Shewanella Alga BrY Research Articles

Bioleaching of silica sand using bioreducing bacteria (<i>Shewanella</i> strains)

Silica sand is generally not pure when mined; often associated with iron hydroxide and oxy-hydroxide impurities which lowers its industrial value and requires purification before use. To join the global community towards environmental consciousness, protection and effective energy utilization, this paper considers microbial purification in a closed non-growth system as an alternative for iron removal. The anaerobic removal of Fe(III)-bearing impurities from the mineral was investigated using small scale microcosm experiments conducted with Fe(III) in silica sand as the sole electron acceptor and lactate as the electron donor in the presence of pure cultures of Shewanella strains (S. putrefaciens CIP8040, S. putrefaciens CN32, S. oneidensis MR-1, S. algae BrY and S. loihica) and anthraquinone 2,6 disulphonate (AQDS) serving as electron shuttling mediator. The reduction of Fe(III) from silica sand was measured as the production of Fe(II) in HCl extracts using modified p-Phenanthroline techniques. Absorbance was measured using UV vis-spectrophotometry at 510nm. Up to 17.6% of the iron bearing impurities (~117mg of bioreducible Fe2O3 per 100g of silica sand) was successfully removed after 15 days. Addition of AQDS as electron transport mediator enhanced the rate and extent of bioreduction by facilitating the exchange of electrons between the iron reducing bacteria and the iron-bearing phase in the mineral. This study revealed that all the selected commercially available Shewanella strains tested were able to reduce and leach iron (III) from Silica sand by coupling oxidation of lactate to anaerobic respiration except Shewanella loihica whereas Shewanella algae BrY was the most efficient. Keywords : Silica sand, iron impurities, iron reducing bacteria, Shewanella strains bioreduction.

Bacterial Pu(V) reduction in the absence and presence of Fe(III)–NTA: modeling and experimental approach

Plutonium (Pu), a key contaminant at sites associated with the manufacture of nuclear weapons and with nuclear-energy wastes, can be precipitated to "immobilized" plutonium phases in systems that promote bioreduction. Ferric iron (Fe(3+)) is often present in contaminated sites, and its bioreduction to ferrous iron (Fe(2+)) may be involved in the reduction of Pu to forms that precipitate. Alternately, Pu can be reduced directly by the bacteria. Besides Fe, contaminated sites often contain strong complexing ligands, such as nitrilotriacetic acid (NTA). We used biogeochemical modeling to interpret the experimental fate of Pu in the absence and presence of ferric iron (Fe(3+)) and NTA under anaerobic conditions. In all cases, Shewanella alga BrY (S. alga) reduced Pu(V)(PuO(2) (+)) to Pu(III), and experimental evidence indicates that Pu(III) precipitated as PuPO(4(am).) In the absence of Fe(3+) and NTA, reduction of PuO(2) (+) was directly biotic, but modeling simulations support that PuO(2) (+) reduction in the presence of Fe(3+) and NTA was due to an abiotic stepwise reduction of PuO(2) (+) to Pu(4+), followed by reduction of Pu(4+) to Pu(3+), both through biogenically produced Fe(2+). This means that PuO(2) (+) reduction was slowed by first having Fe(3+) reduced to Fe(2+). Modeling results also show that the degree of PuPO(4(am)) precipitation depends on the NTA concentration. While precipitation out-competes complexation when NTA is present at the same or lower concentration than Pu, excess NTA can prevent precipitation of PuPO(4(am)).

Determination of ferrous and ferric iron in aqueous biological solutions

A solvent extraction method was employed to determine ferrous and ferric iron in aqueous samples. Fe(3+) is selectively extracted into the organic phase (n-heptane) using HDEHP (bis(2-ethylhexyl) hydrogen phosphate) and is then stripped using a strong acid. After separation, both oxidation states and the total iron content were determined directly by ICP-MS analysis. This extraction method was refined to allow determination of both iron oxidation states in the presence of strong complexing ligands, such as citrate, NTA and EDTA. The accuracy of the method was verified by crosschecking using a refinement of the ferrozine assay. Presented results demonstrate the ability of the extraction method to work in a microbiological system in the presence of strong chelating agents following the bioreduction of Fe(3+) by the Shewanella alga BrY. Based on the results we report, a robust approach was defined to separately analyze Fe(3+) and Fe(2+) under a wide range of potential scenarios in subsurface environments where radionuclide/metal contamination may coexist with strongly complexing organic contaminants.

