Vibrio natriegens is sensitive to its acidic fermentation products

Vibrio natriegens is sensitive to its acidic fermentation products | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Vibrio natriegens is sensitive to its acidic fermentation products View ORCID Profile Nicholas W. Haas , View ORCID Profile James B. McKinlay doi: https://doi.org/10.1101/2025.08.29.673078 Nicholas W. Haas 1 Department of Biology, Indiana University , Bloomington, IN Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicholas W. Haas James B. McKinlay 1 Department of Biology, Indiana University , Bloomington, IN Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for James B. McKinlay For correspondence: jmckinla{at}iu.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Vibrio natriegens is an emerging bacterial platform for a range of biotechnological applications due to its rapid growth and ease of genetic manipulation. Whereas much has been learned about V. natriegens aerobic physiology, comparatively little is known about its anaerobic fermentative physiology, despite the relevance to many industrial conditions. We compared the metabolic parameters of V. natriegens versus another biotechnologically relevant bacterium Escherichia coli under fermentative conditions. Both species excreted a similar array of fermentation products but V. natriegens consumed less glucose and had a lower product titer. V. natriegens also exhibited rapid death, reaching extinction within 12 hours after the growth phase, 3 days sooner than E. coli . Rapid V. natriegens death was avoided, and glucose consumption and product titers improved, by increasing the buffering capacity of the growth medium, indicating that V. natriegens is comparatively sensitive to its organic acid fermentation products. Indeed, the V. natriegens genome lacks nearly all the acid resistance genes that have been characterized in E. coli . Our findings thus highlight an acid sensitivity that will need to be considered when designing fermentative applications of V. natriegens . IMPORTANCE Bioprocessing, the biological conversion of renewable resources into value-added chemicals, is poised to meet an increasing demand for sustainable alternatives to petroleum-based products. Many examples of bioprocessing feature anoxic fermentations that naturally maximize product formation relative to growth of the microbial catalyst. Vibrio natriegens is a facultatively fermentative bacterium that has gained attention for bioprocessing due to its rapid growth rate and ease of genetic engineering. However, the fermentative properties of V. natriegens have not been compared to traditional bioprocessing workhorses like Escherichia coli . We revealed that V. natriegens is comparatively sensitive to its own acidic fermentation products, likely because V. natriegens lacks acid resistance mechanisms possessed by E. coli . Thus, fermentative applications must address this sensitivity either by buffering the fermentations, engineering resistance mechanisms, or bypassing the sensitivity by engineering V. natriegens to produce neutral products. INTRODUCTION Bioprocessing utilizes native or engineered microbes to generate value-added products from renewable resources, offering sustainable alternatives to petroleum-based counterparts ( 1 – 5 ). A diverse set of microbes have been proposed as platforms, or chassis, for bioprocessing ( 6 ). The marine bacterium Vibrio natriegens has increasingly gained attention as a chassis for bioprocessing primarily due to its rapid growth and metabolism ( 7 – 9 ) and the relative ease with which combinatorial genetics can be performed ( 10 – 13 ). Proof-of-concept studies have demonstrated that V. natriegens can been engineered to produce a variety of value-added products from a range of carbon substrates ( 9 ). However, whereas many bioprocesses use anoxic fermentative conditions to maximize product yields and minimize microbial growth, most V. natriegens studies have used oxic conditions, with a few exceptions ( 8 , 14 , 15 ). Under anoxic conditions with glucose, V. natriegens carries out a mixed-acid fermentation, excreting ethanol and a variety of organic acids ( Fig 1 ) ( 8 ). The accumulation of organic acids acidifies the medium. In general, acid stress negatively impacts metabolism, viability, and product yield ( 16 ). Even under aerobic conditions, V. natriegens growth is impacted by acidification from acetic acid that accumulates due to overflow metabolism ( 17 ). Thus, it is expected that negative impacts would be exacerbated under anoxic fermentative conditions where there is a greater accumulation of organic acids. Download figure Open in new tab Fig 1. V. natriegens fermentative metabolism. Reactions are based on NCBI gene annotations. Red, acidic products (named here for their carboxylate forms); green, neutral products. Whereas other Vibrio s like V. cholerae