Optimization and mechanism of diacetyl accumulation by <Emphasis Type="Italic">Enterobacter</Emphasis><Emphasis Type="Italic">aerogenes</Emphasis> mutant UV-3

A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of α-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0–7.5, and temperature between 36 and 39°C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l⁻¹. Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%. Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH₄)₂SO₄, 0.1% citric acid, 0.2% MnSO₄ and 0.2% MgSO_4, and this was determined by one-factor-at-a-time approach. Data from the five level central composite designs demonstrated that initial pH of 7.0, temperature of 37°C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl production. The maximum yield of diacetyl could reach 1.35 g l⁻¹ in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of α-acetolactate and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3.     

Quantification of indole-3-acetic acid from plant associated <Emphasis Type="Italic">Bacillus</Emphasis> spp. and their phytostimulatory effect on <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Sixteen Bacillus strains isolated from rhizosphere, histoplane and phyllosphere of different plant species were identified by 16S rDNA gene sequencing and evaluated for in vitro auxin production as well as growth stimulation of Vigna radiata (L.) Wilczek. Auxin production by Bacillus spp. in L-broth medium supplemented with 1,000 μg ml⁻¹ L-tryptophan ranges from 0.60 to 3.0 μg IAA ml⁻¹ as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. Rhizospheric isolates exhibit relatively more IAA synthesis than histoplane and phyllosphere isolates. Plant microbe interaction experiments conducted under gnotobiotic conditions recorded 55.55, 46.46 and 46.20% increase in shoot length with Bacillus megaterium MiR-4, B. pumilus NpR-1 and B. subtilis TpP-1, respectively, over control. Bacillus inoculations also increased shoot fresh weight with B. megaterium MiR-4 (60.94%) and B. pumilus NpR-1 (37.76%). Highly significant positive correlation between auxin production analyzed by GC–MS and shoot length (r = 0.687**, P = 0.01) and shoot fresh weight (r = 0.703**, P = 0.01) was noted under gnotobiotic conditions. Similarly, significant correlation was also found between auxin production by Bacillus spp. (GC–MS analysis) and different growth parameters such as shoot length (r = 0.495*, P = 0.05), number of pods (r = 0.498*, P = 0.05) and grain weight (r = 0.537*, P = 0.05) at full maturity under natural wire house conditions. Results showed that auxin production potential of plant associated Bacillus spp. can be effectively exploited to enhance the growth and yield of V. radiata.     

Hydrogen and Polyhydroxybutyrate Producing Abilities of <Emphasis Type="Italic">Bacillus</Emphasis> spp. From Glucose in Two Stage System

Metabolic activities of four Bacillus strains to transform glucose into hydrogen (H₂) and polyhydroxybutyrate (PHB) in two stages were investigated in this study. Under batch culture conditions, Bacillus thuringiensis EGU45 and Bacillus cereus EGU44 evolved 1.67–1.92 mol H₂/mol glucose, respectively during the initial 3 days of incubation at 37°C. In the next 2 days, the residual glucose solutions along with B. thuringiensis EGU45 shaken at 200 rpm was found to produce PHB yield of 11.3% of dry cell mass. This is the first report among the non-photosynthetic microbes, where the Bacillus spp.—B. thuringiensis and B. cereus strains have been shown to produce H₂ and PHB in same medium under different conditions.     

Biodegradation of benzene by pure and mixed cultures of <Emphasis Type="Italic">Bacillus</Emphasis> spp.

Benzene has a wide range of industrial applications, but it is also a major source of environmental pollution. The most eco-friendly/cost-effective method of remediation is biodegradation. In the present study, we used a variety of microbial strains in different combinations on a selection of substrate concentrations to determine the most effective degradation processes. Bacterial strains of pure culture (L₄, N₃, and N₆) were isolated from oil sludge in both Luria–Bertani buffer (LB) and nutrient broth media, and identified by 16S-rRNA analysis (≥98% similarity). The degradation experiments were performed using different combinations of bacterial strains (L₄, N₃, N₆, L₄ + N₃, L₄ + N₆, N₃ + N₆, and L₄ + N₃ + N₆) in modified carbon-free media with different concentrations of benzene as a carbon source (60, 100, and 160 mg l⁻¹) at 30 °C. The isolates of L₄ (Acc no: FJ686821), N₃ (FJ686825) and N₆ (FJ868628) were identified as Bacillus spp. using 16S-rRNA gene sequence analysis. All combinations of isolates have the capacity to degrade benzene. However, the L₄ + N₃ combination was more efficient than the other mixed or single cultures. In the presence of N₆ isolate, the degradation rate of benzene decreased, possibly due to inter- and/or intra species interaction amongst the bacteria. The kinetic parameters ‘K _m’ of the Lineweaver–Burk regressions conducted as part of this experiment showed that the lower the level of K _m was, the better the biodegradation achieved. The results of this study showed that the use of Bacillus strains in benzene decomposition is feasible. In addition, different strain combinations exhibited different degradation patterns, which are attributed to the most efficient mixed cultures of Bacillus spp.     

