Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:307–315, 2015     

Changes in the species composition of a thermotolerant community of acidophilic chemolithotrophic microorganisms upon switching to the oxidation of a new energy substrate

Construction and analysis of the 16S rDNA clone libraries was used to investigate the species composition of two thermotolerant communities of acidophilic chemolithotrophic microorganisms (ACM) isolated from the pulp of laboratory reactors used for oxidation of different gold-containing ore concentrates. The first community was formed during oxidation of the pyrite-arsenopyrite ore concentrate from the Kyuchus deposit. The clones of the bacterial component of this community belonged to the genera Sulfobacillus (32 clones) and Leptospirillum (33 clones). The Sulfobacillus clones belonged to three groups: Sb. thermosulfidooxidans, Sb. benefaciens, and Sb. thermotolerans. All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. The microorganisms of this community were used as inoculum for biooxidation of a different mineral concentrate, the pyrrhotite-containing pyrite-arsenopyrite ore concentrate from the Olympiadinskoe deposit, and the structure of the community formed in the process was investigated. The clones of the bacterial component of the second community also belonged to the genera Sulfobacillus (14 clones) and Leptospirillum (48 clones). The Sulfobacillus clones belonged to the species Sb. thermosulfidooxidans (13 clones) and Sb. thermotolerans (1 clone). All Leptospirillum clones were closely related to L. ferriphilum. All clones of the archaeal component belonged to Ferroplasma acidiphilum. During the adaptation of the community to a new oxidized mineral substrate, both the composition and the ratio of the constituent microbial species changed.     

Species composition of the association of acidophilic chemolithotrophic microorganisms participating in the oxidation of gold-arsenic ore concentrate

The species composition of the microbial association involved in industrial tank biooxidation of the concentrate of refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore of the Olympiadinskoe deposit at 39°C was studied by cultural and molecular biological techniques. Pure microbial cultures were isolated, their physiological characteristics were investigated, and their taxonomic position was determined by 16S rRNA gene sequencing. The library of 16S rRNA gene clones obtained from the total DNA isolated from the biomass of the pulp of industrial reactors was analyzed. The diversity of microorganisms revealed by cultural techniques in the association of acidophilic chemolithotrophs (Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, Ferroplasma acidiphilum, Alicyclobacillus tolerans, and Acidiphilium cryptum) was higher than the diversity of the 16S rDNA clone library (At. ferrooxidans, L. ferriphilum, and F. acidiphilum). The combination of microbiological and molecular biological techniques for the investigation of the biodiversity in natural and anthropogenic microbial communities promotes detection of new phylogenetic microbial groups in these communities.     

Ferroplasma cupricumulans sp. nov., a novel moderately thermophilic, acidophilic archaeon isolated from an industrial-scale chalcocite bioleach heap

A new species of Archaea was isolated from an industrial mineral sulphide bioleach heap. Strain BH2, a non-motile pleomorphic coccus, was capable of chemomixotrophic growth on ferrous sulphate and yeast extract. Growth was not supported in the absence of yeast extract. Phylogenetic analysis based on the 16S rRNA gene showed that strain BH2 was most closely related to the species Ferroplasma acidiphilum; however, it showed only 95% sequence similarity with this species. Strain BH2 had a temperature optimum of 53.6°C and a temperature range for growth between 22 and 63°C. Thus, it is the first moderately thermophilic member of the genus Ferroplasma. The optimum pH for the growth of the strain occurred between pH 1.0 and 1.2 and the lowest pH at which growth was observed was 0.4. Based on 16S rRNA gene sequence analysis and other physiological characteristics, strain BH2 constitutes a new species within the genus Ferroplasma. The name Ferroplasma cupricumulans is proposed for the new species and strain BH2 (DSM 16651) is proposed as the type strain.     

Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA

To compare oxidative dissolution rates of chalcopyrite by different consortia of moderately thermophilic acidophiles, various defined mixed cultures of three bacteria Acidithiobacillus caldus s2, Leptospirillum ferriphilum YSK, and Sulfobacillus sp. LN and one archaeon Ferroplasma thermophilum L1 were studied in batch shake flask cultures incubated at 45 °C. Chalcopyrite dissolution was determined by measuring variations of soluble copper, ferric iron, and pH. Microbial population dynamics involved in bioleaching process were monitored using real-time quantitative polymerase chain reaction (PCR) technology. The complex consortia containing both chemoautotrophic (L. ferriphilum and At. caldus) and chemomixotrophic (Sulfobacillus LN and F. thermophilum) moderate thermophiles were found to be the most efficient in all of those tested. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transformations of iron and sulfur and transfer of organic compound, were considered to play a critical role in promoting chalcopyrite dissolution. The real-time PCR assay was reliable to analyze population dynamics of moderate thermophiles in bioleaching systems, and the analysis results were consistent with physiological characteristics of these strains.     

A Moderately Thermophilic Mixed Microbial Culture for Bioleaching of Chalcopyrite Concentrate at High Pulp Density

Three kinds of samples (acid mine drainage, coal mine wastewater, and thermal spring) derived from different sites were collected in China. Thereafter, these samples were combined and then inoculated into a basal salts solution in which different substrates (ferrous sulfate, elemental sulfur, and chalcopyrite) served as energy sources. After that, the mixed cultures growing on different substrates were pooled equally, resulting in a final mixed culture. After being adapted to gradually increasing pulp densities of chalcopyrite concentrate by serial subculturing for more than 2 years, the final culture was able to efficiently leach the chalcopyrite at a pulp density of 20% (wt/vol). At that pulp density, the culture extracted 60.4% of copper from the chalcopyrite in 25 days. The bacterial and archaeal diversities during adaptation were analyzed by denaturing gradient gel electrophoresis and constructing clone libraries of the 16S rRNA gene. The results show that the culture consisted mainly of four species, including Leptospirillum ferriphilum, Acidithiobacillus caldus, Sulfobacillus acidophilus, and Ferroplasma thermophilum, before adapting to a pulp density of 4%. However, L. ferriphilum could not be detected when the pulp density was greater than 4%. Real-time quantitative PCR was employed to monitor the microbial dynamics during bioleaching at a pulp density of 20%. The results show that A. caldus was the predominant species in the initial stage, while S. acidophilus rather than A. caldus became the predominant species in the middle stage. F. thermophilum accounted for the greatest proportion in the final stage.     

Physiological and Morphological Characteristics of Acidophilic Bacteria <Emphasis Type="Italic">Leptospirillum ferriphilum</Emphasis> and <Emphasis Type="Italic">Acidithiobacillus thiooxidans</Emphasis>, Members of a Chemolithotrophic Microbial Consortium

A thermoacidophilic consortium of chemolithotrophic microorganisms oxidizing the concentrate of high-pyrrhotite pyrite?arsenopyrite ore at 38–40°C was isolated. The most active members of the consortium were identified as Leptospirillum ferriphilum, Acidithiobacillus thiooxidans, Ferroplasma acidiphilum, and Sulfobacillus thermotolerans. Leptospirillum and Thiobacillus species were the most numerous members of the consortium and had the highest activity of ferrous iron and sulfur oxidation, respectively. The optimal temperature values for the growth of both isolates were within 35–38°C. The optimal ranges of initial pH were 1.0–1.2 and 1.75–1.85 for leptospirilla and 1.7–3.3 for thiobacilli with the pH optimum of 1.9. Significant polymorphism and specific cyclic growth with formation of vibrios, spirilla, rods with different end shape, spiral filaments, numerous “pseudococci,” and densely packed spiral filaments surrounded by a sheath were revealed for the Leptospirillum isolate. Two latter morphoforms of L. ferriphilum were not previously described. Differences in ability of the morphoforms to oxidize Fe²⁺ were revealed. For the first time, the possibility of growth in the presence of organic substances was demonstrated for A. thiooxidans. The rates of growth and substrate oxidation, cell size, and the maximal cell yield decreased insignificantly in comparison with the lithoautotrophic strain.     

