal acetate concentration (Roe et al. 1998). Glutamate, along with potassium ions, is an important regulator of cell turgor (Wood 2011) which is expected to increase as a result of the accumulation of acetate in the internal environment. No change in potassium pools was observed in response to less than 40 mM acetate; however, when cells were exposed to higher concentrations, accumulation of K+ was, in fact, observed (Roe et al. 1998; Diez-Gonzalez and Russell 1997). At lower external pH values, E. coli K12 showed a larger deficit in ATP content for acetate-challenged cells relative to the corresponding noacetate control (Bakker and Mangerich 1983), indicating that acetic acid stress might lead to energy depletion in this bacterium. Inhibition of the methionine biosynthetic pathway Observations that methionine supplementation provide a protective effect for E. coli against acetate (Han et al. 1993) (described below in the BOther environmental components^ section) inspired the observation that homocysteine accumulates during acetate challenge. While control cultures had intracellular homocysteine pools of 0.1 mM, cells challenged with 8 mM acetate at pH 6.0 exhibited pools of 1.6 mM (Roe et al. 2002). Homocysteine is toxic in its own right, and a pool of 1.6 mM is associated with a 3-fold reduction in specific growth rate (Roe et al. 2002). Homocysteine is an intermediate of methionine biosynthesis, and this accumulation is thought to be due to inhibition of methionine biosynthesis
Appl Microbiol Biotechnol
enzymes, though the mechanism remains unknown. Methionine supplementation presumably provides protection due to feedback inhibition of the entire methionine biosynthetic pathway, preventing production and therefore resulting in accumulation of homocysteine. Improvement of acetate tolerance in E. coli via modification of the methionine biosynthesis enzymes has been performed through the isolation of improved versions of MetA and MetE enzymes which catalyze, respectively, the synthesis of methionine production from homoserine and the conversion of homocysteine to methionine. Strains expressing an improved metA showed increased tolerance of 30 mM acetic acid and also showed improved thermotolerance (Mordukhova et al. 2008), while strains expressing the improved metE showed improved tolerance of acetate and propionate, but not benzoate (Mordukhova and Pan 2013).
Environmental factors In addition to genetic factors that contribute to enhance tolerance to acetic acid, environmental conditions may also play a crucial role in mitigating the deleterious effects of this weak acid or in potentiating them. External pH Expectedly, environmental pH is critical for acetate toxicity due to its influence on the distribution between the protonated and non-protonated forms of the acid. Since teasing out the difference between acetate dose and pH has proven problematic in some studies, here we only describe experiments where the total amount of acetic acid provided was maintained constant and the external pH was changed. The increased toxicity of acetic acid at lower external pH values has been reported for a variety of organisms including E. coli O157:H7 (Oh et al. 2009), ethanologenic E. coli strains (Takahashi et al. 1999; Zaldivar and Ingram 1999), rumen organisms like Streptococcus bovis (Russell 1991b), and Megasphaera elsdenii (Miyazaki et al. 1991). The intracellular acidification caused by acetic acid in different organisms is presented in Table 1. Studies of the pH effect have gone beyond inhibition of growth. During challenge of E. coli BL21 at a variety of acetate concentrations, the intracellular acetate concentration was reduced in half when cells were grown at pH 7.5 instead of 6.5 (Wang et al. 2014), confirming that acetate internal accumulation depends of the diffusion of the undissociated form. The external pH had a roughly linear influence on the proton motive force and total transmembrane chemical gradient of Streptococcus bovis during acetic acid challenge (Russell 1991b).