Impact of different pH control agents on biopesticidal activity of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> during the fermentation of starch industry wastewater

Different pH control agents (NaOH/H₂SO₄—SodSulp, NaOH/CH₃COOH—SodAcet, NH₄OH/CH₃COOH—AmmoAcet and NH₄OH/H₂SO₄—AmmoSulp) were used to investigate their effects on growth, enzyme production (alkaline protease and amylase), and entomotoxicity of Bacillus thuringiensis var. kurstaki HD-1 (Btk) against eastern spruce budworm larvae (Choristoneura fumiferana) using starch industry wastewater (SIW) as a raw material in a 15-l fermentor. AmmoSulp and SodSulp were found to be the best pH control agents for alkaline protease and amylase production, respectively; whereas, the fermented broth obtained by using SodAcet as pH control agents recorded the highest delta-endotoxin production of 1043.0 mg/l and entomotoxicity value 18.4 × 10⁹ SBU/l. Entomotoxicity of re-suspended centrifuged pellet in one-tenth of original volume in case of SodAcet as pH control agents was 26.7 × 10⁹ SBU/l and was the highest value compared to three other pH control agents.     

Role of nitrite reductase in the ammonia-oxidizing pathway of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Metabolism of ammonia (NH₃) and hydroxylamine (NH₂OH) by wild-type and a nitrite reductase (nirK) deficient mutant of Nitrosomonas europaea was investigated to clarify the role of NirK in the NH₃ oxidation pathway. NirK-deficient N. europaea grew more slowly, consumed less NH₃, had a lower rate of nitrite (NO₂ ⁻) production, and a significantly higher rate of nitrous oxide (N₂O) production than the wild-type when incubated with NH₃ under high O₂ tension. In incubations with NH₃ under low O₂ tension, NirK-deficient N. europaea grew more slowly, but had only modest differences in NH₃ oxidation and product formation rates relative to the wild-type. In contrast, the nirK mutant oxidized NH₂OH to NO₂ ⁻ at consistently slower rates than the wild-type, especially under low O₂ tension, and lost a significant pool of NH₂OH–N to products other than NO₂ ⁻ and N₂O. The rate of N₂O production by the nirK mutant was ca. three times higher than the wild-type during hydrazine-dependent NO₂ ⁻ reduction under both high and low O₂ tension. Together, the results indicate that NirK activity supports growth of N. europaea by supporting the oxidation of NH₃ to NO₂ ⁻ via NH₂OH, and stimulation of hydrazine-dependent NO₂ ⁻ reduction by NirK-deficient N. europaea indicated the presence of an alternative, enzymatic pathway for N₂O production.     

The Influence of Ammonium Nitrate,pH and Indole Butyric Acid on Root Induction and Survival in Soil of Micropropagated <Emphasis Type="Italic">Eucalyptus globulus</Emphasis>

Rooting of Eucalyptus globulus shoots was influenced by the concentration of the indole butyric acid (IBA) and NH₄ ⁺ in the root-induction medium. Optimum plantlet vigor and survival were achieved using low concentrations (1 – 2.5 μM) of IBA and when NH₄NO₃ was removed. Removal of NH₄ ⁺ also had a significant effect on medium pH, its presence caused a decrease in pH as the culture period proceeded. When different nitrate compounds (excluding NH₄NO₃) were used as the nitrogen source, the medium pH was more stable and this was associated with higher root production. The higher root production, in association with appropriate IBA concentrations, produced plantlets with higher survival and better growth on transfer to soil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced 2,3-butanediol production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> SDM

Enhanced 2,3-butanediol (BD) production was carried out by Klebsiella pneumoniae SDM. The nutritional requirements for BD production by K. pneumoniae SDM were optimized statistically in shake flask fermentations. Corn steep liquor powder and (NH₄)₂HPO₄ were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform fed-batch fermentations with K. pneumoniae SDM. BD production was then studied in a 5-l bioreactor applying different fed-batch strategies, including pulse fed batch, constant feed rate fed batch, constant residual glucose concentration fed batch, and exponential fed batch. The maximum BD concentration of 150 g/l at 38 h with a diol productivity of 4.21 g/l h was obtained by the constant residual glucose concentration feeding strategy. To the best of our knowledge, these results were new records on BD fermentation. Cuiqing Ma and Ailong Wang contributed equally to this work.     