Effect of the aeration mode and yeast extract on the oxidation of high-pyrrhotite sulfide ore flotation concentrate and on the composition of the acidophilic chemolithotrophic microbial community

Optimal aeration conditions were determined and the effect of yeast extract on biooxidation of high-pyrrhotite sulfide ore flotation concentrate in the course of continuous cultivation of an acidophilic chemolithotrophic microbial community was studied in a line of four sequential laboratory reactors; the aeration rate was 3 L/(L min), yeast extract concentration was 0.02%. The gold recovery level was 96.45% at 2.23% elemental sulfur content in the solid residue. The dominant strains identified in the community responsible for biooxidation were Acidithiobacillus caldus OL13-1, At. caldus OL13-3 = At. caldus OL12-3, and an ‘Acidiferrobacter’ strain. Strains Sulfobacillus thermosulfidooxidans OL13-2 = S. thermosulfidooxidans OL12-1 and Ferroplasma acidiphilum OL13-4 = F. acidiphilum OL12-4 were isolated in pure culture and identified.     

Selection of a community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate

A community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate was selected. The Acidithiobacillus caldus OP-1 and Ferroplasma acidiphilum OP-2 cultures were identified to be dominating members. The presence of the Acidithiobacillus ferrooxidans OP-3, Leptospirillum ferriphilum OP-4, and Sulfobacillus thermosulfidooxidans OP-5 cultures in the community’s composition was also mentioned. The analysis results of solid residues of the process showed a greater elemental sulfur oxidation level and gold recovery when the initial pH value in tank I was maintained at a level of 1.8–2.0 (90.5%) rather than 1.6–1.8 (86.3%).     

Succession of Acidophilic Bacterial Community During Bio-oxidation of Refractory Gold-Containing Sulfides

To optimize the rate of bio-oxidation to recover gold from sulfide minerals, it is important to understand the dynamic change of acidophilic bacteria involved in this process. In this study, a batch bio-oxidation experiment was set up to bioleach Au from refractory pyrite and arsenopyrite using a mixed acidophilic culture over the duration of eight days. The 16S rRNA gene clone library and denaturing gradient gel electrophoresis approaches (DGGE) were used to monitor the dynamic succession of the acidophilic bacterial population. The results showed that there were five bacteria in the bio-oxidation reactor: Leptospirillum ferriphilum, Acidithiobacillus caldus, Sulfobacillus thermotolerans, Alicyclobacillus sp. and a heterotrophic iron-oxidizing bacterium. The overall succession pattern was that Acidithiobacillus caldus, a sulfur oxidizer, and Sulfobacillus thermotolerans, a sulfur-iron oxidizer, were predominant at the beginning of the bio-oxidation process, but they were replaced by iron oxidizer L. ferriphilum at a later stage. The competitive advantage of At. caldus and Sb. thermotolerans over L. ferriphilum at the early stage was availability of abundant inorganic sulfur compounds, but lower pH, higher redox potential, and ferrous iron favored L. ferriphilum growth at a later stage. These results have important implications for understanding the role of acidophilic bacterial population in bio-oxidation of refractory gold-containing sulfides.     

Growth of Leptospirillum ferriphilum in sulfur medium in co-culture with Acidithiobacillus caldus

Leptospirillum ferriphilum and Acidithiobacillus caldus are both thermotolerant acidophilic bacteria that frequently co-exist in natural and man-made environments, such as biomining sites. Both are aerobic chemolithotrophs; L. ferriphilum is known only to use ferrous iron as electron donor, while A. caldus can use zero-valent and reduced sulfur, and also hydrogen, as electron donors. It has recently been demonstrated that A. caldus reduces ferric iron to ferrous when grown aerobically on sulfur. Experiments were carried out which demonstrated that this allowed L. ferriphilum to be sustained for protracted periods in media containing very little soluble iron, implying that dynamic cycling of iron occurred in aerobic mixed cultures of these two bacteria. In contrast, numbers of viable L. ferriphilum rapidly declined in mixed cultures that did not contain sulfur. Data also indicated that growth of A. caldus was partially inhibited in the presence of L. ferriphilum. This was shown to be due to greater sensitivity of the sulfur-oxidizer to ferric than to ferrous iron, and to highly positive redox potentials, which are characteristic of cultures containing Leptospirillum spp. The implications of these results in the microbial ecology of extremely acidic environments and in commercial bioprocessing applications are discussed.