can switch to neutral fermentation products in response to acidic conditions ( 18 ), production of neutral fermentation products by V. natriegens has not been observed ( 8 ). Fermentative bacteria like Escherichia coli have an array of mechanisms to deal with acid stress ( 19 , 20 ). For example, E. coli can offset acidosis by converting formic acid into H2 and CO2 using formate hydrogen lyase (FHL), converting an organic acid into two gasses ( 21 ). In the presence of certain amino acids, E. coli can also eliminate cytoplasmic protons via amino acid decarboxylation reactions ( 22 ). As the medium acidifies during fermentation, E. coli also shifts its fermentation profile towards lactic acid ( 23 ), which might also offset acidosis by producing a single acid instead of the combination of acetic and formic acid ( 24 ). The extent to which V. natriegens can use these or other acid resistance mechanisms during mixed acid fermentation is unknown. Here, we compared the growth and metabolic parameters of V. natriegens versus E. coli under anoxic fermentative conditions. Whereas both organisms produced similar fermentation products, E. coli cultures consumed more glucose, accumulated more organic acids, and exhibited longer stationary phase viability. We conclude that V. natriegens is comparatively sensitive to fermentative organic acids due to a lack of acid resistance mechanisms. RESULTS and DISCUSSION V. natriegens and E. coli have similar fermentative product yields V. natriegens is often compared to E. coli , a bacterium that is well-established in synthetic biology, as a potential platform for bioprocessing ( 7 ). However, a direct comparison of growth and metabolic parameters under fermentative conditions has not been reported. We grew E. coli MG1655 and V. natriegens ATCC14048 in their respective anoxic minimal media with 25 mM glucose as the sole carbon source. The media only differed by a higher NaCl concentration for V. natriegens , and an optimum growth temperature of 30°C for V. natriegens versus 37°C for E. coli ( Fig 2 ). Despite the lower growth temperature, V. natriegens grew 1.3-times faster than E. coli ( Fig 2A-C ). Although V. natriegens reached a higher turbidity (OD660; Fig 2D ), E. coli has a higher ratio of CFU ml -1 : OD660 ( Fig 2E ) and was thus estimated to achieve a 1.2-fold higher actual cell density (CFU ml -1 ) than V. natriegens ( Fig 2F ). Download figure Open in new tab Fig 2. V. natriegens grows faster but to a lower cell density while producing a similar array of fermentation products as E. coli . E. coli (A) and V. natriegens (B) were grown in 60 ml of anoxic minimal media at their optimum growth temperatures and growth, glucose, and fermentation products were monitored. The same cultures were used to determine exponential growth rates (C) and final (highest) cell densities (D) . Standard curves to convert turbidity (OD600) measurements of cell density to CFU ml -1 (E) suggests that the difference in final cell densities between the two species is opposite when considering turbidity (D) versus CFU ml -1 (F) . Error bars, SD; n = 3. Statistical differences were determined using an unpaired, two-tailed t test; ***, P < 0.001; ****, P < 0.0001. Late stationary phase (36 h) fermentation product yields between E. coli and V. natriegens were either statistically similar or exhibited small differences for the most abundant fermentation products: formic acid, no difference; ethanol, no difference; lactic acid, 1.06-fold higher for V. natriegens ; acetic acid, 1.20-fold higher for E. coli ( Fig 3A ). Both species produced lactic acid as they exited exponential phase, with continued production into stationary phase ( Fig 2A-B ). Greater fold-differences were observed for minor fermentation product concentrations ( Fig 2A , B) and yields ( Fig 3A ): (i) E. coli produced ∼ 1.3 mM succinic acid whereas levels from V. natriegens were near the detection limit, (ii) V. natriegens produced ∼ 1.7 mM pyruvate that was stable in the supernatant whereas E. coli transiently produced as much as 0.8 mM pyruvate, and (iii) E. coli produced ∼0.8 mM α-ketoglutarate, whereas none was detected in V. natriegens supernatants ( Fig 2A-B ). Another key difference was that V. natriegens did not produce H2 whereas E. coli is known to produce H2 as the pH drops below neutral ( 25 , 26 ). We thus omitted gaseous fermentation products from this study. Download figure Open in new tab Fig 3. V. natriegens consumes less glucose and produces less organic acids than E. coli . Fermentation product yields (A) , glucose consumed (B) , and total organic acid yields (C) , concentrations (D) , and concentrations normalized for cell density (E) , were determined using stationary phase (36 h) samples from Fig 2 cultures. Error bars, SD; n = 3. Statistical differences were determined using an unpaired, two-tailed t test; ns, not