Effects of macro elements and nitrogen source on biomass accumulation and bacoside A production from adventitious shoot cultures of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.)

The present work deals with optimization of adventitious shoot culture of Bacopa monnieri for the production of biomass and bacoside A and has investigated the effects of macro elements (NH₄NO_3, KNO_3, CaCl_2, MgSO₄ and KH₂PO₄) and nitrogen source [NH₄ ⁺/NO₃ ⁻] of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) on accumulation of biomass and bacoside A content. Optimum number of adventitious shoots (99.33 shoots explant⁻¹), fresh weight (1.841 g) and dry weight (0.150 g) were obtained in the medium with 2.0× strength of NH₄NO₃. The highest production of bacoside A content was also recorded in the medium of 2.0× NH₄NO₃, which produced 17.935 mg g⁻¹ DW. The number of adventitious shoot biomass and bacoside A content were optimum when the NO₃ ⁻ concentration was higher than that of NH₄ ⁺. Maximum number of shoots (70.00 shoots explant⁻¹), biomass (fresh weight 1.137 g and dry weight 0.080 g) and also bacoside A content (27.106 mg g⁻¹ DW) were obtained at NH₄ ⁺/NO₃ ⁻ ratio of 14.38/37.60 mM. Overall, MS medium supplemented with 2.0× NH₄NO₃ is recommended for most efficient bacoside A production.     

Phosphate accumulation of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis>

The cells of Acetobacter xylinum decreased phosphate concentration in the medium from 5 to 2.5 or 0.3 mM during incubation in the presence of Mg²⁺ and glucose, or Mg²⁺ and casamino acids, respectively. The prevalence of orthophosphate or polyphosphate in the biomass of A. xylinum depends on the medium composition. Under phosphate uptake in the presence of glucose, the content of orthophosphate in the biomass changed little, while that of polyphosphate increased fourfold. At incubation with casamino acids, the content of orthophosphate increased 15 times, while that of polyphosphate increased only 2.5 times. Some part of orthophosphate in this case seems to be bound with the cell surface. The polyphosphate chain length in the cells of A. xylinim increases under phosphate uptake. This increase is more noticeable in the presence of glucose. Casamino acids can be replaced by α-ketoglutaric acid in combination with (NH₄)₂SO₄, or arginine, or glutamine, the catabolism of which results in formation of NH₄ ⁺ and α-ketoglutarate.     

Natronobacillus azotifigens gen. nov., sp. nov., an anaerobic diazotrophic haloalkaliphile from soda-rich habitats

Gram-positive bacteria capable of nitrogen fixation were obtained in microoxic enrichments from soda soils in south-western Siberia, north-eastern Mongolia, and the Lybian desert (Egypt). The same organisms were obtained in anoxic enrichments with glucose from soda lake sediments in the Kulunda Steppe (Altai, Russia) using nitrogen-free alkaline medium of pH 10. The isolates were represented by thin motile rods forming terminal round endospores. They are strictly fermentative saccharolytic anaerobes but tolerate high oxygen concentrations, probably due to a high catalase activity. All of the strains are obligately alkaliphilic and highly salt-tolerant natronophiles (chloride-independent sodaphiles). Growth was possible within a pH range from 7.5 to 10.6, with an optimum at 9.5–10, and within a salt range from 0.2 to 4 M Na⁺, with an optimum at 0.5–1.5 M for the different strains. The nitrogenase activity in the whole cells also had an alkaline pH optimum but was much more sensitive to high salt concentrations compared to the growing cells. The isolates formed a compact genetic group with a high level of DNA similarity. Phylogenetic analysis based on 16S-rRNA gene sequences placed the isolates into Bacillus rRNA group 1 as a separate lineage with Amphibacillus tropicus as the nearest relative. In all isolates the key functional nitrogenase gene nifH was detected. A new genus and species, Natronobacillus azotifigens gen. nov., sp. nov., is proposed to accommodate the novel diazotrophic haloalkaliphiles. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the 16S rRNA gene of the novel strains are EU143681-EU143690 and EU850814-EU850816; for the nifH gene the accession numbers are EU542601, EU563380-EU563386 and EU850817-EU850819.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Kinetics of kojic acid fermentation by Aspergillus flavus using different types and concentrations of carbon and nitrogen sources

