Growth of Acetogens at Different pH Levels with Varying Carbon and Energy Sources Jacob W. Cohen MBL Microbial Diversity 2014 Harvard University [email protected] Abstract Organisms that produce acetate via the acetyl-­‐CoA pathway are broadly known as acetogens. Here, attempts were made to isolate an acetogen from an enrichment culture. The resulting semi-­‐pure culture was then subjected to different growth conditions varying by pH level and carbon/energy source provided. Growth was measured by OD420 and organic acid production and consumption was measured via high-­‐pressure liquid chromatography. Quantification of all carbon by mass balance was attempted for heterotrophic cultures. Based on amount of acetate produced and speed of switching metabolisms from autotrophic to heterotrophic, it seems that the organisms studied herein grow best at circumneutral pH. Not all carbon could be accounted for by mass balance, possibly due to the presence of an acetoclastic methanogen. Introduction Acetogens are a phylogenetically diverse group of organisms that are grouped together based on the metabolic pathway they share: the acetyl-­‐CoA pathway, also called the Wood-­‐Ljungdahl pathway. This can be used both to conserve energy and fix CO2 to acetate, which can then be assimilated into biomass or excreted. The acetyl-­‐CoA pathway can also function during heterotrophic growth, where its primary function is to remove excess cellular reductant, similar to fermentation. Also like fermentation, the acetyl-­‐CoA pathway only operates under anaerobic conditions, as many of the enzymes involved in the pathway are very sensitive to oxygen stress. The terminal electron acceptor in the acetyl-­‐CoA pathway is CO2 however, distinguishing it from classical fermentations, which use an organic acid as a terminal electron acceptor (Drake et al., 2006). All organisms have an optimum pH for growth. Bacterial cells can survive in differing pH levels by keeping their internal pH constant (usually between pH 5 and pH 8). However, at pH levels other than their optimum, it becomes harder for cells to grow, as they must expend energy to maintain that internal pH. At pH levels too acidic or alkaline it can become too difficult for a cell to maintain the proper pH and the cell will die (Slonczewski and Foster, 2011). In this project, an acetogenic bacterium was grown as the major member of a mostly purified enrichment culture. This bacterium was subjected to conditions promoting either autotrophic or heterotrophic growth at pH levels that were acidic, neutral, and alkaline. Its growth, production of acetate, and consumption of organic acids were monitored over a period of about five days to determine what effect, if any, differing pH levels had. Methods Media for Culturing Acetogenic Bacteria Basal media for the initial enrichment of acetogens, as well as for pH 7.2 cultures, contained 342.2 mM NaCl, 14.8 mM MgCl2·6H2O, 1 mM CaCl2·2H2O, 6.71 mM KCl, 5 mM NH4Cl, 1 mM KH2PO4 buffer (pH 7.2), 5 mM MOPS buffer (pH 7.2), to which 1 ml 1000X trace elements solution and 100 µl 1% resazurin solution were added per liter. This solution was then boiled for 10 minutes in a round bottom flask while flushing the headspace with a N2/CO2 (80%/20%) stream. The flask was then cooled under the same gas stream and, after cooling, 10 ml of 1000X 13-­‐vitamin solution and 70 ml 1M NaHCO3 were added per liter. The round bottom flask was then transferred to an anaerobic chamber and 5 ml 0.2 M H2S and 10 ml 1 M anoxic bromomethanesulfonate were added per liter. Media was then anaerobically dispensed into serum vials. Initial enrichment vials contained 25 ml and vials for purified cultures contained 50 ml. The vials were then stoppered, crimped, and autoclaved. Agar plates for isolating single colonies were made from the same basal media containing 0.14% agarose and were poured in an anaerobic chamber. Acetogen media at pH 5.5 was made the same way as the basal media, except it contained 5 mM MES buffer (pH 5.5) instead of MOPS buffer and 0.136 g KH2PO4 per liter instead of KH2PO4 buffer. Media at pH 8 was also the same as the basal media, except it contained 5 mM Trizma hydrochloride buffer (pH 8) instead of MOPS buffer and 0.174 g K2HPO4 instead of KH2PO4 buffer. For testing growth on lactate or formate, 0.5 ml 1 M anoxic lactate solution or 1 M anoxic formate solution was added to vials containing 50 ml media. Initial Enrichment and Isolation A vial with 25 ml of pH 7.2 acetogen media was inoculated in an anaerobic chamber with about 1 g of anoxic sediment taken from Trunk River, located in Woods Hole, MA. The headspace of the vial was then replaced with H2/CO2 (80%/20%) and it was incubated in the dark at 30˚C. After the culture became turbid, 1 ml was passaged to a new vial. This was repeated when the first passage also became turbid. About 10 µl of the second passage was spotted