significant; *, P < 0.05; **, P < 0.01. For, formic acid; EtOH, ethanol; Lac, lactic acid; Ace, acetic acid; Suc, succinic acid; Pyr, pyruvate. V. natriegens is more sensitive to acid than E. coli Neither strain fully consumed the glucose provided ( Fig 2A , B) but E. coli consumed 1.2-times more glucose than V. natriegens ( Fig 3B ). A possible reason is that each species reached their acid tolerance threshold and V. natriegens has a lower acid tolerance. Indeed, although each species had a similar total organic acid yield ( Fig 3C ), E. coli accumulated 1.2-fold more organic acids ( Fig 3D ) and 1.3-fold more organic acids per cell ( Fig 3E ). To compare the acid tolerance between the two species, we tracked cell viability by colony forming units (CFUs) during stationary phase ( Fig 4A ). Although cell death was not evident from turbidity measurements, CFU measurements indicated that both species ultimately went extinct. However, whereas viable E. coli cells were detected up to 82 h after inoculation, no viable V. natriegens cells were detected 24 h after inoculation, about 12 h after the end of the exponential growth phase ( Fig 4A ). Download figure Open in new tab Fig 4. V. natriegens is sensitive to acidic fermentation products compared to E. coli . (A) Culture turbidity (solid lines; OD660) was tracked in minimal media with 25 mM glucose over the entire experiment whereas viable cell density (dotted lines; CFU ml -1 ) was tracked from the end of the growth phase (∼12 h). All data points from the duplicate cultures are shown. (B) Viable cell counts were monitored in cell suspensions inoculated in late exponential phase to defined media lacking glucose with different concentrations of a 1:1 solution of acetic and formic acid. Data points, mean ± SD; n=3. ( B,C ) The limit of detection is 10 2 CFU ml -1 . (C) Death rates determined from the slope of a semi-log plot fitted to the data from panel B and two other similar experiments using different starting cell densities and incubation times. Horizonal line, mean ± SD; n=5-7. Statistical differences were determined using unpaired, two-tailed t tests; ns, not significant; *, P < 0.05. To verify that organic acids were a primary driver of V. natriegens cell death, we inoculated late exponential phase cultures ( V. natriegens , 0.65-0.69 OD660 ; E. coli , 0.82-0.87 OD660) to media lacking glucose but with different concentrations of a 1:1 mixture of acetic and formic acid. The cultures used to prepare these suspensions were grown with less glucose (15 mM) to ensure less acid exposure prior to the assay. Without added organic acids, both species showed little decline in cell viability ( Fig 4B , C). E. coli viability was also unaffected by 20 and 40 mM organic acids ( Fig 4B , C). In contrast, increasing the organic acid concentration progressively decreased V. natriegens viability ( Fig 4B ) and increased the death rate ( Fig 4C ). We then verified that the V. natriegens sensitivity to organic acids is dependent on the pH by increasing the buffering capacity of medium with either 100 or 200 mM 4-morpholinepropanesulfonic acid (MOPS) buffer. In each case, viable cell counts exceeded 10 8 CFU mL -1 at the onset of stationary phase, even though the turbidity was notably lower with 200 mM MOPS compared to the other conditions ( Fig 5A ). Unlike the rapid extinction observed when MOPS was excluded, 100 mM MOPS delayed extinction by 24 h and 200 mM MOPS avoided extinction within the 100-h time course ( Fig 5A ). Even though cells still went extinct with 100 mM MOPS, glucose was fully consumed by 24 h, with continued production of most fermentation products, especially lactic acid ( Fig 5B , C). Thus, we conclude that the rapid extinction of V. natriegens upon reaching stationary phase under fermentative cultures is due to acid sensitivity. Download figure Open in new tab Fig 5. Increasing the buffer concentration of the growth medium alleviates V. natriegens organic acid sensitivity. (A) V. natriegens culture turbidity (solid lines; OD660) was tracked in minimal media with different concentrations of glucose and MOPS over the entire experiment whereas viable cell density (dotted lines; CFU ml -1 ) was tracked from the end of the growth phase (∼12 h). The ‘0 mM MOPS’ data is the same as in Fig 4A . Each data point represents the mean ± range; n=2. The limit of detection is 10 2 CFU ml -1 . (B,C) Remaining glucose and accumulated fermentation products from V. natriegens cultures grown with 25 mM glucose in ‘early’ stationary phase ( B , ∼ 12 h) and ‘late’ stationary phase ( c , ∼ 24 h). Error bars, mean ± SD; n=3. Statistical differences were determined using unpaired, two-tailed t tests; ns, not significant; *, P < 0.05. Glc, glucose; For, formic acid; EtOH, ethanol; Lac, lactic acid; Ace, acetic acid; Suc, succinic acid; Pyr, pyruvate. V. natriegens lacks acid resistance