Microalgal adaptation to a stressful environment (acidic,metal-rich mine waters) could be due to selection of pre-selective mutants originating in non-extreme environments

At least six species of eukaryotic microalgae inhabit the acidic (pH 2.4–2.7), metal-rich mine waters from ponds in the copper mine district of Mynydd Parys (N Wales, UK). Consequently, these ponds constitute interesting natural laboratories for analysis of adaptation by microalgae to extremely stressful conditions. To distinguish between the pre-selective and post-selective origin of adaptation processes that allow the existence of microalgae in these ponds, a Luria-Delbrück fluctuation analysis was performed with the chlorophycean Dictyosphaerium chlorelloides isolated from non-acidic waters. In this analysis, natural Mynydd Parys pond water (MPW) was used as selective factor. Pre-selective, resistant D. chlorelloides cells appeared with a frequency of 1.6 × 10^?6 per cell per generation. MPW-resistant mutants, with a diminished Malthusian fitness, are maintained in non-extreme waters as the result of a balance between new MPW-resistant cells arising by mutation and MPW-resistant mutants eliminated by natural selection (equilibrium at ca. 19 MPW-resistant per 10⁷ wild-type cells). We propose that the microalgae inhabiting these stressful ponds could be the descendents of chance mutants that arrived in the past or are even arriving at the present.     

Enhanced Yields of Iron-Oxidizing Bacteria by In Situ Electrochemical Reduction of Soluble Iron in the Growth Medium

An electrochemical apparatus for culturing chemolithotrophic bacteria that respire aerobically on ferrous ions is described. Enhanced yields of the bacteria were achieved by the in situ electrochemical reduction of soluble iron in the growth medium. When subjected to a direct current of 30 A for 60 days, a 45-liter culture of Thiobacillus ferrooxidans grew from 6 × 10⁷ to 9.5 × 10⁹ cells per ml. Growth of the bacterium within the electrolytic bioreactor was linear with time. A final cell density corresponding to 4.7 g of wet cell paste per liter was achieved, and a total of 320 g of wet cell paste was harvested from one culture. The apparatus was designed to deliver protons concomitantly with electrons; therefore, the pH of the culture remained stable at 1.6 ± 0.1 for the duration of growth. This laboratory-scale apparatus may be readily adapted to pilot or production scale. It is thus anticipated that abundant numbers of iron-oxidizing bacteria may be obtained for both fundamental and applied studies.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

A critical stage in the formation of acid mine drainage: Colonization of pyrite by Acidithiobacillus ferrooxidans under pH-neutral conditions

A dominant Acidithiobacillus ferrooxidans ssp. was isolated from the supergene copper deposit in Morenci, Arizona, USA. Washed bacterial suspensions (10⁸ MPN per treatment), in pH‐neutral buffer, were inoculated onto pyrite cubes for 24 h. Heterogeneous bacterial absorption onto the pyrite removed approximately 90% of the viable bacteria from the inoculum. At T = 0, the bacteria were observed primarily in regions enriched in phosphorus. Over 30 days, the bacterial population on the pyrite cubes increased from 1.3 × 10⁷ to 2.9 × 10⁸ bacteria cm^?2. During this growth stage, low levels of thiobacilli (228 ± 167 MPN mL^?1) were also recovered from the fluid phase; however, this population decreased to zero within 30 days. Growth on pyrite occurred as micrometre‐scale planar microcolonies, a biofilm, coating the mineral surfaces. These microcolonies possessed viable thiobacilli, even after 4 months at ‘circumneutral pH’. Imaging the pyrite cubes using SEM‐EDS and scanning force microscopy demonstrated that the thiobacilli grew as iron oxy‐hydroxide‐cemented cells, leading to the formation of mineralized microcolonies. Removing the iron oxy‐hydroxides with oxalic acid did not dislodge the bacteria, demonstrating that the secondary minerals were not responsible for ‘gluing’ the bacteria to the pyrite surface. Removing organic material, i.e. the cells, by an oxygen plasma treatment revealed the presence of corrosion pits the size and shape of bacteria. Because of the inherent geochemical constraints on pyrite oxidation at neutral pH, the colonization of pyrite under circumneutral pH conditions must be facilitated by the development of an acidic nanoenvironment between the bacteria and the pyrite mineral surface.     