Oxygen availability Different results have been reported in different studies, thereby remaining to be fully elucidated on how oxygen availability modulates bacterial tolerance to acetic acid. Results obtained in various enterobacteria and in Listeria monocytogenes show that when these cells are subjected at 30 °C for 5 days to acetic acid stress at a range of external pH values, oxygen availability seems to decrease acetate toxicity (Ostling and Lindgren 1993). Similarly, 24-h survival of E. coli O157:H7 in the presence of acetic acid (provided at 32 °C at a pH 3.5 or 4.0) increased in shaken cultures relative to static cultures, though this difference was not observed in cultures previously adapted to acetic acid stress (Brudzinski and Harrison 1998). Contrastingly, survival of E. coli O157:H7 isolates challenged with 400 mM acetic acid at pH 3.3 and 30 °C for 25 min was significantly increased under anaerobic conditions relative to aerobic (Oh et al. 2009). This difference in trends could be due to the different experimental conditions, particularly in what regards to the different acid exposure times. Survival of Salmonella typhimurium anaerobic cultures adapted to 100 mM acetic acid for 1 h and then subjected to 1 h of HCl challenge at pH 3.0 and 37 °C increased relative to aerobic cultures subjected to the same procedure (Kwon and Ricke 1998), though this could be more informative regarding acid tolerance than acetic acid tolerance. Viability studies of A. aceti in the presence of 55 g/L acetic acid showed a 4fold increase in cell viability with 1 ppm oxygen relative to 15 ppm (Park et al. 1989). These same studies concluded that the optimum dissolved oxygen level for production of more than 45 g/L acetic acid was 1–3 ppm (Park et al. 1989).
Temperature The effect of temperature on acetic acid toxicity has mainly been studied in E. coli O157:H7, though also with mixed results. Studies performed at pH 3.5 and 3.3 showed that acetic acid toxicity increased when growth temperatures of 30 °C or greater were used (Brudzinski and Harrison 1998; Oh et al. 2009). Similarly, studies performed in cucumber puree with acetic acid at pH
MINI-REVIEW
Adaptation and tolerance of bacteria against acetic acid Janja Trček 1,2 & Nuno Pereira Mira 3 & Laura R. Jarboe 4,5
Received: 10 March 2015 / Revised: 5 June 2015 / Accepted: 15 June 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Acetic acid is a weak organic acid exerting a toxic effect to most microorganisms at concentrations as low as 0.5 wt%. This toxic effect results mostly from acetic acid dissociation inside microbial cells, causing a decrease of intracellular pH and metabolic disturbance by the anion, among other deleterious effects. These microbial inhibition mechanisms enable acetic acid to be used as a preservative, although its usefulness is limited by the emergence of highly tolerant spoilage strains. Several biotechnological processes are also inhibited by the accumulation of acetic acid in the growth medium including production of bioethanol from lignocellulosics, wine making, and microbe-based production of acetic acid itself. To design better preservation strategies based on acetic acid and to improve the robustness of industrial biotechnological processes limited by this acid’s toxicity, it is essential to deepen the understanding of the underlying toxicity
* Janja Trček [email protected] * Laura R. Jarboe [email protected]
mechanisms. In this sense, adaptive responses that improve tolerance to acetic acid have been well studied in Escherichia coli and Saccharomyces cerevisiae. Strains highly tolerant to acetic acid, either isolated from natural environments or specifically engineered for this effect, represent a unique reservoir of information that could increase our understanding of acetic acid tolerance and contribute to the design of additional tolerance mechanisms. In this article, the mechanisms underlying the acetic acid tolerance exhibited by several bacterial strains are reviewed, with emphasis on the knowledge gathered in acetic acid bacteria and E. coli. A comparison of how these bacterial adaptive responses to acetic acid stress fit to those described in the yeast Saccharomyces cerevisiae is also performed. A systematic comparison of the similarities and dissimilarities of the ways by which different microbial systems surpass the deleterious effects of acetic acid toxicity has not been performed so far, although such exchange of knowledge can open the door to the design of novel approaches aiming the development of acetic acid-tolerant strains with increased industrial robustness in a synthetic biology perspective. Keywords Acetic acid tolerance . Acetic acid bacteria . Acetic acid/acetate transporters . Intracellular pH . Food preservatives . Vinegar . Biomass fermentation
1
Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroška cesta 160, 2000 Maribor, Slovenia
2
Faculty of Chemistry and Chemical Engineering, University of Maribor, Maribor, Slovenia
Introduction: why is the study of acetic acid tolerance important?