onto agar plates and allowed to partially sink into the agar. This spot was then streaked for isolation. Plates were incubated in a Wolfe incubator at 37˚C under an atmosphere of H2/CO2/H2S (79.9%/20%/0.1%). After a week, single colonies were selected and, for each, a sterile 1 ml syringe was pushed through the agar surrounding the colony, which was removed as a plug. A needle was then placed on the syringe and the agar plug was injected into a vial of pH 7.2 acetogen media, which had its headspace replaced with H2/CO2 (80%/20%) and was then incubated at 30˚C in the dark. Carbon/Energy Source and pH Regimes Growth of the isolated acetogen was measured in seven different regimes, varying by the source of carbon/energy and pH. For each pH (5.5, 7.2, and 8), cultures were grown with either 10 mM lactate or H2/CO2. In addition, cultures were also grown at pH 7.2 with 10 mM formate. 0.5 ml of a turbid culture of the isolated acetogen was used to inoculate each vial. The headspace was then replaced with either N2/CO2 (80%/20%) for the lactate and formate cultures or H2/CO2 (80%/20%) for vials without an additional carbon source. Each regime was tested in four replicates. Reading of Optical Density for Growth Measurements Due to resazurin dye having a wavelength of maximal absorbance of about 600 nm, OD600 could not be used to measure bacterial growth. A spectral scan of uninoculated media showed that resazurin does not absorb wavelengths of 400-­‐500 well (Fig. 1). Therefore, all OD measurements were taken at 420 nm, after Bose et al., 2006. Absorbance spectra of media containing resazurin 0.2 0.18 0.16 Absorbance 0.14 0.12 0.1 0.08 pH 5.5 Absorbance 0.06 pH 7.2 Absorbance 0.04 pH 8 Absorbance 0.02 0 -­‐0.02 0 200 400 600 800 1000 1200 Wavelength (nm) Figure 1. Spectra of the different media used in this this study. Resazurin also acts as an indicator of pH, so each growth medium was measured separately to find a wavelength far from any peaks at which to measure OD. A 1 ml sample was taken from each vial for OD420 measurements every 12-­‐16 hours starting at 24.5 hours. The first 4 samples were frozen at -­‐80˚C before OD420 measurements were made, but subsequent samples were read immediately in efforts to minimize further error caused by cells lysing due to freezing. Samples were read on a Thermo Scientific™ SPECTRONIC 200 spectrophotometer. The averages of these readings were used to calculate a generation time for both the autotrophic cultures and the cultures grown on lactate Determination of Acetate Production and Organic Acid Consumption One vial from each regime was selected as a representative culture for monitoring organic acid production and consumption. The pH 7.2/lactate vial was changed after the 7th measurement, as it did not seem to be growing and had very low OD420 measurements. An additional 1 ml sample was taken from each of these representative vials and filtered through a 0.2 µl filter. 900 µl of each sample was then acidified with 100 µl 5N H2SO4 before being run on a Shimadzu high-­‐pressure liquid chromatograph. Organic acid concentrations were determined from UV absorbance readings based on standards for a variety of organic acids and alcohols. Biomass Calculations For cultures grown on lactate, the total biomass produced was calculated by first measuring the absorbance spectrum of a turbid culture (Fig. 2). This spectrum was used to make a conversion factor from OD420 to OD630. The OD420 of vials used for acetate measurements at the last time point was then converted to OD630 and total biomass concentration was calculated assuming 0.79 mM biomass/OD630 (Hanselmann et al., 1995). These numbers were used to determine how much acetate was incorporated into biomass and how much was lost as CO2 via the following stoichiometry: 21.75 CH! COO! + 21.75 H ! → < C!" H!" O!" > + 4.5 CO! + 17.5 H! O This was also used to determine how much of the total carbon supplied by lactate could be accounted for by biomass, acetate, and CO2 production. For the pH 7.2 culture, since the initial concentration of lactate was unknown because which vial was measured changed, the initial lactate concentration was assumed to be the average of all three measured starting lactate concentrations. Figure 2. Curve used for conversion of OD420 to OD630. The absorbance of a turbid culture grown in resazurin-­‐containing media was measured at each wavelength. The ratio of OD420/OD630 from this curve was then used to make a conversion factor, which was multiplied by each OD420 value to approximate OD630 values. Results and Discussion Isolation Microscopy showed that the culture used as inoculum was mostly composed of short rods, some of which were joined end-­‐to-­‐end (Fig. 3). This is the typical morphology of Acetobacterium spp (Balch et al., 1977, Drake et al., 2006). The culture was not entirely pure, however, which was expected as it was only