Cream fermentation by immobilized lactic acid bacteria

Summary Mesophilic lactic acid bacteria were immobilized in calcium alginate gel and added to pasteurized 15% fat cream at a 1.6x10⁹ bacteria per ml inoculation level. A pH of 5.5 was obtained in only two hours while it took 4 hours under classical conditions. Once the immobilized cells were removed, the cream contained 6.1x10⁶ lactic bacteria per ml which was almost 400 times less than the bacterial population obtained at pH 5.5 under a classical fermentation. The fermented cream obtained from immobilized cells showed much less overacidification under subsequent refrigeration.     

Bacterial Communities in Acidic and Circumneutral Streams

The relationship between pH and the abundance and activity of bacteria in streams was examined as part of a study of the effect of acidification on stream communities. Of the bacterial communities examined, the epilithic community appeared to be the most significantly affected by acidification. Microbial biomass, as quantified by measuring the ATP level, on rock surfaces was significantly correlated with pH. Also, bacterial production by the epilithic bacteria, indicated by incorporation of tritiated thymidine into DNA, was always higher at high-pH sites than at low-pH sites of the same stream order and elevation. Bacterioplankton concentrations varied between 0.53 × 10⁵ and 9.42 × 10⁵ cells · ml⁻¹ in the first- to fourth-order streams examined. The bacterioplankton concentration in one sample from a spring was 0.17 × 10⁵ cells · ml⁻¹. Bacterioplankton concentrations were not correlated with pH but were significantly correlated with seston concentrations. The correlation with seston is a result of increases in particle-associated bacteria at high seston concentrations. The proportion of bacterioplankton attached to particles varied from 0 to 70%. Bacterial numbers and production in the sediments were significantly correlated with the organic content of the sediment rather than with the pH of the overlying water. Thus, reduced abundance and activity of bacteria as a result of acidification could be detected only for the relatively active community on rock surfaces; this community was exposed to the low pH because of the unbuffered nature of its environment.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.     

The Synthesis of Cyclotene and Its Related Compounds by the Acyloin Condensation

A mutant which required glutamate for growth as the sole nitrogen source was derived from alkalophilic Bacillus No. 8–1 by UV irradiation. The relationship was examined between cell growth and glutamate transport into cells.

Cell growth and glutamate transport into cells were dependent on extracellular pH in the presence of Na⁺, and both were maximum between pH 9 and 10. The quantitative relation between specific growth rate and glutamate uptake rate indicated that the amount of glutamate required for growth at pH 7 and 9 was consistent with that of glutamate transported at pH 7 and 9, respectively. But the amount of glutamate transported at pH 7 was not sufficient to support growth at pH 9. The glutamate transport system of this mutant strain evidently had an effect on growth.     

Conversion of Glycerol to Dihydroxyacetone by Immobilized Whole Cells of Acetobacter xylinum

Enzymatic production of dihydroxyacetone (DHA) was studied by immobilization of the whole cells of acetic acid bacteria capable of oxidizing glycerol to DHA. Acetobacter xylinum A-9 cells immobilized in a polyacrylamide gel were selected as the most favorable enzyme preparation. The enzymatic properties of immobilized cells converting glycerol to DHA were investigated and compared with those of intact cells. The optimum pH for the immobilized cells was broad (4.0 to 5.5), whereas the intact cells had a narrow pH optimum at 5.5. The thermal stability of the immobilized cells was somewhat higher than that of the intact cells. Apparent K_m values for glycerol with both intact and immobilized cells were about equal, 6.3 × 10⁻² to 6.5 × 10⁻² M. The complete conversion of glycerol to DHA was achieved within 40 h under optimum conditions, and pure crystalline DHA was readily isolated from the reaction mixture with over 80% yield.     