3
Department of Bioengineering, Instituto Superior Técnico, Institute of Bioengineering and Biosciences, Universidade de Lisboa, Avenida Rovisco Pais, 1049-001 Lisbon, Portugal
Food production and spoilage
4
Department of Chemical and Biological Engineering, Iowa State University, Sweeney Hall, Ames, IA 50011, USA
5
Interdepartmental Microbiology Program, Iowa State University, Ames, IA, USA
Humanity has long relied on acetic acid as an antimicrobial agent to enable food storage: acetic acid is the main product of acetic acid bacteria during production of certain foods and drinks, such as vinegar, kombucha beverage, nata de coco,
Appl Microbiol Biotechnol
kefir, and cocoa bean fermentation (Sievers and Swings 2005). Depending on the strain of acetic acid bacterium and the process used (one or two steps), a final concentration of acetic acid up to 20.5 vol% can be achieved today during industrial vinegar production (Heinrich Frings Information). Beside the high productivity, a high acetic acid resistance is still the most appreciated feature of starter cultures to be used in vinegar production (Trček et al. 2006a, b, 2007). However, acetic acid can also play an undesirable role during food production and storage when the ethanol in wine, beer, juice, and fruits is converted to acetic acid, thereby resulting in undesirable alterations of the sensorial properties of the drinks (Sievers and Swings 2005).
Food safety and public health Our reliance on acetic acid and on other weak organic acids that are also used as food preservatives (such as benzoic and sorbic acids (Brul and Coote 1999)) is hindered by the emergence of spoilage bacterial and fungal strains highly tolerant to these acids, this being particularly relevant in the context of pathogenic microbes. For example, enterohemorrhagic Escherichia coli O157:H7 is more acetate-resistant than the non-pathogenic K12 strain (Diez-Gonzalez and Russell 1997) and E. coli isolates associated with outbreaks were also found to be more acetate-resistant than strains simply isolated from fermented foods or other sources (McKellar and Knight 1999). It should be noted that acetic acid is also present in the human gastrointestinal and genitourinary tracts and in the blood stream, often being more than twice as abundant as other short-chain fatty acids (Cummings et al. 1987; Owen and Katz 1999). Thus, increased tolerance to acetic acid could possibly enable increased survival of microbes in these environments. In fact, it has been demonstrated that invasive candidiasis caused by the pathogenic yeast Candida albicans in the mice gut is restrained by the presence of bacteria that produce acetic acid (Yamaguchi et al. 2005).
By-product in microbial processes Acetic acid is also produced as a by-product or overflow metabolite of many biotechnological processes, and therefore, it is desirable for the production organism to be tolerant of acetic acid (Kleman and Strohl 1994). At concentrations as low as 5 g/L, these acidic overflow products already begin to affect cell growth and productivity almost equally in Gram-positive and Gram-negative bacteria (Russell 1991a). Specifically, the average MIC value (mmol/L) is 65 for Gram-positive bacteria and 58 for Gram-negative bacteria (Russell 1991a). Acetic acid is also produced as a by-product during wine fermentations, presumably due to the activity of fermenting yeasts and/ or contaminating bacteria. The final concentration of acetic acid attained at the end of the fermentation has a strong effect in the sensorial properties of the wine produced, and it may reduce fermentation yields by deleteriously affecting the metabolic activity of fermenting yeast cells, particularly in the later stages of the fermentation when other toxic metabolites, such as ethanol, also accumulate in the acidic growth medium (Gibson et al. 2007; Paraggio and Fiore 2004; Romano et al. 2003). Here, we give a short review on acetic acid tolerance mechanisms in bacteria with a focus on the knowledge gathered in acetic acid bacteria, the most well-known bacteria tolerant to acetic acid. Knowledge gathered in other tolerant bacterial strains engineered to cope with increased concentrations of acetic acid will also be described, as well, as the environmental factors that influence tolerance to acetic acid and the toxic effects exerted by acetic acid on physiology of bacterial cells. Furthermore, a comparative analysis of these tolerance mechanisms with those active in Saccharomyces cerevisiae will also be performed, taking advantage of the extensive amount of knowledge that has been gathered on the adaptive responses and tolerance mechanisms to acetic acid stress in this yeast species (reviewed in Mira et al. 2010c; Mollapour et al. 2008; Piper et al. 2001).