Kinetics of kojic acid fermentation by Aspergillus flavus Link 44-1 using various sources of carbon [glucose, xylose, sucrose, starch, maltose, lactose or fructose] and nitrogen [NH₄Cl, (NH₄)₂S₂O₈, (NH₄)₂NO₃, yeast extract or peptone] were analyzed using models based on logistic and Luedeking–Piret equations. The highest kojic acid production (39.90 g l⁻¹) in submerged batch fermentation was obtained when 100 g l⁻¹ glucose was used as a carbon source. Organic nitrogen sources such as peptone and yeast extract were favorable for kojic acid production as compared to inorganic nitrogen sources. Yeast extract at 5 g l⁻¹ was optimal. The optimal carbon to nitrogen (C/N) ratio for kojic acid fermentation was 93.3. In a resuspended cell system, the rate of glucose conversion to kojic acid by cell-bound enzymes increased with increasing glucose concentration up to 70 g l⁻¹, suggesting that the reaction followed the Michaelis–Menten enzyme kinetic model. The value of K _m and V _max for the reaction was 18.47 g l⁻¹ glucose and 0.154 g l⁻¹ h⁻¹, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 20–24. Received 13 October 1999/ Accepted in revised form 02 April 2000     

Optimization of polyhydroxybutyrate production by <Emphasis Type="Italic">Bacillus</Emphasis> sp. CFR 256 with corn steep liquor as a nitrogen source

Polyhydroxyalkanotes (PHAs), the eco-friendly biopolymers produced by many bacteria, are gaining importance in curtailing the environmental pollution by replacing the non-biodegradable plastics derived from petroleum. The present study was carried out to economize the polyhydroxybutyrate (PHB) production by optimizing the fermentation medium using corn steep liquor (CSL), a by-product of starch processing industry, as a cheap nitrogen source, by Bacillus sp. CFR 256. Response surface methodology (RSM) was used to optimize the fermentation medium using the variables such as corn steep liquor (5–25 g l⁻¹), Na₂HPO₄ 2H₂O (2.2–6.2 g l⁻¹), KH₂PO₄ (0.5–2.5 g l⁻¹), sucrose (5–55 g l⁻¹) and inoculum concentration (1–25 ml l⁻¹). Central composite rotatable design (CCRD) experiments were carried out to study the complex interactions of the variables. The optimum conditions for maximum PHB production were (g l⁻¹): CSL-25, Na₂HPO₄ 2H₂O-2.2, KH₂PO₄ − 0.5, sucrose − 55 and inoculum − 10 (ml l⁻¹). After 72 h of fermentation, the amount of PHA produced was 8.20 g l⁻¹ (51.20% of dry cell biomass). It is the first report on optimization of fermentation medium using CSL as a nitrogen source, for PHB production by Bacillus sp.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.     

Optimization of a corn steep medium for production of ethanol from synthesis gas fermentation by <Emphasis Type="Italic">Clostridium ragsdalei</Emphasis>

Fermentation of biomass derived synthesis gas to ethanol is a sustainable approach that can provide more usable energy and environmental benefits than food-based biofuels. The effects of various medium components on ethanol production by Clostridium ragsdalei utilizing syngas components (CO:CO₂) were investigated, and corn steep liquor (CSL) was used as an inexpensive nutrient source for ethanol production by C. ragsdalei. Elimination of Mg²⁺, NH₄ ⁺ and PO₄ ³⁻ decreased ethanol production from 38 to 3.7, 23 and 5.93 mM, respectively. Eliminating Na⁺, Ca²⁺, and K⁺ or increasing Ca²⁺, Mg²⁺, K⁺, NH₄ ⁺ and PO₄ ³⁻ concentrations had no effect on ethanol production. However, increased Na⁺ concentration (171 mM) inhibited growth and ethanol production. Yeast extract (0.5 g l⁻¹) and trace metals were necessary for growth of C. ragsdalei. CSL alone did not support growth and ethanol production. Nutrients limiting in CSL were trace metals, NH₄ ⁺ and reducing agent (Cys: cysteine sulfide). Supplementation of trace metals, NH₄ ⁺ and CyS to CSL (20 g l⁻¹, wet weight basis) yielded better growth and similar ethanol production as compared to control medium. Using 10 g l⁻¹, the nutritional limitation led to reduced ethanol production. Higher concentrations of CSL (50 and 100 g l⁻¹) were inhibitory for cell growth and ethanol production. The CSL could replace yeast extract, vitamins and minerals (excluding NH₄ ⁺). The optimized CSL medium produced 120 and 50 mM of ethanol and acetate, respectively. The CSL could provide as an inexpensive source of most of the nutrients required for the syngas fermentation, and thus could improve the economics of ethanol production from biomass derived synthesis gas by C. ragsdalei.     