     

Bioleaching of chalcopyrite by pure and mixed cultures of Acidithiobacillus spp. and Leptospirillum ferriphilum

Bioleaching is an economical method for the recovery of metals that requires low investment and operation costs. Furthermore, it is generally more environmentally friendly than many physicochemical metal extraction processes. The bioleaching of chalcopyrite in shake flasks was investigated with pure and mixed cultures of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, and Leptospirillum ferriphilum. The mixed cultures containing both iron- and sulfur-oxidizing bacteria were more efficient than the pure culture alone. The presence of sulfur-oxidizing bacteria positively increased the dissolution rate and the percentage recovery of copper from chalcopyrite. Mixed cultures consisting of moderately thermophilic L. ferriphilum and A. caldus leached chalcopyrite more effectively than mesophilic A. ferrooxidans pure and mixed cultures. The decrease of the chalcopyrite dissolution rate in leaching systems containing A. ferrooxidans after 12–16 days coincided with the formation of jarosite precipitation as a passivation layer on the mineral surface during bioleaching. Low pH significantly reduces jarosite formation in pure and mixed cultures of L. ferriphilum and A. caldus.     

The effect of the introduction of exogenous strain Acidithiobacillus thiooxidans A01 on functional gene expression, structure and function of indigenous consortium during pyrite bioleaching

Acidithiobacillus thiooxidans A01 was added to a consortium of bioleaching bacteria including Acidithiobacilluscaldus, Leptospirillumferriphilum, Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans, Acidiphilium spp., and Ferroplasma thermophilum cultured in modified 9 K medium containing 0.5% (w/v) pyrite, and 10.7% increase of bioleaching rate was observed. Changes in community structure and gene expression were monitored with real-time PCR and functional gene arrays (FGAs). Real-time PCR showed that addition of At. thiooxidans caused increased numbers of all consortium members except At. caldus, and At. caldus, L. ferriphilum, and F. thermophilum remained dominant in this community. FGAs results showed that after addition of At. thiooxidans, most genes involved in iron, sulfur, carbon, and nitrogen metabolisms, metal resistance, electron transport, and extracellular polymeric substances of L. ferriphilum, F. thermophilum, and Acidiphilium spp., were up-regulated while most of these genes were down-regulated at 70-78 h in At. caldus and up-regulated in At. ferrooxidans, then down-regulated at 82-86 h.     

Competitive adsorption of binary mixture of <Emphasis Type="Italic">Leptospirillum ferriphilum</Emphasis> and <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis> onto pyrite

Leptospirillum ferriphilum and Acidithiobacillus caldus are two important acidophilic microorganisms involved in iron and sulfur oxidation during bioleaching. Cell adsorption to mineral surfaces is important for the direct leaching or contact leaching of minerals. In this study, we report the competitive adsorption of binary mixtures of L. ferriphilum LF-104 and A. caldus MTH-04 onto pyrite surfaces. The Langmuir adsorption parameter (C_Am) indicated that these two bacteria underwent competitive adsorption to pyrite. Real-time quantitive PCR was used to quantify the relative amounts of L. ferriphilum and A. caldus adsorbed onto the surfaces of pyrite following exposure to a mixture of these two organisms. The adsorption of L. ferriphilum was not affected by A. caldus. However, adsorption of A. caldus was greatly affected by the presence of L. ferriphilum. Zeta-potential measurements and FT-IR spectroscopic studies showed that L. ferriphilum had a higher electrostatic attraction towards pyrite when compared to A. caldus. Based on the above results, we propose a competitive adsorption model to explain the mechanism by which L. ferriphilum and A. caldus compete in their adsorption to pyrite, although L. ferriphilum dominated in the competitive adsorption process. This work provides a better understanding of the adsorption behavior of mixed microbial populations onto mineral surfaces.     

