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 0.14 0.12 0.1 0.08 0.06 pH 5.5 Absorbance Absorbance 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.