Influence of ethanol on the hopanoid content and the fatty acid pattern in batch and continuous cultures of Zymomonas mobilis

By thin layer chromatographic, gas-liquid chromatographic, and mass spectrometric methods 1,2,3,4-tetrahydroxypentane-29-hopane (THBH) was shown to occur in Zymomonas mobilis. This compound contributed up to 20% to the total lipids.The fatty acid pattern and the content of hopanoids (hopene, hopanol, and THBH) were determined in batch and continuous cultures. In late exponential cells from batch cultures the relative amount of palmitic acid was increased partially at the expense of cis-vaccenic acid, when the initial glucose concentrations were increased. In a batch culture, THBH reached a maximum value in the early exponential growth phase.In an anaerobic continuous culture with a low glucose feed concentration, the THBH content and the relative amount of cis-vaccenic acid were low. The contribution of both compounds increased strongly with increasing glucose feed concentrations (i.e. at higher steady-state ethanol concentrations). The same result was found with aerobic continuous cultures which produced significant amounts of acetaldehyde and acetic acid, in addition to ethanol and carbon dioxide.It was concluded that stability and permeability of the cytoplasmic membrane of the ethanol producing bacterium Z. mobilis was regulated by variations in the distribution of hopanoids and fatty acids.Abbreviations 14:0 myristic acid - 16:0 palmitic acid - 18:1 cisvaccenic acid - THBH 1,2,3,4-tetrahydroxypentane-29-hopane     

Continuous ethanol production by zymomonas mobilis on an industrial scale

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was lower in the presence of oxygen than under anaerobic conditions. Aerobically, considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide. This bacterium contains considerable amounts of pentacyclic triterpenoids, mainly 1, 2, 3, 4-tetrahydroxypentane-29-hopane. It seems that stability and permeability of the cytoplasmic membrane of this rather ethanol tolerant organism is achieved by the hopanoid content. A continuous culture of Zymomonas mobilis produced 60 g/l ethanol over a test period of 39 days. This strain was used for ethanol production from an enzymatically hydrolyzed wheat starch fraction on an industrial scale of 100 m³.     

Properties of hopanoids and phosphatidylcholines containing ω-cyclohexane fatty acid in monolayer and liposome experiments

1,2,3,4-Tetrahydroxypentane-29-hopane (THBH) and a glycolipid derived from it are associated with ω-cyclohexane fatty acids-containing lipids in the membrane of Bacillus acidocaldarius. In order to elucidate the function of these lipids we studied mixed monolayer films and compared these with cholesterol-containing films. The hopanoids are able to condense a liquid-expanded film of di-ω-cyclohexyldodecanoylphosphatidylcholine (DCDPC). The condensing effect of THBH is smaller than that of cholesterol. Hopane glycolipid in comparison shows only little condensation. In a more condensed film, at lower temperatures, THBH slightly decreases while hopane glycolipid increases the molecular area. In egg phosphatidylcholine liposomes, 22-hydroxyhopane (diplopterol) and hopane glycolipid reduce the glycerol permeability to a smaller extent than cholesterol. In DCDPC liposomes, the effect of 22-hydroxyhopane is similar to that of cholesterol, while the hopane glycolipid shows only a weak reduction of the permeability. The results demonstrate that hepanoids have a cholesterol-like function in membranes. This function is also discussed in the context of membrane adaptation of a thermoacidophilic bacterium.     

The influence of hopanoids on growth of Mycoplasma mycoides

Hopane glycolipid, isolated from Bacillus acidocaldarius, was studied in its ability to support growth from Mycoplasma mycoides var. capri as substitute for sterols. All attempts to culture Mycoplasma on hopane glycolipid failed. When added together with cholesterol, the effect of HGL was a reduction in growth rate.The hopanoid diplopterol moderately supported growth of Mycoplasma. This effect was strikingly enhanced in diplopterol adapted cultures. Application of diplopterol via dipalmitoyl-phosphatidylcholine liposomes resulted in an improved growth rate. These results suggested that diplopterol can fullfill sterol function in Mycoplasma membranes.Abbreviations HGL Hopane glycolipid 1-(O--N-acylglucosaminyl)-2,3,4-tetrahydroxypentane-29-hopane - diplopterol 22-hydroxy-hopane - DPPC dipalmitoyl-phosphatidylcholine - PC phosphatidylcholine     