Isolation and characterization of a Pseudomonas strain that degrades 4-acetamidophenol and 4-aminophenol

Though many microorganisms that are capable of using phenol as sole sourceof carbon have been isolated and characterized, only a few organisms degradingsubstituted phenols have been described to date. In this study, one strain ofmicroorganism that is capable of using phenol (3000 ppm), 4-aminophenol(4000 ppm) and 4-acetamidophenol (4000 ppm) as sole source of carbon andenergy was isolated and characterized. This strain was obtained by enrichmentculture from a site contaminated with compounds like 4-acetamidophenol,4-aminophenol and phenol in Pakistan at Bhai Pheru. The contaminated siteis able to support large bacterial community as indicated by the viable cellcounts (2 × 10⁴–5 × 10⁸) per gram of soil. Detailed taxonomic studies identified the organisms as Pseudomonas species designated as strain STI. The isolate also showed growth on other organic compounds like aniline, benzene, benzyl alcohol, benzyl bromide, toluene, -cresol, trichloroethylene and o-xylene. Optimum growth temperature and pH were found to be 30 °C and 7, respectively, while growth at 4, 25 and 35 °C and at pH 8 and 9 was also observed. Non growing suspended cells of strain ST1 degraded 68, 96 and 76.8% of 4-aminophenol (1000 ppm), phenol (500 ppm) and 4-acetamidophenol (1000 ppm), respectively, in 72 hrs. The isolation and characterization of Pseudomonas speciesstrain ST1, may contribute to efforts on phenolic bioremediation, particularly in anenvironment with very high levels of 4-acetamidophenol and 4-aminophenol.     

Antibacterial Action of Spermine: Effect on Urinary Tract Pathogens

The basic polyamine spermine was tested for antibacterial activity at two pH levels by the modified cup method against a variety of gram-positive and gram-negative organisms isolated from urine. At pH 6.4, with concentrations ranging from 39 to 2,500 mug per 0.1 ml, there were no clear zones of inhibition seen with any of the gram-negative test organisms, although some adverse effect on growth within the area of the cylinder was noted in 36%. Three of 17 gram-positive strains were inhibited at this pH. Spermine was more active at pH 7.4, but even at the highest concentrations only 16% of the gram-negative and 47% of the gram-positive bacteria tested showed definite zones of inhibition. It is concluded that spermine probably plays little, if any, role in natural resistance to urinary tract infections in vivo.     

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

Aims: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Methods and Results: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram‐negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth‐associated. Although it was not an absolute requirement, 20 g l^?1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. Conclusions: A new PHB‐accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. Significance and Impact of the Study: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.     

Grazing of nonindigenous bacteria by nano-sized protozoa in a natural coastal system

Mesocosms (4.5 m³) situated in a closed bay area were used to investigate the effect of protozoan predation on nonindigenous bacteria. Pseudomonas fluorescens strain Agl was released into mesocosms as a single inoculum of 1 × 10⁵ cells ml^–1 (final concentration) or as four inocula (same concentration each) at intervals of 3 days. Mesocosms that had received growth media corresponding to the inoculum served as controls. Numbers of P. fluorescens Ag1 decreased rapidly whether released as single or multiple inocula. Direct estimation of protozoan predation using fluorescently labeled P. fluorescens from log phase and starved cultures, respectively, revealed that natural populations of heterotrophic nanoflagellates consumed substantial amounts of the nonindigenous bacterial strain. The volume of fluorescently labeled cells prepared from starved cells was 68% of log phase cell volume, but the individual clearance of the small cells was five to seven times higher than that of the log phase bacteria. The natural populations of nanoflagellates consumed 34–62% of P. fluorescens Ag1 daily if starved bacteria were offered as food, and 3–13% if the cells were in the logarithmic growth phase. This suggests that the effect of protozoan predation on nonindigenous bacterial strains is substantial because cultured bacteria are likely to starve in natural environments. The addition of P. fluorescens Ag1 and the growth medium enhanced the abundance of natural bacteria, chlorophyll a, heterotrophic nanoflagellates, and ciliates, but it did not improve the growth conditions for the released strain. The effects on the indigenous populations were more pronounced after addition of fresh medium than following inoculation with cells, which possibly was due to the lower nutrient content of spent medium. However, these results, based on direct estimation of protozoan predation on log phase and starved nonindigenous bacteria, point to the conclusion that mortality induced by bacterivorous predators is the key factor determining removal of nonindigenous bacteria introduced in natural aquatic systems. Correspondence to: K. Christoffersen.     