Biogenic Formation of As-S Nanotubes by Diverse Shewanella Strains

Shewanella sp. strain HN-41 was previously shown to produce novel, photoactive, As-S nanotubes via the reduction of As(V) and S(2)O(3)(2-) under anaerobic conditions. To determine if this ability was unique to this bacterium, 10 different Shewanella strains, including Shewanella sp. strain HN-41, Shewanella sp. strain PV-4, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were examined for production of As-S nanotubes under standardized conditions. Of the 10 strains examined, three formed As-S nanotubes like those of strain HN-41. While Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly formed As-S precipitates in 7 days, strains S. alga BrY and S. oneidensis MR-1 reduced As(V) at a much lower rate and formed yellow As-S after 30 days. Electron microscopy, energy-dispersive X-ray spectroscopy, and extended X-ray absorption fine-structure spectroscopy analyses showed that the morphological and chemical properties of As-S formed by strains S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 were similar to those previously determined for Shewanella sp. strain HN-41 As-S nanotubes. These studies indicated that the formation of As-S nanotubes is widespread among Shewanella strains and is closely related to bacterial growth and the reduction rate of As(V) and thiosulfate.

Degeneration of biogenic superparamagnetic magnetite

Magnetite crystals precipitated as a consequence of Fe(III) reduction by Shewanella algae BrY after 265 h incubation and 5-year anaerobic storage were investigated with transmission electron microscopy, Mössbauer spectroscopy and X-ray diffraction. The magnetite crystals were typically superparamagnetic with an approximate size of 13 nm. The lattice constants of the 265 h and 5-year crystals are 8.4164A and 8.3774A, respectively. The Mössbauer spectra indicated that the 265 h magnetite had excess Fe(II) in its crystal-chemistry (Fe(3+) (1.990)Fe(2+) (1.015)O(4)) but the 5-year magnetite was Fe(II)-deficient in stoichiometry (Fe(3+) (2.388)Fe(2+) (0.419)O(4)). Such crystal-chemical changes may be indicative of the degeneration of superparamagnetic magnetite through the aqueous oxidization of Fe(II) anaerobically, and the concomitant oxidation of the organic phases (fatty acid methyl esters) that were present during the initial formation of the magnetite. The observation of a corona structure on the aged magnetite corroborates the anaerobic oxidation of Fe(II) on the outer layers of magnetite crystals. These results suggest that there may be a possible link between the enzymatic activity of the bacteria and the stability of Fe(II)-excess magnetite, which may help explain why stable nano-magnetite grains are seldom preserved in natural environments.

Pyomelanin is produced by Shewanella algae BrY and affected by exogenous iron

Melanin production by Shewanella algae BrY occurred during late- and (or) post-exponential growth in lactate basal salts liquid medium supplemented with tyrosine or phenylalanine. The antioxidant ascorbate inhibited melanin production but not production of the melanin precursor homogentisic acid. In the absence of ascorbate, melanin production was inhibited by the 4-hydroxyphenylpyruvate dioxygenase inhibitor sulcotrione and by concentrations of Fe >or= 0.38 mmol L(-1). These data support the hypothesis that pigment production by S. algae BrY was a result of the conversion of tyrosine or phenylalanine to homogentisic acid, which was excreted, auto-oxidized, and self-polymerized to form pyomelanin. Pyomelanin production by S. algae BrY may play an important role in the biogeochemical cycling of Fe in the environment.

Regeneration of iron for trichloroethylene reduction by Shewanella alga BrY

Zero valent iron (ZVI), the primary reactive material in several permeable reactive barriers, is often oxidized to ferrous or ferric iron, resulting in decreased reactivity with time. Iron reducing bacteria can reconvert the ferric iron to its ferrous form, prolonging the reduction of chlorinated organic contaminants. In this study, the reduction of Fe(II,III) oxide and Fe(III) oxide by a strain of iron reducing bacteria of the group Shewanella alga BrY(S. alga BrY) was observed in both aqueous and solid phases. S. alga BrY preferentially reduced dissolved ferric iron over the solid ferric iron. In the presence of iron oxide the Fe(II) ions reduced by S. alga BrY efficiently reduced trichloroethylene (TCE). On the other hand, Fe(II) produced by S. alga BrY covered the reactive surfaces of ZVI iron filings and inhibited the reduction of TCE by ZVI. The formation of precipitates on the iron oxide or Fe0 surface was confirmed by scanning electron microscopy. The results suggest that iron-reducing bacteria in the oxidized Fe0 barriers can enhance the removal rate of chlorinated organic compounds and influence on the long-term performance of Fe0 reactive barriers.