Influence of ethanol on the activities of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase and squalene-hopene-cyclase in Zymomonas mobilis

Summary The influence of different primary aliphatic alcohols on the activities of two key enzymes in hopanoid biosynthesis of Zymomonas mobilis was investigated. By use of ¹⁴C- and ³H-labelled substrates the enzymes 3-hydroxy-3-methylglutaryl-CoA-reductase and squalene-hopenecyclase were detected with activities of 1.6 pmol x (min x mg protein)^-1 and 2.3 pmol x- (min x mg protein)^-1, respectively. Cells grown in the presence of 6% (v/v) ethanol did not show higher activities of these enzymes than cells grown in the presence of 1% (v/v) ethanol. Furthermore, 3-hydroxy-3-methylglutaryl-CoA-reductase was not activated by ethanol. However, ethanol activated the squalene-hopene-cyclase when added to the enzyme test system. Besides ethanol, propanol also had a positive effect on the squalene-hopene-cyclase: the enzyme's activity increased 1.7-fold in the presence of either alcohol at a concentration of 6% (v/v). This corresponded with a similar increase of hopanoid content of whole cells when grown in the presence of 6% (v/v) added ethanol or propanol. These results indicated that the squalene-hopene-cyclase has a regulatory function in the alcohol dependent hopanoid biosynthesis of Z. mobilis.Abbreviation HMG-CoA-reductase 3-hydroxy-3-methylglutaryl-coenzyme A-reductase     

Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis

Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial‐derived liposomes showed that hopanoids protect against several ethanol‐driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter‐lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.     

Isolation of Zymomonas mobilis Mutant with Increased Tetrahydroxybacteriohopane Content

A mutant of Zymomonas mobilis with an increased content of tetrahydroxybacteriohopane (THBH) was isolated. From comparisons of hopanoids of THBH, a glucosamine and an ether derivative of THBH between the parent strain, THBH-decreased and THBH-increased mutants, the biosynthetic pathway of the side-chain of these hopanoids is discussed.     

Growth of a thermotolerant ethanol-producing strain of Kluyveromyces marxianus on cellobiose containing media

Summary The thermotolerant yeast strain, Kluyveromyces marxianus 1MB 3, was shown to be capable of limited growth on cellobiose containing media at 45°C. Growth, sugar utilization and ethanol production were shown to increase in the presence of exogenously added thermostable fungal -glucosidase. During active growth of the organism on cellobiose-containing media, -glucosidase activity was detected in cell lysate preparations with only minor amounts of activity present in the extracellular culture filtrate. The results suggest that limitations in ethanol production by this organism during growth on cellobiose containing media may be overcome by addition of exogenously added -glucosidase which results in increased substrate access to the biocatalytic unit.     

Content and composition of hopanoids in Zymomonas mobilis under various growth conditions.

By using a new method for quantification of the different hopanoid derivatives, a total hopanoid content of about 30 mg/g (dry cell weight) was observed in Zymomonas mobilis. This value is the highest reported for bacteria so far. The major hopanoids in Z. mobilis were the ether and glycosidic derivatives of tetrahydroxy-bacteriohopane, constituting about 41 and 49% of the total hopanoids. Tetrahydroxybacteriohopane itself, diplopterol, and hopene made up about 6, 3, and 1%, respectively. Only minor changes in hopanoid composition were observed with changes in growth conditions. Earlier reports on a correlation between hopanoid content and ethanol concentration in the medium could not be confirmed. Over a wide range of ethanol concentrations (5 to 60 g/liter), growth rates (0.08 to 0.25 h-1), and temperatures (25 to 37 degrees C), the molar ratio of hopanoids to phospholipids in the cells amounted to about 0.7. Only at growth rates of greater than 0.30 h-1 did the molar ratio increase to about 1.     