pH dependency of cell attachment and growth at both clonal and subculture densities of cultured lepidopteran cells

Summary TN-368 cells were seeded at 10⁶ per flask in TNM-FH medium adjusted to a variety of pH levels which ranged from approximately 5.9 to 6.8. In general, growth was similar from pH 6.2 to nearly 6.7. The medium pH increased with time in culture to a maximum near 7.0 for all pH levels. Similar results for growth and pH increase were also obtained when the cells were plated at densities of 10⁴ and 10⁵ per flask. Both the fraction of attached cells and the relative intensity of attachment increased with seeding pH. Cells seeded near pH 6.7 or above frequently required vigrrous procedures such as trypsinization to detach them. DNA synthesis was measured and found to be similar for cells seeded in medium between pH 6.2 and 6.7. Colony forming efficiency increased from approximately 27% at pH 5.9 to 39% at 6.2, remained in the region of 40% between 6.2 and 6.7 with a peak of 48% at 6.6, and plunged abruptly to a few percent just above 6.7 and was near zero above 6.8. Colony morphology was optimal near pH 6.6. This work was supported by USPHS grant R01 CA34158, awarded by the National Cancer Institute, DHHS, Bethesda, MD.     

Thermophilic Iron-Oxidizing Bacteria Found in Copper Leaching Dumps

Rod-shaped bacteria capable of oxidizing ferrous iron at 55°C were cultured from samples of a copper mine leach dump. Yeast extract or cysteine was required by these Thiobacillus-like bacteria for growth, using ferrous iron as an energy source.     

Microbiological Comparison of Surface Soil and Unsaturated Subsurface Soil from a Semiarid High Desert

Thirty-two chemoheterotrophic bacteria were isolated from unsaturated subsurface soil samples obtained from ca. 70 m below land surface in a high desert in southeastern Idaho. Most isolates were gram positive (84%) and strict aerobes (79%). Acridine orange direct counts of microbes in one subsurface sample showed lower numbers than similar counts performed on surface soils from the same location (ca. 5 × 10⁵ versus 2 × 10⁶ cells per g [dry weight] of soil), but higher numbers than those from plate counts performed on the subsurface material. Another sample taken from the same depth at another location showed no evidence of colonies under identical conditions. Soil analyses indicated that subsurface sediments versus surface soils were slightly alkaline (pH 7.9 versus 7.4), had a higher water content (25.7 versus 6.3%), and had lower organic carbon concentrations (0.05 to 0.17 versus 0.25% of soil dry weight). Analyses of biologically relevant gases from the unsaturated subsurface indicated an aerobic environment. As in other unsaturated soil environments, either a high proportion of bacteria in these subsurface sediments are not viable or they are incapable of growth on conventional media under aerobic conditions. The presence and numbers of bacteria in these deep sediments may be influenced by colonization opportunities afforded by periodic percolation of surface water through fractures in overlying strata.     