Acetic acid-tolerant bacteria Acetic acid bacteria
Biorenewables Acetate is both a hindrance and a substrate in terms of microbial production of fuels and chemicals from biomass. It is a hindrance due to its presence at inhibitory concentrations in biomass hydrolysate (Martinez et al. 2001; Mills et al. 2009; Taylor et al. 2012), though at low concentrations, acetate has been observed to slightly increase product titers (FernándezSandoval et al. 2012). It is used as a substrate in the production of polyhydroxyalkanoates, such as poly-3-hydroxybutyrate (PHB) (Wang and Yu 2000).
Acetic acid bacteria are the most prominent acetic acidtolerant microorganisms. They are Gram-negative aerobic rods of the α-Proteobacteria that are presently classified into 19 genera. The taxonomy of acetic acid bacteria has been updated many times over the past few years (Cleenwerck and De Vos 2008; Trček and Barja 2015). The species with the highest acetic acid tolerance, such as Gluconacetobacter e u ro p a e u s, G l u c o na c e t o b a ct e r o b ed i e n s, a n d Gluconacetobacter intermedius (Trček et al. 2000, 2006a), have been recently reclassified from genus
Appl Microbiol Biotechnol
Gluconacetobacter to Komagataeibacter (Yamada et al. 2012). These species are responsible for industrial production of vinegar in submerged bioreactors with final concentration of acetic acid above 10 % (Boesch et al. 1998; Sievers et al. 1992; Trček et al. 1997). These submerged bioreactors differ from bioreactors with static cultures in having the bacteria dispersed throughout the whole bioreactor and continually aerated with fine bubbles. Also, the species of genus Acetobacter, such as Acetobacter aceti, Acetobacter pasteurianus, and Acetobacter pomorum, are used for vinegar production but mainly for the production of vinegars with final concentration of acetic acid not exceeding 6 %, such as in cider, malt, and rice vinegar (González et al. 2004; Gullo et al. 2006; Nanda et al. 2001; Slapšak et al. 2013; Sokollek et al. 1998; Wu et al. 2010). These bioprocesses are usually performed with immobilized or surface cultures (Ghommidh et al. 1982; Osuga et al. 1984). Acetic acid bacteria oxidize ethanol to acetic acid in two sequential reactions catalyzed by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), both of these enzymes being located on the periplasmic side of the cytoplasmic membrane and linked to the respiratory chain (Matsushita et al. 1994). Durine et al. (1979) suggested that the ADH of acetic acid bacteria was possibly a quinoprotein. Further on, it was confirmed by Ameyama et al. (1981) that ADH and ALDH of acetic acid bacteria had the pyrroloquinoline quinone (PQQ) prosthetic group. Later, it was revoked that PQQ is a cofactor of ALDH (Takemura et al. 1994). Thurner et al. (1997) proposed that the putative cofactor of ALDH complex of Komagataeibacter europaeus is the molybdopterin. Besides oxidizing ethanol to acetic acid, some genera of acetic acid bacteria, such as Gluconacetobacter, Acetobacter, and Komagataeibacter, can also oxidize acetic acid to CO2 and H2O (Sievers and Swings 2005). This phenomenon is called overoxidation and results in diauxic growth of acetic acid bacteria. The overoxidation of acetate is enabled by enzymes of the tricarboxylic acid (TCA) cycle and acetyl-CoA synthetase. Between these two exponential phases, a stationary phase appears whose length differs among strains of acetic acid bacteria. During the overoxidation process, the glyoxylate cycle could play an important role by supplying oxaloacetate to the TCA cycle (Saeki et al. 1999; Sakurai et al. 2013). Other bacteria Acetic acid can also be produced anaerobically from H2 and CO2 by acetogenic bacteria. Among this group of bacteria, the most well-known species are Clostridium aceticum, Acetobacterium woodii, and Moorella thermoacetica (Diekert 1992). They utilize the acetyl-CoA WoodLjungdahl pathway for CO2 reduction with electrons coming from H2. Most acetogens have pH optima at pH >6 and are
sensitive to acetate and acetic acid. Novel species, such as Clostridium drakei, that produces acetate at moderately low pH might have also higher tolerance to acetic acid (Drake et al. 2008). An example of highly acetate-resistant Gram-positive bacterium is Staphylococcus capitis (Lasko et al. 2000), a close relative to Staphylococcus epidermidis and Staphylococcus aureus, whose natural ecological niche is the skin (Naber 2009). Evolution as a means to improve acetic acid tolerance In addition to bacteria that naturally have a high acetic acid tolerance, other bacteria have been evolved or engineered for increased tolerance. Classical evolution methods involving strain enrichment under selective pressure have been used with the goal of improving acetate tolerance in order to improve acetate production by Clostridium thermoaceticum (Reed et al. 1987) and A. aceti (Steiner and Sauer 2003a). This technique has also been used to increase acetate tolerance of Zymomonas mobilis (Joachimsthal et al. 1998) and E. coli (Steiner and Sauer 2003b; Fernández-Sandoval et al. 2012). Improving acetate tolerance in E. coli has also been addressed through the use of global transcription machinery engineering (gTME) (Chong et al. 2013) and scalar analysis of library enrichments (SCALES) (Sandoval et al. 2011). Insights into the mechanisms for increased tolerance by these strains are discussed below in the BMechanisms of tolerance to acetic acid stress^ section.
Things that acetate does Acetate leads to cross protection to other stresses As with many inhibitors, adaptation to acetate primes tolerance to further acetate challenge; this has been reported for E. coli O157:H7 (Brudzinski and Harrison 1998) and Acetobacter senegalensis (Shafiei et al. 2013). However, short-term adaptation to acetate can also prime tolerance to a variety of other stressors. In the case of Salmonella typhimurium, 1 h of adaptation to 100 mM acetate at pH 7.0 increased survival of this bacterium during challenge with HCl at pH 3.0 (Kwon and Ricke 1998). Similar experiments performed with E. coli K12 and E. coli O157:H7 showed that pre-exposure to acetic acid stress increased survival during challenge with citric acid at pH 3.0 (Arnold et al. 2001). E. coli’s adaptation to acetate challenge also enabled increased tolerance to oxidative stress and thermal challenge (Arnold et al. 2001). It was also found that adaptation of Pseudomonas putida S12 to acetate challenge increased tolerance to several organic solvents, including decane, styrene, and toluene (Weber et al. 1993).