streaked for isolation once due to time constraints. Spirilla or spirochetes and small vibrios were also observed, but were in the vast minority. Figure 3. Picture of the major organism found in this enrichment culture. Smaller organisms of different morphologies also appeared but were much more rare. Growth Curves Semilog plots of growth curves were greatly affected by error (Fig. 4). In the cultures grown autotrophically, there seemed to be a short period of exponential growth up to about 40 hours (Fig. 4A). Unfortunately, the exponential growth phase of the pH 7.2 culture was mostly missed due to time points taken before 24 hours being near the detection limit of the spectrophotometer and not very accurate. Therefore generation times could only be calculated for pH 5.5 and pH 8, which were 6.5 hours and 6.4 hours respectively. This is very close to the literature value of 6 hours for Acetobacterium woodii (Balch et al., 1977). Figure 4. Growth curves. Determining an exact generation time was impossible from the measured growth curves. Only pH 5.5 and pH 8 H2/CO2 cultures had sections that were approximately exponential. Vials were sampled every 12-­‐16 hours. Values shown are the average of four replicates. The cultures grown on formate did not grow to an OD420 much greater than the detection limit of the spectrophotometer (Fig. 4B). This is because the concentration formate that was supplied was consumed very quickly and likely did not contribute much energy for growth (Fig. 5). It was therefore impossible to tell where exponential growth occurred, as there was a large degree of fluctuation curve. Formate Concentration (mM) Formate Consumption at pH 7.2 12 10 8 6 4 2 0 0 20 40 60 80 100 120 Time (Hours) Figure 5. Formate concentrations measured over time. Formate consumption seems to have started at about 30 hours. An initial increase in formate may be due to byproducts of the acetyl-­‐CoA pathway. There was also a large amount of fluctuation in the growth of the cultures grown on lactate (Fig 4C). In the pH 5.5 curve there seems to have been an initial period of exponential growth and then a large drop in OD420 followed by another period of exponential growth. This may have been due to the culture not being totally pure and another organism growing on lactate before the putative Acetobacterium could produce the enzymes necessary to metabolize it. The drop in OD420 is likely due to some oxygen being introduced to the culture, causing cell death. At pH 8 there also seems to be initial exponential growth, followed by a plateau, and then another period of exponential growth. This may again be due to another organism in the culture consuming the lactate until the putative Acetobacterium could switch over to being able to metabolize it. The same seems to be true for pH 7.2, and there is also a drop in this growth curve which is probably again due to oxygen being introduced into the cultures. Acetate Production and Lactate Production When grown on H2/CO2 and formate, acetate production started very quickly, coinciding with (in the case of H2/CO2 cultures) the start of exponential growth (Fig 6A & 6B). This makes sense, as the culture used as an inoculum was grown on H2/CO2, so these cells already possessed all of the enzymes necessary for the acetyl-­‐CoA pathway. Formate is an intermediate in this pathway, so existing enzymes could also metabolize it immediately (Fig 5 & 6B). Figure 6. Acetate production curves. Note the differences in scale between different curves. This was done to make the data more visible for each different condition. Cultures grown on lactate, however, took much longer to consume lactate and produce an appreciable amount of acetate (Fig. 6C & 7). It is well known that bacteria will only induce production of an enzyme if the substrate for that enzyme is present (Novick and Weiner, 1957). Therefore, since the acetogens in this experiment had until now grown entirely autotrophically, they did not possess any of the enzymes required for metabolizing lactate. It seems that it took this organism approximately 80 hours to synthesize these enzymes, at which point acetate production and lactate consumption increased rapidly. It is worth noting that the start of acetate production was likely missed in the pH 7.2 culture, since the culture it was measured from stopped growing before switching to a different bottle. Acetate production from lactate consumption probably started in the pH 7.2 culture before the pH 5.5 and pH 8 cultures. Lactate Concentration (mM) Lactate Consumption 16 14 pH 5.5 12 pH 7.2 10 pH 8 8 6 4 2 0 -­‐2 0 20 40 60 80 100 120 Time (Hours) Figure 7. Lactate concentrations measured over time. It is unknown why there was some fluctuation in the initial lactate concentration. The vial from which measurements were made was changed after the 7th time point, as the old vial appeared to have stopped growing. Acetate