mechanisms We sought an explanation for the stark difference between V. natriegens and E. coli survival in acidic conditions. Bacteria like E. coli and V. cholerae have mechanisms to deal with acid stress both during fermentative conditions and to transit the highly acidic stomach ( 19 , 20 ). To determine the acid resistance repertoire of V. natriegens , we performed BLASTp searches ( 27 ) using E. coli and V. cholerae acid resistance proteins as query sequences ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. V. natriegens acid tolerance proteins Based on our homolog threshold criteria of 25% identity over 50% of the query sequence, V. natriegens likely has several proteins to respond to cell envelope stress, which could include acid stress, but also other stressors, like heat. For example, we identified a σ E homolog and associated proteins that, in E. coli , activates a regulon in response to denatured outer membrane proteins, and the Cpx two component system that responds to periplasmic and inner membrane stress signals ( 28 ). Among genes of the E. coli Cpx regulon, V. natriegens also has cfa , which encodes a protein that produces cyclopropane fatty acids that can protect against various stressors, including acidity ( 29 ). However, V. natriegens appears to lack many of the chaperones and proteases (i.e., degP , hdeABD , skp ) that are crucial in E. coli for the proper folding or degradation of cell envelope proteins under stress conditions ( 30 – 32 ). When considering specific responses to acid stress, the V. natriegens genome suggests that it is even less well-equipped. FHL, a complex of formate dehydrogenase (FDH) and hydrogenase, converts formic acid into CO2 and H2, thereby converting an acidic fermentation product into two gasses ( 21 , 25 ). Whereas V. natriegens has FDH protein homologs, V. natriegens does not have hydrogenase genes. In agreement with this bioinformatic result, no H2 was detected in V. natriegens fermentative cultures by gas chromatography. V. natriegens also lacks amino acid decarboxylases that can eliminate cytoplasmic protons ( 19 , 20 , 22 ), but it has one of two H + /Cl - antiporters that help prevent hyperpolarization of the membrane during the use of amino acid decarboxylases ( 33 ). Regardless, we would not expect amino acid decarboxylases to be effective in our minimal medium that did not contain amino acids. Lactic acid production prolongs growth and metabolism but does not improve survival Organisms that produce multiple fermentation products can alter their fermentation profile to respond to environmental conditions. Unlike V. cholerae , V. natriegens lacks genes for producing neutral fermentation products ( Table 1 ) ( 18 ). However, like E. coli , V. natriegens produced lactic acid starting in late exponential phase and into stationary phase ( Fig 2A-B ). In E. coli , cytoplasmic lactate dehydrogenase (LDH) expression increases in response to acidic pH ( 23 ), suggesting that lactic acid production might decrease acid stress ( 24 ). Shifting to lactic acid production would result in less ATP but also less acid overall to maintain electron balance ( Fig 6A ); 2 pyruvate could either go to 2 lactatic acid (pKa, 3.86) or to 1 ethanol + 2 formic acid (pKa, 3.75) + 1 acetate (pKa, 4.76). The latter pathway would generate more organic acids, one of which is acetic acid that would more readily deprotonate due to its high pKa, and which is potentially more toxic than lactic acid in general ( 34 ). Download figure Open in new tab Fig 6. V. natriegens LDH extends fermentative metabolism and growth but not viability. (A) Lactic acid production can hypothetically achieve electron balance while producing less acid (2 lactic acid; Lac; left) via LDH compared to producing ethanol, formic acid, and acetic acid (right). Comparison of ‘late’ (24 h) fermentation product yields (B) , glucose consumed (C) , exponential growth rate (D) , final (highest) cell density (E) , and total organic acid yield (F) between the parent strain (WT Δ dns ::Spec R ) and the LDH mutant (Δ dldh ) in a minimal medium with 25 mM glucose and no MOPS. For, formic acid; EtOH, ethanol; Lac, lactic acid; Ace, acetic acid; Suc, succinic acid; Pyr, pyruvate. (C,E,F) Early, 9 h exponential phase samples; Late, 24 h stationary phase samples. (G) Turbidity (solid lines; OD660) and viable cell density (dotted lines; CFU ml -1 ) were tracked into stationary phase in minimal medium with 25 mM glucose plus 100 mM MOPS; each data point is the mean ± SD. The limit of detection is 10 2 CFU ml -1 . (B-G) Error bars, SD; n=3. Statistical differences were determined using unpaired, two-tailed t tests; ns, not significant; *, P < 0.05; **, P < 0.01. To test whether lactic acid production contributes to V. natriegens survival under fermentative conditions, we first created an LDH mutant. V. natriegens has three different