Effects of starvation on bacterial transport through porous media

A major problem preventing widespread implementation of microbial injection strategies for bioremediation and/or microbially enhanced oil recovery is the tendency of bacteria to strongly adhere to surfaces in the immediate vicinity of the injection point. Long term (weeks to months) nutrient starvation of bacteria prior to injection can decrease attachment and enhance transport through porous media. This paper summarizes results of starvation-enhanced transport experiments in sand columns of 30 cm, 3 m, and 16 m in length. The 16 m column experiments compared transport, breakthrough and distribution of adhered cells for starved and vegetative cultures of Klebsiella oxytoca, a copious biofilm producer. Results from these experiments were subsequently used to design and construct a field-scale biofilm barrier using starved Pseudomonas fluorescens. The 30 cm and 3 m sand columns experiments investigated starvation-enhanced transport of Shewanella algae BrY, a dissimilatory metal-reducing bacterium. In both cases the vegetative cells adsorbed onto the sand in higher numbers than the starved cells, especially near the entrance of the column. These results, taken together with studies cited in the literature, indicate that starved cells penetrate farther (i.e. higher breakthrough concentration) and adsorb more uniformly along the flow path than vegetative cells.

Biological activation of hydrous ferric oxide for reduction of hexavalent chromium in the presence of different anions

Shewanella alga BrY, a dissimilatory iron reducing bacterium (DIRB), transformed inert ferric oxides that are common in sediments, aquifer material and passivated permeable reactive iron barriers (PRBs), producing dissolved and sorbed Fe(II) capable of rapidly reducing and immobilizing Cr(VI). The effect of groundwater chemistry on the formation and reactivity of such microbial-produced, abiotic reductants was investigated. Batch reactors with high carbonate concentration (10 mM) were the most reactive, removing 66.0% ± 2.8 of Cr (VI) (76 mg/l) from liquid phase within 5 min. Treatments with high concentrations of sulfate (5.2 mM), chloride (10 mM), phosphate (1 mM) or silica (0.75 mM) were less reactive (about 40% removal). Loss of reactivity was observed possibly due to oxidation of Fe(II) (sorbed and dissolved) by Cr(VI). Normalization of Cr(VI) removal to the mass of biogenic solid present showed the following molar Cr/Fe ratios in solid phase: 0.185 ± 0.041 (carbonate), 0.146 ± 0.013 (sulfate), 0.092 ± 0.010 (silica), 0.075 ± 0.012 (phosphate) and 0.062 ± 0.012 (chloride). Overall, these results show that bacterial transformation of inert ferric oxides can contribute to the (abiotic) natural attenuation of Cr(VI) in and around PRBs, and that groundwater chemistry is an important determinant of biogenic solids reactivity.

Sustained and Complete Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Degradation in Zero-Valent Iron Simulated Barriers under Different Microbial Conditions

Flow-through columns packed with "aged" zero-valent iron (ZVI) between layers of soil and sand were constructed to mimic a one-dimensional permeable reactive iron barrier (PRB). The columns were continuously fed RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine, ca. 18 mg l−1) for over one year. Two columns were bioaugmented with dissimilatory iron reducing bacteria (DIRB) Shewanella algae BrY or Geobacter metallireducens GS-15 to investigate their potential to enhance the reactivity of aged iron by reductive dissolution of passivating iron oxides or via production of biogenic reactive minerals. A third column was not bioaugmented to evaluate colonization by indigenous soil microorganisms. [14C]-RDX was completely removed in all columns at the start of the iron layer, and concentration profiles showed rapid and sustainable RDX removal over one year; however, a phylogenetic profile conducted after one year using DGGE analysis of recovered DNA did not detect S. algae BrY or G. metallireducens in their respective columns. Bacterial DNA was recovered from within the ZVI. Several unidentified 14C-labeled byproducts were present in the effluent of all columns. Dissolved 14C removal and the detection of dissolved inorganic 14C in these columns (but not in the sterile control) suggest microbial-mediated mineralization of RDX and sorption/precipitation of degradation products. Enhanced RDX mineralization in bioaugmented columns was temporary relative to the indigenously colonized column. However, shorter acclimation periods associated with bioaugmented PRBs may be desirable for rapid RDX mineralization, thereby preventing breakthrough of potentially undesirable byproducts. Overall, these results show that high RDX removal efficiency by ZVI-PRBs is achievable and sustainable and that the efficacy and start-up of ZVI-PRBs might be enhanced by bioaugmentation.