Patterns of growth and oxidation of natural pyrites by the representatives of acidophilic chemolithotrophic microorganisms

The patterns of the growth and oxidation of different types of natural pyrites were studied for the three microbial species adapted to these substrates and belonging to phylogenetically remote groups: gram-negative bacterium Acidithiobacillus ferrooxidans, gram-positive bacterium Sulfobacillus thermotolerans, and the archaeon Ferroplasma acidiphilum. For both A. ferrooxidans strains, TFV-1 and TFBk, pyrite 4 appeared to be the most difficult to oxidize and grow; pyrite 5 was oxidized by both strains at an average rate, and pyrite 3 was the most readily oxidized. On each of the three pyrites, growth and oxidation by TFBk were more active than by TFV-1. The effectiveness of the adaptation of S. thermotolerans Kr1^T was low compared to the A. ferrooxidans strains; however, the adapted strain Kr1^T showed the highest growth rate on pyrite 3 among all the cultures studied. No adaptation of strain Kr1^T to pyrite 5 was observed; the rates of growth and pyrite oxidation in the third transfer were lower than in the first transfer. The strain F. acidiphilum Y^T was not adapted to pyrites 3 and 5; the rates of growth and pyrite oxidation were the same in the first five transfers. The strains of three species of the microorganisms studied, A. ferrooxidans, S. thermotolerans, and F. acidiphilum, grew on pyrite 3 (holetype (p) conductivity) and oxidized it better than pyrite 5 (mixed-type (n-p) conductivity). The most readily oxidized were the pyrites with a density of 5.6–5.7 g/cm³ and high resistance values (ln R = 8.8). The pyrite oxidation rate did not depend on the type of conductivity. Changes in the chromosomal DNA structure were revealed in strain TFBk on adaptation to pyrites 3 and 4 and in the TFV-1 plasmid profile on adaptation to pyrite 3. Correlation between genetic variability and adaptive capabilities was shown for A. ferrooxidans. No changes in the chromosomal DNA structure were found in S. thermotolerans Kr1^T and F. acidiphilum Y^T on adaptation to pyrites 3 and 5. Plasmids were absent in the cells of these cultures.     

Characterization of Ferroplasma Isolates and Ferroplasma acidarmanus sp. nov., Extreme Acidophiles from Acid Mine Drainage and Industrial Bioleaching Environments

Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y^T by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate “Ferroplasma acidarmanus” Fer1^T required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42°C and the pH optima were 1.0 to 1.7, and “F. acidarmanus” Fer1^T was capable of growing at pH 0. The optimum yeast extract concentration for “F. acidarmanus” Fer1^T was higher than that for the other three isolates. Phenotypic results suggested that isolate “F. acidarmanus” Fer1^T is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y^T group as a single species. “F. acidarmanus” Fer1^T groups separately, and we propose the new species “F. acidarmanus” Fer1^T sp. nov.     

Optimization of Mixed Cultivation of the Moderate Thermophilic Bioleaching Microorganisms for High Cell Density Using Statistical Methodology