putative LDH genes, BA890_15740 , BA890_20630 ( dldh ), and BA890_20645 ( lldh ). The latter two genes had been deleted by another group and had little effect on fermentative lactic acid production ( 8 ). However, the conditions in that study favored succinic acid production over lactic acid production whereas we observed the opposite. Using the fermentative LDH gene from E. coli MG1655 (P52643) as a BLASTp query, we hypothesized that dldh was most likely the V. natriegens fermentative LDH (100% coverage, 62% identity); the other two candidates did not show significant sequence identity to the query sequence. Indeed, when we deleted V. natriegens dldh , the lactic acid yield was close to zero at 24 h ( Fig 6B ). Compared to the parent strain, the Δ dldh mutant produced 1.2-times more formic acid and 1.3-times more ethanol ( Fig 6B ), a route that would replace the electron balancing role of lactic acid production ( Fig 6A ). The Δ dldh mutant also produced 2.8-times more pyruvate, suggesting that a bottleneck was created in the absence of LDH. The Δ dldh mutant consumed less glucose than the parent ( Fig 6C ), which did not affect the growth rate ( Fig 6D ), but the final Δ dldh mutant turbidity was 90% of that of the parent ( Fig 6E ). Still, the total organic acid yield was not significantly different between the two strains, suggesting that even without lactic acid production, V. natriegens faced comparable acid exposure ( Fig 6F ). We thus examined the effect of the LDH deletion on V. natriegens stationary phase survival under fermentative conditions. Anticipating that the rapid death rate would make a comparative analysis difficult, we added 100 mM MOPS to slow the death rate; MOPS does not affect stationary phase lactic acid production ( Fig 5B , C). Stationary-phase survival was similar between the parent and Δ dldh strains ( Fig 6G ). Thus, whereas lactic acid production improves growth ( Fig. 6E, G ) and extends glucose consumption into stationary phase, it does not contribute to acid tolerance under fermentative conditions. Below we speculate why lactic acid might be produced during the transition out of exponential growth and into stationary phase. One possibility is that acetate and ethanol production become less available as an option. For example, as the pH drops and growth slows, the biosynthetic demand for ATP would also slow. This might limit ADP availability for acetate production and create a bottleneck at pyruvate ( Fig 6A ). LDH offers a carbon and electron sink to maintain metabolic flow and help alleviate this bottleneck ( Fig 6A ); a role for LDH in addressing this bottleneck would also explain the elevated pyruvate excretion in the Δ dldh mutant ( Fig 6B ). If we assume similar kinetic parameters to E. coli enzymes, LDH is poised to respond to pyruvate accumulation and low pH; as the pH drops from neutral to 5, LDH activity increases 1.6-fold and the relatively low affinity (Km) for pyruvate improves from 7.2 to 5.0 mM. Although PFL has a higher affinity for pyruvate (Km, 2.0 mM) the optimal pH for activity is 7.5, and would thus worsen if the cytoplasm begins to acidify ( 35 – 37 ). Conclusion As interest grows in V. natriegens as a bioprocessing strain, one must weigh its favorable attributes of rapid growth and genetic tractability against its less favorable attributes. Here we report that, relative to E. coli , V. natriegens is comparatively sensitive to the acidic conditions that arise during mixed acid fermentations. This difference in acid tolerance is likely due to V. natriegens lacking most of the acid resistance mechanisms that have been characterized in E. coli . Such acid-resistance mechanisms could potentially be engineered to improve V. natriegens acid tolerance. Increasing the buffering capacity of the fermentation medium would also avert acid sensitivity, but one would need to consider the cost of buffer under industrial conditions. Thus, acid sensitivity will need to be considered when engineering V. natriegens for bioprocessing, unless the goal is to engineer V. natriegens to produce neutral products. MATERIALS AND METHODS Strains Experiments used the wild-type strains Escherichia coli MG1655 ( 38 ) and Vibrio natriegens ATCC 14048 ( 10 ) or its derivatives. V. natriegens mutants were made as described ( 10 ), except the transformation mixtures of cells and PCR products were incubated statically at 30°C for 12-16 h before outgrowth, and selection plates used 250 µg mL -1 spectinomycin. The V. natriegens Δ dldh mutant was made in V. natriegens TND1964, which is the wild-type strain with pMMB- tfoX that allows for IPTG-inducible competency ( 10 ). Briefly, a Δ dldh (Δ20530) construct was made by PCR amplifying (Q5 DNA polymerase; New England Biolabs) 3kb upstream and downstream fragments. The two fragments were then combined by splicing-by-overlap extension PCR using the