The structure of the core region of the lipopolysaccharide from Shewanella algae BrY, containing 8-amino-3,8-dideoxy- d- manno-oct-2-ulosonic acid

The structure of the carbohydrate backbone of the lipid A–core region of the LPS from Shewanella algae strain BrY was analysed. The LPS was N, O-deacylated to give three products, which were isolated and studied by chemical methods, NMR and mass spectrometry: ▪ where R=Ac or H α-Glc-(1→4) -β-Gal-(1→4) -α- dd Hep-(1→5) -α-8-amino-Kdo4P -(2→6) -β-GlcN4P -(1→6) -α-GlcN1P All monosaccharides except l-rhamnose had the d-configuration. This LPS presents a second example (after S. oneidensis) of the structure with a novel linking unit between the core and lipid A moieties, 8-amino-3,8-dideoxy- d- manno-oct-2-ulosonic acid (8-amino-Kdo).

Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell-associated melanin

Shewanella algae BrY uses insoluble mineral oxides as terminal electron acceptors, but the mechanism of electron transfer from cell surface to mineral surface is not well understood. We tested the hypothesis that cell-associated melanin produced by S. algae BrY serves as an electron conduit for bacterial–mineral reduction. Results from Fourier transform infrared spectroscopy and cell surface hydrophobicity assays indicated that extracellular melanin was associated with the cell surface. With H 2 as electron donor, washed cell suspensions of melanin-coated S. algae BrY reduced hydrous ferric oxide (HFO) 10 times faster than cells without melanin. The addition of melanin (20 μg ml −1) to these melanin-free cells increased their HFO reduction rate two-fold. These results suggest that cell-associated melanin acts as an electron conduit for iron mineral reduction by S. algae BrY.

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain l-rhamnose, 2-acetamido-4-[ d-3-hydroxybutyramido)]-2,4,6-trideoxy- d-glucose ( d-BacNAc4NHbu), and 2-amino-2,6-dideoxy- l-galactose, N-acylated by the 4-carboxyl group of l-malic acid ( l-malyl-(4→2)-α- l-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established: -3)- α- d - BacNAc4NHbu-(1 →3)- α- l - Rha-(1 →2)- α- l - Rha-(1 →2)- l - malyl-(4 →2)- α- l - FucN-(1- The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.

Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY.

Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced by Shewanella algae BrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced by S. algae BrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth of S. algae BrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures of S. algae BrY. Melanin produced by S. algae BrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.

Degradation of TCE, Cr(VI), sulfate, and nitrate mixtures by granular iron in flow-through columns under different microbial conditions

Flow-through aquifer columns packed with a middle layer of granular iron (Fe 0) were used to study the applicability and limitations of bio-enhanced Fe 0 barriers for the treatment of contaminant mixtures in groundwater. Concentration profiles along the columns showed extensive degradation of hexavalent chromium Cr(VI), nitrate, sulfate, and trichloroethene (TCE), mainly in the Fe 0 layer. One column was bioaugmented with Shewanella algae BRY, an iron-reducing bacterium that could enhance Fe 0 reactivity by reductive dissolution of passivating iron oxides. This strain did not enhance Cr(VI), which was rapidly reduced by iron, leaving little room for improvement by microbial participation. Nevertheless, BRY-enhanced nitrate removal (from 15% to 80%), partly because this strain has a wide range of electron acceptors, including nitrate. Sulfate was removed (55%) only in a column that was bioaugmented with a mixed culture containing sulfate-reducing bacteria. Apparently, these bacteria used H 2 (produced by Fe 0 corrosion) as electron donor to respire sulfate. Most of the TCE was degraded in the zone containing Fe 0 (50–70%), and bioaugmentation with BRY slightly increased the removal efficiency to about 80%. Microbial colonization of the Fe 0 surface was confirmed by scanning electron microscopy.