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This activation of a variety of stress response systems is consistent with the observed increase in expression of stress response pathways in E. coli following acetate exposure and adaptation (Arnold et al. 2001; Kirkpatrick et al. 2001; Polen et al. 2003). Specifically, the activated pathways in response to acetic acid were found to be regulated by the alternative sigma factor RpoS. Thus, it is not surprising that deletion of rpoS prevents priming of acid tolerance during acetate challenge (Arnold et al. 2001). Diffusion of acetic acid through the cell membrane Acetic acid is a weak one carbon-chain carboxylic acid with a moderate pKa value of 4.75. The acid dissociation constant directly connects the amount of protonated and ionic forms of acetic acid as a function of the pH value of the cultivation medium. This function is mathematically described by the Henderson-Hasselbalch equation (Hasselbalch 1917): pH ¼ pKa þ log
½A‐ ½HA
According to the Henderson-Hasselbalch equation, 50 % of the acid is protonated at the pH of pKa value. Thus, at a pH below 4.75, most of the acetic acid is in its undissociated form (CH3COOH), 90 % of it being protonated at a pH of 3.75 and 99 % protonated when the pH drops to 2.75. Due to their lipophilicity, the undissociated acetic acid molecules can permeate the cell membrane simply by passive diffusion, thereby dissociating directly in the cytosol. This is a marked difference from what occurs with strong acids like HCl, which acidify the growth medium but are not efficient in the acidification of microbial cytosol as protons diffuse poorly through the cell envelope. The accumulation of protons in the cytosol causes a decrease of internal pH to values that may not be tolerable to the cell, thereby leading to a strong reduction of metabolic activity and, eventually, to cell death (Axe and Bailey 1995; Russell 1991a). As described below, another consequence of cytosol acidification is dissipation of the electrochemical gradient maintained across the cell membrane, this being an essential demand for secondary transport. Various effects of anion accumulation As described above, one of the most dramatic effects of acetic acid entry into the cell is the acidification resulting from the protons released upon chemical dissociation of the acid. However, the acetate that is released upon dissociation of acetic acid also accumulates at high values inside microbial cells, this event being impactful on cell physiology. When held at a constant external pH, E. coli K12 accumulates
intracellular acetate in a roughly linear response to increasing acetate dosage (Diez-Gonzalez and Russell 1997). Intracellular pools of 500 mM were measured in the presence of 80 mM acetate at pH 5.9 (Diez-Gonzalez and Russell 1997) and greater than 3 M for 340 mM external acetate at pH 6.5 or pH 5.5 (Steiner and Sauer 2003a). A detailed characterization during challenge with 8 mM acetate at pH 6.0 showed that the internal acetate pool can reach 170 mM within just 2 min (Roe et al. 1998). While E. coli O157:H7 pool sizes were similar to E. coli K12 at low external acetate concentrations, the intracellular concentration leveled off at approximately 300 mM when the external concentration surpassed 40 mM (DiezGonzalez and Russell 1997). Not surprisingly, this acetate anion accumulation results in increased membrane potential; a dose-dependent response was observed during E. coli K12 challenge with increasing amounts of acetate at pH 6.8 (Bakker and Mangerich 1983). The accumulation of acetate is accompanied by perturbations of other anion pools. Most notably, glutamate is depleted in E. coli K12 cells under acetic acid stress (Roe et al. 1998). In specific, E. coli cells challenged with 8 mM acetate at pH 6.0 exhibited internal glutamate pools of 10 mM, comparing to a 60 mM pool of glutamate registered in unstressed cells (Roe et al. 1998). The drop in glutamate internal concentration occurred concomitantly with the increase in internal acetate concentration (Roe et al. 1998). Glutamate, along with potassium ions, is an important regulator of cell turgor (Wood 2011) which is expected to increase as a result of the accumulation of acetate in the internal environment. No change in potassium pools was observed in response to less than 40 mM acetate; however, when cells were exposed to higher concentrations, accumulation of K+ was, in fact, observed (Roe et al. 1998; Diez-Gonzalez and Russell 1997). At lower external pH values, E. coli K12 showed a larger deficit in ATP content for acetate-challenged cells relative to the corresponding noacetate control (Bakker and Mangerich 1983), indicating that acetic acid stress might lead to energy depletion in this bacterium. Inhibition of the methionine biosynthetic pathway Observations that methionine supplementation provide a protective effect for E. coli against acetate (Han et al. 1993) (described below in the BOther environmental components^ section) inspired the observation that homocysteine accumulates during acetate challenge. While control cultures had intracellular homocysteine pools of 0.1 mM, cells challenged with 8 mM acetate at pH 6.0 exhibited pools of 1.6 mM (Roe et al. 2002). Homocysteine is toxic in its own right, and a pool of 1.6 mM is associated with a 3-fold reduction in specific growth rate (Roe et al. 2002). Homocysteine is an intermediate of methionine biosynthesis, and this accumulation is thought to be due to inhibition of methionine biosynthesis
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enzymes, though the mechanism remains unknown. Methionine supplementation presumably provides protection due to feedback inhibition of the entire methionine biosynthetic pathway, preventing production and therefore resulting in accumulation of homocysteine. Improvement of acetate tolerance in E. coli via modification of the methionine biosynthesis enzymes has been performed through the isolation of improved versions of MetA and MetE enzymes which catalyze, respectively, the synthesis of methionine production from homoserine and the conversion of homocysteine to methionine. Strains expressing an improved metA showed increased tolerance of 30 mM acetic acid and also showed improved thermotolerance (Mordukhova et al. 2008), while strains expressing the improved metE showed improved tolerance of acetate and propionate, but not benzoate (Mordukhova and Pan 2013).