outermost primers. The Δ dldh construct (1 µg) was then co-transformed into TND1964 with 50 ng of a DNA fragment containing a spectinomycin resistance cassette flanked by ∼1 kb of homology to the upstream and downstream regions of the dns locus ( BA890_12415 )amplified from V. natriegens SAD1306 genomic DNA ( 10 ). The ‘parent’ strain (NWH004) used as the control strain in experiments with the Δ dldh mutant was derived from TND1964 by introducing a spectinomycin resistance cassette at the dns locus. Colonies were screened by PCR using OneTaq DNA polymerase (New England Biolabs). Mutations were confirmed by Sanger sequencing. Primers are in Table 2 . View this table: View inline View popup Download powerpoint Table 2. Primers used in this study Growth conditions Colonies were grown from 25% glycerol frozen stocks on agar plates of either lysogeny broth medium (LB; E. coli ), or the media supplemented with an additional 20 g NaCl L -1 (LB3; V. natriegens ). LB3 was supplemented with either 100 µg mL -1 carbenicillin, 250 µg mL -1 spectinomycin, or 100 µM IPTG when appropriate. LB3 was also used routinely for strain construction. For all other experiments anoxic minimal M9-derived coculture medium (MDC) ( 39 ) for E. coli or MDC with an additional 80 mM NaCl for V. natriegens (MDC80) was prepared by aliquoting 10 ml media into 27-ml anaerobic tubes or 60 ml media into 150-ml serum vials, bubbling with N2, then sealing with rubber stoppers and aluminum crimps before autoclaving. Media supplements were then added by syringe to the following final concentrations from anoxic 100X stock solutions: glucose, 25 mM; NH4Cl, 10mM; cation solution 1% v/v (1 mM MgSO4 and 0.1 mM CaCl2). A 2 M anoxic MOPS stock solution (pH 7.0) was filter sterilized into a serum vial and then flushed with filter-sterilized N2 and then added to cultures to the indicated concentration after autoclaving, with sterile ultrapure water also added where necessary to have common dilution effects between cultures. Starter cultures were inoculated to MDC or MDC80 from single colonies. Late-exponential phase starter cultures (0.65-0.85 OD660) were then used to inoculate test media to an initial cell density of ∼0.01 OD660. Tubes were incubated horizontally at either 30°C ( V. natriegens ) or 37°C ( E. coli ) with shaking at 150 rpm. Analytical procedures A Genesys 20 spectrophotometer (Thermo-Fisher) was used to measure cell density at 660 nm (OD660) either directly in the 27-ml culture tubes or in 1-ml samples in cuvettes when 150-ml serum vials were used. Growth rates were determined by fitting an exponential trendline to turbidity measurements taken between 0.1-0.8 OD660 using GraphPad Prism v6. Glucose and fermentation products were quantified in supernatant samples by HPLC (Shimadzu) as described ( 40 ). Temporal assessment of fermentations was performed in 60-mL cultures in 150-mL serum vials. Samples (1 ml) were taken at regular intervals; air contamination was minimized by flushing a 1-mL syringe and needle with filter-sterilized N2 before sampling. To check for H2, 0.1 ml of headspace gas was sampled using a gas-tight syringe and injected into a Shimadzu GC-2014 gas chromatograph with a thermal conductivity detector as described ( 41 ). Viable cell counts were quantified as CFUs using the track plate method ( 42 ). Samples (0.4-0.5 ml) were then removed from 10-ml cultures using an N2-flushed syringe and serially diluted in oxic MDC or MDC80 in a 96-well plate. The last six dilutions were spotted (10 µL) onto a LB or LB3 agar using a multichannel pipette. A minimum of 1 colony a cutoff, making the limit of detection 100 CFU mL -1 for an undiluted sample. For experiments using nongrowing cells, late-exponential phase cultures (0.65-0.85 OD660) were inoculated to anoxic MDC or MDC80 with all supplements except glucose. The target inoculum was 10 7 cells mL -1 . The pH was decreased by adding the appropriate volume of a solution containing 1 M glacial acetic acid plus 1 M formic acid by syringe. Cell suspensions were then sampled for CFU measurements. Death rates were determined from the slope of a semi-log equation (linear X, log Y) fitted to experimental data using GraphPad Prism v6. Bioinformatics NCBI’s BLASTp ( 27 ) was used to identify putative acid-resistance genes in V. natriegens ATCC14048 (NCBI RefSeq: GCF_001456255.1) using amino acid sequences from E. coli MG1655 (NCBI RefSeq: GCF_000005845.2) or V. cholerae (GCF_000969265.1). V. natriegens proteins that had >25% identity with >50% query coverage were considered putative homologs. Statistics. GraphPad Prism v6 was used for all statistical analyses. Acknowledgments This work was supported in part by the National Science Foundation CAREER award MCB-1749489. NWH was supported by a IU College of Arts and Sciences John and Wendy Kindig Fellowship. We thank A. Dalia and the Dalia lab for bacterial strains, resources, and guidance for V. natriegens genetics. We also thank C. Fuqua, C. Landeta, and the McKinlay lab for helpful discussions. We declare no conflicts of interest. Funder Information Declared U.S. National Science Foundation, https://ror.org/021nxhr62 , MCB-1749489 REFERENCES 1. ↵ Epps TH , 3rd . , Korley LTJ , Yan T , Beers KL , Burt TM . 