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral reduction.

Biogeochemical Elimination of Chromium (VI) from Contaminated Water

Ferrous iron [Fe(II)] reductively transforms heavy metals in contaminated groundwater, and the bacterial reduction of indigenous ferric iron [Fe(III)] to Fe(II) has been proposed as a means of establishing redox reactive barriers in the subsurface. The reduction of Fe(III) to Fe(II) can be accomplished by stimulation of indigenous dissimilatory metal-reducing bacteria (DMRB) or injection of DMRB into the subsurface. The microbially produced Fe(II) can chemically react with contaminants such as Cr(VI) to form insoluble Cr(III) precipitates. The DMRB Shewanella algae BrY reduced surface-associated Fe(III) to Fe(II), which in batch and column experiments chemically reduced highly soluble Cr(VI) to insoluble Cr(III). Once the chemical Cr(VI) reduction capacity of the Fe(II)/Fe(III) couple in the experimental systems was exhausted, the addition of S. algae BrY allowed for the repeated reduction of Fe(III) to Fe(II), which again reduced Cr(VI) to Cr(III). The research presented herein indicates that a biological process using DMRB allows the establishment of a biogeochemical cycle that facilitates chromium precipitation. Such a system could provide a means for establishing and maintaining remedial redox reactive zones in Fe(III)-bearing subsurface environments.

Dissimilatory Iron-Reducing Bacteria Can Influence the Reduction of Carbon Tetrachloride by Iron Metal

Little is known about the long-term performance of zerovalent iron (Fe0) subsurface barriers. Groundwater exposure induces corrosion processes that can passivate the Fe0 surface and decrease barrier reaction rates. We present evidence that dissimilatory iron-reducing bacteria (DIRB) can stimulate the rate of carbon tetrachloride (CT) transformation in the presence of corroded iron. The DIRB, Shewanella alga BrY, adhered to the corroded Fe surfaces that showed little or no capacity to transform CT. The addition of BrY to these systems with decreased CT transformation rates resulted in increased ferrous iron concentrations and increased CT transformation to chloroform (CF). The results suggest that DIRB can have an influence on the long-term performance of Fe0 barriers.

Influence of microbial activity on the stability of activated sludge flocs

The influence of the microbial activity on the strength of activated sludge flocs was studied in short term experiments (0–3 h). Increased floc strength was generally obtained when the aerobic microbial activity was stimulated by adding substrate. Deflocculation was observed when the aerobic microbial activity was inhibited by (i) anaerobic conditions; (ii) addition of the metabolic inhibitors azide and chloramphenicol; and (iii) reduction of the temperature to 4°C. Furthermore, addition of nitrate as electron acceptor under anaerobic conditions partly prevented deflocculation from taking place. These results strongly suggested that microorganisms using oxygen and/or nitrate as electron acceptors were important for maintaining the floc strength. The increase in turbidity under deflocculation was well correlated with the number of bacteria and concentration of protein, humic substances and carbohydrates in the supernatant. However, only approximately 1–2% of the total amount of sludge deflocculated, so the deflocculation could be understood as an erosion of small particles from the larger flocs. The extent of deflocculation under anaerobic conditions could be enhanced by stimulation of the anaerobic biological activity. When anaerobic conditions prevailed, a microbial iron reduction immediately started with iron reduction rates of 4–150 μmol/gVS·h. Subsequently, a decrease in floc strength was observed which could also be observed when the iron-reducing bacterium Shewanella alga BrY was added to the activated sludge. Furthermore, the importance of Fe(III) for the floc strength was illustrated by removal of Fe(III) from the sludge matrix by adding sulphide, which resulted in strong deflocculation. Thus, the deflocculation observed could be either directly due to lack of aerobic microbial activity or indirectly due to change in the local physico-chemical conditions mediated by anaerobic microbial activity.

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry.

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.