Effect of temperature on secondary metabolites production and antioxidant enzyme activities in Eleutherococcus senticosus somatic embryos

Somatic embryos of Eleutherococcus senticosus were exposed at 12, 16, 24 and 30 °C for duration of 45 days in bioreactor. The effects of such treatments on the growth, eleutheroside B, E, E₁, total phenolics, flavonoids, chlorogenic acid concentrations and antioxidant enzymes activities were investigated. The results revealed that low (12 and 18 °C) and high (30 °C) temperature caused significant decrease in fresh weight (FW), dry weight (DW), total phenolics, flavonoids and total eleutheroside accumulation, while low temperature increased eleutheroside E accumulation in somatic embryos. Low temperature significantly increased superoxide dismutase (SOD), catalase (CAT), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities whereas a strong increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activity was obtained at 12 °C grown somatic embryos. In contrast, high temperature significantly decreased antioxidant enzymes activities and even guaiacol peroxidase (G-POD) activity also decreased at low temperature in comparison to 24 °C grown embryos. These data suggest that low and high temperature treatment provoked an oxidative stress in E. senticosus embryos, as shown by the increase in lipid peroxidation. The increase in lipid peroxidation was paralleled by a rise in lipoxygenase (LOX) activity and hydrogen peroxide (H₂O₂) content. However, this stress was more prominent at high temperature than low temperature grown embryos. This result suggests that the reduced growth of embryo at 30 °C was concomitant with reduced efficiency of these protective enzymes. On the other hand, increases in antioxidant activities at 12 and 18 °C could also be a response to the cellular damage; however, this increase could not stop the deleterious effects of low temperature, but reduced stress severity thus allowing embryo growth to occur.     

Production of ethanol by the thermotolerant yeastKluyveromyces marxianus IMB3 during growth on lactose-containing media

Summary The thermotolerant yeast strain,Kluyveromyces marxianus IMB3 was shown to be capable of growth and ethanol production on lactose containing media at 45°C. On media containing 4% (w/v) lactose, ethanol production increased to 6.0g/l within 50h and this represented 29% of theoretical yield. During growth on lactose containing media the organism was shown to produce a cell-associated β-galactosidase and no significant enzyme could be detected in the extracellular culture filtrate. Addition of β-galactosidase, released fromKluyveromyces marxianus IMB3 cells, to active fermentations, resulted in increasing ethanol production to 53% of theoretical yield at 45°C.     

The Role of Antioxidant Systems in the Response of Escherichia colito Heat Shock

Shifting the temperature from 30 to 45°C in an aerobic Escherichia coliculture inhibited the expression of the antioxidant genes katG, katE, sodA, and gor.The expression was evaluated by measuring -galactosidase activity in E. colistrains that contained fusions of the antioxidant gene promoters with the lacZoperon. Heat shock inhibited catalase and glutathione reductase, lowered the intracellular level of glutathione, and increased its extracellular level. It also suppressed the growth of mutants deficient in the katG-encoded catalase HPI, whereas the sensitivity of the wild-type andsodA sodBmutant cells to heat shock was almost the same. In the E. coliculture adapted to growth at 42°C, the content of both intracellular and extracellular glutathione was two times higher than in the culture grown at 30°C. The temperature-adapted cells grown aerobically at 42°C showed an increased ability to express the fused katG–lacZgenes.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

News & Notes: The Effect of Ethanol and Oxygen on the Growth of Zymomonas mobilis and the Levels of Hopanoids and Other Membrane Lipids

Zymomonas mobilis (ATCC 29191) was grown either aerobically or anaerobically in the presence of 2% (wt/vol) glucose and 0, 3, or 6% (vol/vol) ethanol. The rates of growth and the composition of hopanoids, cellular fatty acids, and other lipids in the bacterial membranes were quantitatively analyzed. The bacterium grew in the presence of 3% and 6% ethanol and was more ethanol tolerant when grown anaerobically. In the absence of ethanol, hopanoids comprised about 30% (by mass) of the total cellular lipids. Addition of ethanol to the media caused complex changes in the levels of hopanoids and other lipids. However, there was not a significant increase in any of the hopanoid lipid classes as ethanol concentration was increased. As previously reported, vaccenic acid was the most abundant fatty acid in the lipids of Z. mobilis, and its high constitutive levels were unaffected by the variations in ethanol and oxygen concentrations. A cyclopropane fatty acid accounted for 2.6–6.4 wt % of the total fatty acids in all treatments. Received: 12 November 1996 / Accepted: 25 February 1997     