production under both heterotrophic and autotrophic conditions was lowest in the pH 7.2 cultures. This may mean that the organisms in these cultures have a pH optimum close to 7.2, as less energy would need to be expended to maintain an internal pH. Therefore, less acetate must be excreted for energy production instead of being incorporated into biomass. Additionally, acetate production in the heterotrophic cultures may have started more quickly at pH 7.2, as the energy conserved from not having to maintain pH could have gone to producing the enzymes necessary to metabolize lactate. This makes sense, as the water in Trunk River, where the original enrichment was inoculated from, has a pH that is circumneutral. Accounting for Carbon Based on the equation for conversion of lactate to acetate (2 C! H! O! →
3 C! H! O! ) and the fact that approximately 12 mM lactate was fed to the pH 5.5 and pH 8 heterotrophic cultures, approximately 18 mM acetate should be recovered. However, only about 12 mM acetate was present in each. Attempts to account for this discrepancy based on incorporation of acetate into biomass still showed about 10% and 5% of carbon could not be accounted for in the pH 5.5 and pH 8 cultures respectively (See Appendix). The discrepancy for pH 7.2 was much larger, but it is unknown what the initial concentration of lactate supplied to this culture was, as the original vial that was measured via liquid chromatography stopped growing after about 60 hours and a new vial was selected for measurements in its place. There are a few possibilities for where this carbon could have gone. Based on stoichiometric equations the most likely answer is that an acetoclastic methanogen was present and was resistant to the 2-­‐bromoethanesulfonate in the media, which was intended to inhibit it (Smith and Mah, 1981). This organism would have cleaved acetate to CO2 and methane (C! H! O! → CO! + CH! ), neither of which where actually measured in this experiment (Ferry, 1992). Less likely but still possible is that one or more organisms present in the cultures produced either a hexose sugar or a molecule with the general formula <CH2O>. It is unlikely that HPLC measurements were inaccurate or that the standard it used was so off, so excess acetate is not plausible. Future directions If this experiment were to be repeated exactly as it was, without further purification for acetogens, it should be done in test tubes that can be inserted into a spectrophotometer so that no samples need to be taken out and no oxygen could be introduced. Additionally, possible temperature fluctuations should be kept to a minimum. Measurements for organic acid consumption and production should be made from samples from separate vessels with more replicates, and the headspace should be sampled for methane production. It would also be a good idea to look under a microscope for F420 fluorescence to confirm the presence of methanogens (Reuter et al., 1986). Acknowledgements I am so grateful to have been a part of this course. I would like to thank Dianne and Jared for making the experience so awesome. I also want to thank Kurt for his help with spectra and mass balance and Arpita for her help with pretty much everything else I did here. Finally, I want to say how awesome all of the other students, TAs, and faculty/staff were. You all really helped make this the great time it was with all of your friendship and support. References 1. Balch, W. E., S. Schoberth, R. S. Tanner, and R. S. Wolfe. 1977. Acetobacterium, a New Genus of Hydrogen-­‐Oxidizing, Carbon Dioxide-­‐Reducing, Anaerobic Bacteria. Int. J. Sys. Bacteriol. 27: 355-­‐361. 2. Bose, A., M. A. Pritchard, M. Rother, and W. W. Metcalf. 2006. Differential Regulation of the Three Methanol Methyltransferase Isozymes in Methanosarcina acetivorans C2A. J. Bacteriol. 188(20): 7274-­‐7283. 3. Drake, H. L., K. Küsel, and C. Matthies. 2006. Acetogenic Prokaryotes. The Prokaryotes 2: 354-­‐420. 4. Ferry, J. G. 1992. Methane from Acetate. J. Bacteriol. 174: 5489-­‐5495. 5. Hanselmann, K. W., J. P. Kaiser, M. Wenk, R. Schön, and R. Bachofen. 1995. Growth on methanol and conversion of methoxylated aromatic substrates by Desulfotomaculum orientis in the presence and absence of sulfate. Microbiol. Res. 150: 387-­‐401. 6. Novick, A. and M. Weiner. 1957. Enzyme Induction as an All or Nothing Phenomenon. PNAS 43: 553-­‐566. 7. Reuter, B. W., T. Egeler, H. Schneckenburger, and S. M. Schoberth. 1986. In vivo measurement of F420 fluorescence in cultures of Methanobacterium thermoautotrophicum. J. Biotechnol. 4: 325-­‐332. 8. Slonczewski, J. L. and J. W. Foster. 2011. Microbiology: An Evolving Science. Second ed. 9. Smith, M. R. and R. A. Mah. 1981. 2-­‐Bromoethanesulfonate: A Selective Agent for Isolating Resistant Methanosarcina Mutants. Current Microbiol. 6: 321-­‐326. Appendix – Calculated Carbon Loss and Incorporation into Biomass