2022 . Sustainability of synthetic plastics: considerations in materials life-cycle management . JACS Au 2 : 3 – 11 . OpenUrl PubMed 2. Liao JC , Mi L , Pontrelli S , Luo S . 2016 . Fuelling the future: microbial engineering for the production of sustainable biofuels . Nat Rev Microbiol 14 : 288 – 304 . OpenUrl CrossRef PubMed 3. McKinlay JB , Vieille C , Zeikus JG . 2007 . Prospects for a bio-based succinate industry . Appl Microbiol Biotechnol 76 : 727 – 40 . OpenUrl CrossRef PubMed Web of Science 4. Nielsen J . 2019 . Cell factory engineering for improved production of natural products . Nat Prod Rep 36 : 1233 – 6 . OpenUrl PubMed 5. ↵ Xu X , Liu Y , Du G , Ledesma-Amaro R , Liu L . 2020 . Microbial chassis development for natural product biosynthesis . Trends Biotechnol 38 : 779 – 96 . OpenUrl PubMed 6. ↵ Chi H , Wang X , Shao Y , Qin Y , Deng Z , Wang L , Chen S . 2019 . Engineering and modification of microbial chassis for systems and synthetic biology . Synth Syst Biotechnol 4 : 25 – 33 . OpenUrl PubMed 7. ↵ Weinstock MT , Hesek ED , Wilson CM , Gibson DG . 2016 . Vibrio natriegens as a fast-growing host for molecular biology . Nat Methods 13 : 849 – 51 . OpenUrl CrossRef PubMed 8. ↵ Hoffart E , Grenz S , Lange J , Nitschel R , Muller F , Schwentner A , Feith A , Lenfers-Lucker M , Takors R , Blombach B . 2017 . High substrate uptake rates empower Vibrio natriegens as production host for industrial biotechnology . Appl Environ Microbiol 83 : e01614 – 17 . OpenUrl PubMed 9. ↵ Hädrich M , Schulze C , Hoff J , Blombach B . 2024 . Vibrio natriegens : application of a fast-growing halophilic bacterium , Adv Biochem Eng Biotechnol , 20241112 ed doi: 10.1007/10_2024_271 . Springer , Berlin, Heidelberg . OpenUrl CrossRef 10. ↵ Dalia TN , Hayes CA , Stolyar S , Marx CJ , McKinlay JB , Dalia AB . 2017 . Multiplex genome editing by natural transformation (MuGENT) for synthetic biology in Vibrio natriegens . ACS Synth Biol 6 : 1650 – 5 . OpenUrl CrossRef PubMed 11. Glasgo LD , Lukasiak KL , Zinser ER . 2024 . Expanding the capabilities of MuGENT for large-scale genetic engineering of the fastest-replicating species, Vibrio natriegens . Microbiol Spectr 12 : e0396423 . OpenUrl 12. Wu F , Chen W , Peng Y , Tu R , Lin Y , Xing J , Wang Q . 2020 . Design and reconstruction of regulatory parts for fast-growing Vibrio natriegens synthetic biology . ACS Synth Biol 9 : 2399 – 2409 . OpenUrl CrossRef PubMed 13. ↵ Faber A , Politan RJ , Stukenberg D , Morris KM , Kim R , Jeon E , Inckemann R , Becker A , Thuronyi B , Fritz G . 2025 . Expanding genetic engineering capabilities in Vibrio natriegens with the Vnat Collection . Nucleic Acids Res 53 : gkaf580 . OpenUrl PubMed 14. ↵ Thoma F , Schulze C , Gutierrez-Coto C , Hädrich M , Huber J , Gunkel C , Thoma R , Blombach B . 2022 . Metabolic engineering of Vibrio natriegens for anaerobic succinate production . Microb Biotechnol 15 : 1671 – 84 . OpenUrl PubMed 15. ↵ Schulze C , Hädrich M , Borger J , Rühmann B , Doring M , Sieber V , Thoma F , Blombach B . 2023 . Investigation of exopolysaccharide formation and its impact on anaerobic succinate production with Vibrio natriegens . Microb Biotechnol 17 : e14277 . OpenUrl PubMed 16. ↵ Lund PA , De Biase D , Liran O , Scheler O , Mira NP , Cetecioglu Z , Fernández EN , Bover-Cid S , Hall R , Sauer M , O’Byrne C . 2020 . Understanding how microorganisms respond to acid pH is central to their control and successful exploitation . Front Microbiol 11 : 556140 . OpenUrl PubMed 17. ↵ Forsten E , Gerdes S , Petri R , Büchs J , Magnus J . 2024 . Unraveling the impact of pH, sodium concentration, and medium osmolality on Vibrio natriegens in batch processes . BMC Biotechnol 24 : 63 . OpenUrl PubMed 18. ↵ Hawver LA , Giulietti JM , Baleja JD , Ng WL . 2016 . Quorum sensing coordinates cooperative expression of pyruvate metabolism genes to maintain a sustainable environment for population stability . mBio 7 : e01863 – 16 . OpenUrl PubMed 19. ↵ Yang J , Zhang J , Zhu Z , Du G . 2021 . The challenges and prospects of Escherichia coli as an organic acid production host under acid stress . Appl Microbiol Biotechnol 105 : 8091 – 107 . OpenUrl 20. ↵ Foster JW . 2004 . Escherichia coli acid resistance: tales of an amateur acidophile . Nat Rev Microbiol 2 : 898 – 907 . OpenUrl CrossRef PubMed Web of Science 21. ↵ Noguchi K , Riggins DP , Eldahan KC , Kitko RD , Slonczewski JL . 2010 . Hydrogenase-3 contributes to anaerobic acid resistance of Escherichia coli . PLoS One 5 : e10132 . OpenUrl CrossRef PubMed 22. ↵ Richard H , Foster JW . 