The involvement of trehalose in yeast stress tolerance

Summary A total of 12 yeast strains from various genera were examined for their ability to produce ethanol in the presence of high concentrations of glucose. From these studies, the yeastsTorulaspora delbrueckii andZygosaccharomyces rouxii were observed to the most osmotolerant. These osmotolerant yeast strains were also observed to possess high concentrations of intracellular trehalose. Futhermore, these strains were found to be tolerant to long-term storage at –20°C and to storage at 4°C in beer containing 5% (v/v) ethanol. Cells containing high trehalose levels at the time of freezing or cold storage exhibited the highest cell viabilities. Trehalose concentration was observed to increase during growth on glucose, reaching a maximum after 24–48 h. Increasing the incubation temperature from 21 to 40°C also resulted in an increase in intracellular trehalose content. These results suggest that trehalose plays a role in enhancing yeast survival under environmentally stressful conditions.     

Divergent Effects of Chaperone Overexpression and Ethanol Supplementation on Inclusion Body Formation in RecombinantEscherichia coli

The proper folding of aggregation-prone recombinant proteins inEscherichia colican be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S′-β-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30°C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S′-β-galactosidase 1.5- to 2-fold. When cultures overexpressing thednaKJoperon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S′-β-galactosidase aggregation was almost entirely abolished. Combining DnaK–DnaJ overexpression and growth of the cells at temperatures lower than 30°C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation anddnaKJoverproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S′-β-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S′-β-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.     

Effects of organic carbon sources on cell growth and eicosapentaenoic acid content of <Emphasis Type="BoldItalic">Nannochloropsis</Emphasis> sp.

A strain of Nannochloropsis isolated originally from the East China Sea and obtained from Institute of Hydrobiology, Chinese Academy of Sciences was shown to utilize glucose or ethanol for mixotrophic and heterotrophic growth. The highest cell density, 550 mg L^{− 1} dry weight after culture for 8 days, was obtained during mixotrophic culture with 30 mM glucose. The organic carbon sources had no effect on the net photosynthetic rate, but enhanced the respiratory rate. The addition of an organic carbon source led to an increase in the cell lipid content and a decrease in their eicosapentaenoic acid (EPA) content. The EPA yield was 21.9 mg L^{− 1} using photoautotrophic culture, and 23.4 mg L^{− 1} and 23.0 mg L^{− 1}, respectively, in mixotrophic cultivation with glucose or ethanol as the carbon source.     

The effects of Mn2+ on ethanol production by Kluyveromyces marxianus IMB3 during growth on lactose-containing media at 45°C

Summary It has previously been demonstrated that the thermotolerant yeast strain, K. marxianus IMB3 is capable of growth and ethanol production on lactose containing media at 45°C. Although the organism is capable of producing a -galactosidase, that enzyme has been shown to be extremely thermolabile at 45°C and this has been reflected in reduced efficiencies with respect to conversion of lactose to ethanol. In this paper we demonstrate that addition of Mn²⁺ ions to enzyme preparations contributes significantly to enzyme stability at 45°C. We also demonstrate that addition of Mn²⁺ to fermentations results in increased efficiency of conversion of lactose to ethanol at this temperature.     

Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (≤1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas α-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.     

Endo-polygalacturonase production from untreated lemon peel byAspergillus sp. CH-Y-1043

Summary Endo-polygalacturonase (endo-PG) production byAspergillus sp. CH-Y-1043 using untreated lemon peel as the sole carbon source was investigated. This strain was observed to produce more activity of endo-PG at 37°C than at 29°C. Untreated lemon peel proved to be a beeter substrate than citrus pectin for endo-PG production. Modification of the culture medium and lowering of the initial pH to 2.8 caused a 10-fold increase in the production of endo-PG activity using lemon peel.