Environmental factors In addition to genetic factors that contribute to enhance tolerance to acetic acid, environmental conditions may also play a crucial role in mitigating the deleterious effects of this weak acid or in potentiating them. External pH Expectedly, environmental pH is critical for acetate toxicity due to its influence on the distribution between the protonated and non-protonated forms of the acid. Since teasing out the difference between acetate dose and pH has proven problematic in some studies, here we only describe experiments where the total amount of acetic acid provided was maintained constant and the external pH was changed. The increased toxicity of acetic acid at lower external pH values has been reported for a variety of organisms including E. coli O157:H7 (Oh et al. 2009), ethanologenic E. coli strains (Takahashi et al. 1999; Zaldivar and Ingram 1999), rumen organisms like Streptococcus bovis (Russell 1991b), and Megasphaera elsdenii (Miyazaki et al. 1991). The intracellular acidification caused by acetic acid in different organisms is presented in Table 1. Studies of the pH effect have gone beyond inhibition of growth. During challenge of E. coli BL21 at a variety of acetate concentrations, the intracellular acetate concentration was reduced in half when cells were grown at pH 7.5 instead of 6.5 (Wang et al. 2014), confirming that acetate internal accumulation depends of the diffusion of the undissociated form. The external pH had a roughly linear influence on the proton motive force and total transmembrane chemical gradient of Streptococcus bovis during acetic acid challenge (Russell 1991b).
Oxygen availability Different results have been reported in different studies, thereby remaining to be fully elucidated on how oxygen availability modulates bacterial tolerance to acetic acid. Results obtained in various enterobacteria and in Listeria monocytogenes show that when these cells are subjected at 30 °C for 5 days to acetic acid stress at a range of external pH values, oxygen availability seems to decrease acetate toxicity (Ostling and Lindgren 1993). Similarly, 24-h survival of E. coli O157:H7 in the presence of acetic acid (provided at 32 °C at a pH 3.5 or 4.0) increased in shaken cultures relative to static cultures, though this difference was not observed in cultures previously adapted to acetic acid stress (Brudzinski and Harrison 1998). Contrastingly, survival of E. coli O157:H7 isolates challenged with 400 mM acetic acid at pH 3.3 and 30 °C for 25 min was significantly increased under anaerobic conditions relative to aerobic (Oh et al. 2009). This difference in trends could be due to the different experimental conditions, particularly in what regards to the different acid exposure times. Survival of Salmonella typhimurium anaerobic cultures adapted to 100 mM acetic acid for 1 h and then subjected to 1 h of HCl challenge at pH 3.0 and 37 °C increased relative to aerobic cultures subjected to the same procedure (Kwon and Ricke 1998), though this could be more informative regarding acid tolerance than acetic acid tolerance. Viability studies of A. aceti in the presence of 55 g/L acetic acid showed a 4fold increase in cell viability with 1 ppm oxygen relative to 15 ppm (Park et al. 1989). These same studies concluded that the optimum dissolved oxygen level for production of more than 45 g/L acetic acid was 1–3 ppm (Park et al. 1989).
Temperature The effect of temperature on acetic acid toxicity has mainly been studied in E. coli O157:H7, though also with mixed results. Studies performed at pH 3.5 and 3.3 showed that acetic acid toxicity increased when growth temperatures of 30 °C or greater were used (Brudzinski and Harrison 1998; Oh et al. 2009). Similarly, studies performed in cucumber puree with acetic acid at pH