2004 . Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential . J Bacteriol 186 : 6032 – 41 . OpenUrl Abstract / FREE Full Text 23. ↵ Mat-Jan F , Alam KY , Clark DP . 1989 . Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase . J Bacteriol 171 : 342 – 8 . OpenUrl Abstract / FREE Full Text 24. ↵ Kirkpatrick C , Maurer LM , Oyelakin NE , Yoncheva YN , Maurer R , Slonczewski JL . 2001 . Acetate and formate stress: opposite responses in the proteome of Escherichia coli . J Bacteriol 183 : 6466 – 77 . OpenUrl Abstract / FREE Full Text 25. ↵ Rossmann R , Sawers G , Bock A . 1991 . Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon . Mol Microbiol 5 : 2807 – 14 . OpenUrl CrossRef PubMed Web of Science 26. ↵ Sangani AA , McCully AL , LaSarre B , McKinlay JB . 2019 . Fermentative Escherichia coli makes a substantial contribution to H 2 production in coculture with phototrophic Rhodopseudomonas palustris . FEMS Microbiol Lett 366 : fnz162 . OpenUrl PubMed 27. ↵ Altschul SF , Gish W , Miller W , Myers EW , Lipman DJ . 1990 . Basic local alignment search tool . J Mol Biol 215 : 403 – 10 . OpenUrl CrossRef PubMed Web of Science 28. ↵ Grabowicz M , Silhavy TJ . 2017 . Envelope stress responses: an interconnected safety net . Trends Biochem Sci 42 : 232 – 42 . OpenUrl CrossRef PubMed 29. ↵ Chen YY , Ganzle MG . 2016 . Influence of cyclopropane fatty acids on heat, high pressure, acid and oxidative resistance in Escherichia coli . Int J Food Microbiol 222 : 16 – 22 . OpenUrl CrossRef PubMed 30. ↵ He D , Zhang M , Liu S , Xie X , Chen PR . 2019 . Protease-mediated protein quality control for bacterial acid resistance . Cell Chem Biol 26 : 144 – 150 e3. OpenUrl PubMed 31. Yu XC , Yang C , Ding J , Niu X , Hu Y , Jin C . 2017 . Characterizations of the interactions between Escherichia coli periplasmic chaperone HdeA and its native substrates during acid stress . Biochemistry 56 : 5748 – 57 . OpenUrl CrossRef PubMed 32. ↵ Sklar JG , Wu T , Kahne D , Silhavy TJ . 2007 . Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli . Genes Dev 21 : 2473 – 84 . OpenUrl Abstract / FREE Full Text 33. ↵ Kim M , Choi N , Choi E , Lee EJ . 2023 . ClC chloride channels in Gram-negative bacteria and its role in the acid resistance systems . J Microbiol Biotechnol 33 : 857 – 63 . OpenUrl CrossRef PubMed 34. ↵ King T , Lucchini S , Hinton JC , Gobius K . 2010 . Transcriptomic analysis of Escherichia coli O157:H7 and K-12 cultures exposed to inorganic and organic acids in stationary phase reveals acidulant- and strain-specific acid tolerance responses . Appl Environ Microbiol 76 : 6514 – 28 . OpenUrl Abstract / FREE Full Text 35. ↵ Knappe J , Schacht J , Möckel W , Höpner T , Vetter H , Jr. , Edenharder R . 1969 . Pyruvate formate-lyase reaction in Escherichia coli . The enzymatic system converting an inactive form of the lyase into the catalytically active enzyme . Eur J Biochem 11 : 316 – 27 . OpenUrl CrossRef PubMed Web of Science 36. Chang A , Jeske L , Ulbrich S , Hofmann J , Koblitz J , Schomburg I , Neumann-Schaal M , Jahn D , Schomburg D . 2021 . BRENDA, the ELIXIR core data resource in 2021: new developments and updates . Nucleic Acids Res 49 : D498 – D508 . OpenUrl CrossRef PubMed 37. ↵ Yang D-F , Wei Y-T , Huang R-B . 2007 . Computer-aided design of the stability of pyruvate formate-lyase from Escherichia coli by site-directed mutagenesis . Biosci Biotechnol Biochem 71 : 746 – 53 . OpenUrl PubMed 38. ↵ Blattner FR , Plunkett G , Bloch CA , Perna NT , Burland V , Riley M , Collado-Vides J , Glasner JD , Rode CK , Mayhew GF , Gregor J , Davis NW , Kirkpatrick HA , Goeden MA , Rose DJ , Mau B , Shao Y . 1997 . The complete genome sequence of Escherichia coli K-12 . Science 277 : 1453 – 62 . OpenUrl Abstract / FREE Full Text 39. ↵ LaSarre B , McCully AL , Lennon JT , McKinlay JB . 2017 . Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients . ISME J 11 : 337 – 48 . OpenUrl CrossRef PubMed 40. ↵ McKinlay JB , Zeikus JG , Vieille C . 2005 . Insights into Actinobacillus succinogenes fermentative metabolism in a chemically defined growth medium . Appl Environ Microbiol 71 : 6651 – 56 . OpenUrl Abstract / FREE Full Text 41. ↵ Huang JJ , Heiniger EK , McKinlay JB , Harwood CS . 2010 . Production of hydrogen gas from light and the inorganic electron donor thiosulfate by Rhodopseudomonas palustris . Appl Environ Microbiol 76 : 7717 – 22 . OpenUrl Abstract / FREE Full Text 42. ↵ Jett BD , Hatter KL , Huycke MM , Gilmore MS . 1997 . Simplified agar plate method for quantifying viable bacteria . Biotechniques 23 : 648 – 50 . OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted August 29, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Vibrio natriegens is sensitive to its acidic fermentation products Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 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