Heterotrophic nitrogen removal by <Emphasis Type="Italic">Providencia rettgeri</Emphasis> strain YL

Providencia rettgeri strain YL was found to be efficient in heterotrophic nitrogen removal under aerobic conditions. Maximum removal of NH₄ ⁺–N occurred under the conditions of pH 7 and supplemented with glucose as the carbon source. Inorganic ions such as Mg²⁺, Mn²⁺, and Zn²⁺ largely influenced the growth and nitrogen removal efficiency. A quantitative detection of nitrogen gas by gas chromatography was conducted to evaluate the nitrogen removal by strain YL. From the nitrogen balance during heterotrophic growth with 180 mg/l of NH₄ ⁺–N, 44.5% of NH₄ ⁺–N was in the form of N₂ and 49.7% was found in biomass, with only a trace amount of either nitrite or nitrate. The utilization of nitrite and nitrate during the ammonium removal process demonstrated that the nitrogen removal pathway by strain YL was heterotrophic nitrification-aerobic denitrification. A further enzyme assay of nitrate reductase and nitrite reductase activity under the aerobic condition confirmed this nitrogen removal pathway.     

Screening of bacteria to produce polyhydroxyalkanoates from xylose

Although xylose is a major constituent of lignocellulosic feedstock and the second most abundant sugar in nature, only 22% of 3,152 screened bacterial isolates showed significant growth in xylose in 24 h. Of those 684, only 24% accumulated polyhydroxyalkanoates after 72 h. A mangrove isolate, identified as Bacillus sp. MA3.3, yielded the best results in literature thus far for Gram-positive strains in experiments with glucose and xylose as the sole carbon source. When glucose or xylose were supplied, poly-3-hydroxybutyrate (PHB) contents of cell dry weight were, respectively, 62 and 64%, PHB yield 0.25 and 0.24 g g⁻¹ and PHB productivity (P_PHB) 0.10 and 0.06 g l⁻¹ h⁻¹. This 40% P_PHB difference may be related to the theoretical ATP production per 3-hydroxybutyrate (3HB) monomer calculated as 3 mol mol⁻¹ for xylose, less than half of the ATP/3HB produced from glucose (7 mol mol⁻¹). In PHB production using sugar mixtures, all parameters were strongly reduced due to carbon catabolite repression. PHB production using Gram-positive strains is particularly interesting for medical applications because these bacteria do not produce lipopolysaccharide endotoxins which can induce immunogenic reactions. Moreover, the combination of inexpensive substrates and products of more value may lead to the economical sustainability of industrial PHB production.     

Yeasts isolated from olive mill wastewaters from southern Italy: technological characterization and potential use for phenol removal

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682—C. boidinii and 625, 642, 647, and 941—P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10–30°C) and (NH₄)₂SO₄ (0.0–6.0 g l⁻¹) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10°C with 6.0 g l⁻¹ of (NH₄)₂SO₄ added.     

Biotransformation of menthol and geraniol by hairy root cultures of <Emphasis Type="Italic">Anethum graveolens</Emphasis>: effect on growth and volatile components

Two oxygen-containing monoterpene substrates, menthol or geraniol (25 mg l⁻¹), were added to Anethum graveolens hairy root cultures to evaluate the influence of the biotransformation capacity on growth and production of volatile compounds. Growth was assessed by the dissimilation method and by fresh and dry weight measurement. The volatiles were analyzed by GC and GC–MS. The total constitutive volatile component was composed, in more than 50%, by falcarinol (17–52%), apiole (11–24%), palmitic acid (7–16%), linoleic acid (4–9%), myristicin (4-8%) and n-octanal (2-5%). Substrate addition had no negative influence on growth. The relative amount of menthol quickly decreased 48 h after addition, and the biotransformation product menthyl acetate was concomitantly formed. Likewise, the added geraniol quickly decreased over 48 h alongside with the production of the biotransformation products. The added geraniol was biotransformed in 10 new products, the alcohols linalool, α-terpineol and citronellol, the aldehydes neral and geranial, the esters citronellyl, neryl and geranyl acetates and linalool and nerol oxides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.