Relationship between Succinate Transport and Production of Extracellular Poly(3-Hydroxybutyrate) Depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent K_S0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [¹⁴C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pK_a2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate²⁻ accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

Evidence for a Terpene-Based Food Chain in the Gulf of Alaska

A mixture of ¹⁴C-terpenes was prepared from conifer seedlings and introduced into fresh seawater samples taken near Seward, Alaska. Initial rates of oxidation by the indigenous bacteria were linear and faster than the rates of toluene oxidation. Turnover times were 4 to 19 days. Autoradiographic measurements with ³H-terpenes indicated that at least 10% of the 0.6 × 10⁹ to 2.7 × 10⁹ bacteria per liter present could catabolize terpenes. The rate of terpene oxidation, 24 μg of terpenes per g of cells per h with 3 μg of terpenes added per liter, was a constant function of bacterial biomass. The specific affinity of the process was estimated to be between 8.1 and 81 liters/g of cells per h, indicating a high state of induction and the probable presence of terpenes. Terpene-oxidizing bacteria were grown on [¹⁴C]alanine and added to fresh seawater samples. Transfer of the bacterial radioactivity into larger particles at a rate of 146 pg/liter per h from the 2.3 × 10⁹ organisms added indicated that any terpenes present would participate in the food chain.     

Distribution and activity of bacteria in deep granitic groundwaters of southeastern sweden

This study investigated the distribution of bacteria in groundwater from 16 different levels in five boreholes in granite bedrock down to a maximum of 860 m. Enrichment cultures were used to assay the groups of bacteria present. Autoradiographic studies with¹⁴C- or³H-labeled formate, methanol, acetate, lactate, glucose, sodium bicarbonate, leucine, glutamine, thymidine, orN-acetyl-glucosamine were used to obtain information about bacteria active in substrate uptake. The biofilm formation potential was studied in one borehole. The chemical environment in the groundwater was anaerobic with an E_h between −112 and −383 mV, a pH usually around 8, and a temperature range of 10.2 to 20.5°C, depending on the depth. The organic content ranged between <0.5 and 9.5 mg total organic carbon liter⁻¹. Carbon dioxide, hydrogen, hydrogen sulfide, and methane were present in the water. The nitrate, nitrite, and phosphate concentrations were close to, or below, the detection limits, while there were detectable amounts of NH ₄ ⁺ in the range of 4 to 330 μg liter⁻¹. The average total number of bacteria was 2.6×10⁵ bacteria ml⁻¹, as determined with an acridine organge direct-count (AODC) technique. The average number of bacteria that grew on a medium with 1.5 g liter⁻¹ of organic substrate was 7.7×10³ colony-forming units (CFU) ml⁻¹. The majority of these were facultatively anaerobic, gram-negative, nonfermenting heterotrophs. Enrichment cultures indicated the presence of anaerobic bacteria capable of growth on C-1 compounds and hydrogen, presumably methanogenic bacteria. Most probable number assays with sulfate and lactate revealed up to 5.6×10⁴ viable sulfate-reducing bacteria per ml. A biofilm development experiment indicated an active attached microbial population. Active substrate uptake could not be registered with the bulk water populations, except for an uptake of leucine not associated with growth. The bulk water microbial cells in deep groundwater may be inactive cells detached from active biofilms on the rock surface.     

Zeta potential as a measure of the surface charge of mycobacterial cells

The surface charge of bacteria is closely related to their envelope structure and interactions with surfaces in natural environments. The aim of this study was to estimate the effect of experimental conditions on the zeta (ζ) potential of mycobacterial cells as a measure of their cell-surface charge. We observed that Mycobacterium smegmatis mc²155 cells at physiological conditions displayed a high and stable ζ potential (?42.9?±?5.9 mV) which increased from the late-exponential phase of growth and at pH levels of >8.0. The optimal conditions for estimating the surface charge of mycobacteria using the ζ potential occurred when cells were harvested during the exponential growth phase (OD₅₉₅ 0.3–0.5) and then dispersed in solutions with pH levels of 7.0–10.0. These optimal conditions of ζ potential measurements were useful for differentiating between the virulent M. tuberculosis H37Rv strain and various non-virulent mycobacterial strains at pH 9.8. This study is the first to use zetametry to estimate the cell-surface charge of M. tuberculosis cells. We expect that the experimental conditions presented in this work will have further applications to estimate the cell-surface charge of other wild-type or genetically modified mycobacterial species and thereby further our understanding of the physicochemical interactions of mycobacteria with external surfaces in natural environments.