The low functional microbial population density in the industrial bioleaching process has been a limiting factor for the high leaching efficiency, making the microbial cultivation and continuous inoculation an alternative for sustaining the microbial activity. In the present experiment, the defined mixed cultivation of Leptospirillum ferriphilum YSK, Sulfobacillus acidophilus TPY, Acidithiobacillus caldus S2, and Ferroplasma thermophilum L1 was evaluated and optimized by Statistical Methodology. Going through the Plackett–Burman experimental design, pH value, temperature, and c(MgSO₄·7H₂O) were considered as the most significant factors in the defined range. Then, the relationships were analyzed using the steepest ascent design, the central composite design, and finally the response surface methodology. It was suggested that the optimum parameters were pH 1.38, MgSO₄·7H₂O 0.552?g/L, temperature 44?°C, FeSO₄·7H₂O 40?g/L, sulfur 8?g/L, yeast 0.02% w/v, (NH₄)₂SO₄ 3g/L, K₂HPO₄ 0.5g/L, KCl 0.1g/L, Ca(NO₃)₂ 0.01?g/L, in which allowed total cell density of the microbial community to reach 7.63?×?10⁸ cells/mL in the cultivation period. The lab experiments were routinely undertaken with the expectation that the L. ferriphilum YSK, S. acidophilus TPY, A. caldus S2, F. thermophilum L1 could rapid grown from initial cell density of 0.25?×?10⁷ cells/mL to 2.82?×?10⁸ cells/mL, 1.68?×?10⁸ cells/mL, 2.76?×?10⁸ cells/mL, 2.51?×?10⁷ cells/mL, respectively in 58?h. It demonstrates a possibility to co-culture these microbes in a single reactor, providing an efficient way to regenerate of inoculation for biomining process.     

Phenotypic Features of Ferroplasma acidiphilum Strains YT and Y-2

Earlier, we described a new family of mesophilic, strictly autotrophic Fe²⁺-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and the species F. acidiphilum (strain Y^T) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain Y and a new strain, F. acidiphilum Y-2, isolated from dense pulps during oxidation of gold-containing arsenopyrite/pyrite concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk krai) ore deposits, respectively. The G+C content of DNA from strains Y^T and Y-2 comprised 35.1 and 35.2 mol %, respectively; the level of DNA–DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous iron and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. The strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains Y^T and Y-2 were 34–36 and 40–42°, respectively. Comparative analysis of the total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of the total lipids: lipid I, -D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2 : 1. Thus, it was established that the intraspecies phylogenetic divergence within F. acidiphilum is manifested in the two strains by different temperature optima against a background of similarity in other phenotypic properties.     

Effect of bacteriocinogenic Lactobacillus spp. on the shelf life of fufu, a traditional fermented cassava product

Quantitative determination of antimicrobial compounds produced by the Lactobacillus strains under test was carried out. L. plantarum F1 produced the highest quantity of lactic acid (16.4 g/l) while the lowest amount (0.3 g/l) was produced by L. jensenii F9. All the test organisms produced hydrogen peroxide, with L. brevis OG1 having the highest yield of 0.037 g/l. Diacetyl was also produced by all the organisms, with L. plantarum F1 and L. brevis OG1 having the highest yield of 1.7 g/l, while the lowest producer was L. jensenii F9 (0.86 g/l). Determination of bacteriocin activity was carried out. L. plantarum F1 exhibited 6400 AU/ml bacteriocin activity, while L. brevis OG1 had the lowest activity of 3200 AU/ml using E. coli NCTC10418 as indicator organism. However, L. fermentum F5 and L. jensenii F9 did not produce any detectable bacteriocin. The pH value in the culture supernatant of L. plantarum F1 reached 3.1 within 48 h of incubation, while that of L. jensenii F9 was 5.2. Fufu was prepared using both bacteriocin-producing (BP) L. plantarum F1 and L. brevis OG1, and non-bacteriocin producing L. fermentum F5 and L. jensenii F9. No viable cells of Salmonella typhimurium ATCC13311 and Shigella flexneri AP23498 were detected after 12 h in the cassava products fermented with mixed starter culture of L. plantarum F1 and L. brevis OG1. The rate of survival of enteropathogens in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was much lower when compared to cassava fermented with mixed starter culture of L. fermentum F5 and L. jensenii F9. At 12 h, the viable count of E. coli NCTC10418 in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was 1.1 log₁₀ c.f.u./g whereas in cassava fermented with mixed starter cultures of L. fermentum F5 and L. jensenii F9, 8.5 log₁₀ c.f.u./g was obtained The study revealed that fufu produced with BP mixed starter cultures had a better shelf life and kept for 13 days before spoilage occurred, relative to 5 days observed for fufu produced using non-bacteriocin-producing starter cultures, and 6 days for the traditional fermented fufu.