Several compounds that are formed or released during hydrolysis of lignocellulosic biomass inhibit the fermentation of the hydrolysate. The use of a liquid extractive agent is suggested as a method for removal of these fermentation inhibitors. The method can be applied before or during the fermentation. For a series of alkanes and alcohols, partition coefficients were measured at low concentrations of the inhibiting compounds furfural, hydroxymethyl furfural, vanillin, syringaldehyde, coniferyl aldehyde, acetic acid, as well as for ethanol as the fermentation product. Carbon dioxide production was measured during fermentation in the presence of each organic solvent to indicate its biocompatibility. The feasibility of extractive fermentation of hydrolysate was investigated by ethanolic glucose fermentation in synthetic medium containing several concentrations of furfural and vanillin and in the presence of decanol, oleyl alcohol and oleic acid. Volumetric ethanol productivity with 6 g/L vanillin in the medium increased twofold with 30% volume oleyl alcohol. Decanol showed interesting extractive properties for most fermentation inhibiting compounds, but it is not suitable for in situ application due to its poor biocompatibility. Biotechnol. Bioeng. 2009;102: 1354–1360. © 2008 Wiley Periodicals, Inc.

ABSTRACT Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans , encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m -fluoro- dl -phenylalanine and m -fluoro- l -tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.

Pseudomonas putida MC2 produces 3-methylcatechol from toluene in aqueous medium. A second phase of 1-octanol may improve total product accumulation. To optimise the design of such a biphasic process, a process model was developed, both for one- and two-phase applications. The insights obtained by the model predictions showed the importance of different process parameters (like growth substrate concentration and partition coefficient) on growth of biomass, accumulation of 3-methylcatechol and processing time. For future applications, the process model can be used to ensure enough extraction capacity from aqueous to octanol phase. It is a useful tool to define the optimum process conditions, depending on the desired optimisation parameter: product concentration or processing time.

Toluene-tolerant gram-positive bacteria were isolated and identified to belong to the genus Bacillus. They grew in a medium containing yeast extract and in the presence of a separate phase of toluene or other hydrocarbons, but not when aliphatic alcohols were present. The isolate Bacillus cereus R1 did not metabolise or transform toluene. Toluene accumulation in its cells was rapid, unless the organism was supplied with glucose as energy source. In bacteria adapted to toluene, the amount of toluene accumulating in cells was one-half that in nonadapted bacteria. Valinomycin (K+ ionophore) and o-vanadate (ATPase inhibitor) as inhibitors of energy metabolism partly counteracted the effect of glucose as energy source. These results suggest the presence of an efflux mechanism for toluene in strain R1. The nature of this mechanism and its function in a solvent-tolerant gram-positive strain are discussed.

Compared to the large number of main group Zintl compounds there are only a few that contain transition metals.[1] This is not surprising since the traditional definition of Zintl phases automatically excludes transition metals. However, many such compounds with transition metals can qualify for Zintl phases when a broader definition is used. The latter includes compounds of transition metals with filled or empty d shells, that is the late transition metals of the Ni, Cu, and Zn groups,[2] and the early transition elements of the Ti, V, and Cr groups at maximum formal oxidation states.[3] There are only two Zintl compounds containing a transition metal with partially filled d shell, both based on manganese, (AE)14MnPn11 (AE alkaline-earth metal, Pn pnictogen) and Sr21Mn4Sb18. Perhaps only they should be called true TMtransition metal Zintl phases∫ although the name is contradictory in itself. All but two of the d0 compounds contain isolated tetrahedra [MPn4] (M Nb, Ta, W, Ti).[5] The two exceptions are Na5HfAs3 with dimers of edge-sharing tetrahedra of [Hf2As6] and Rb5TaAs4Tl2 with [TaAs4] tetrahedra, where two Tl atoms bridge opposite edges, ( -Tl)[As2TaAs2]( -Tl)5 .[6] Here we report a new d0 transition metal Zintl phase, Cs7NbIn3As4, which contains an unprecedented anion, [{In3As4Nb} As]7 , a cubane made of three indium, four arsenic, and one niobium atom and a TMhandle∫ composed of an arsenic atom that is multiply bonded to the niobium corner. The title compound was initially made in an attempt to synthesize the recently reported Cs5In3As4 at temperatures higher than the original 500 C.[7] The reaction was carried out in niobium containers at 800 C, at which temperature the arsenic apparently attacked the container and formed the quaternary compound Cs7NbIn3As4. Later it was synthesized in high yield using the corresponding elements in stoichiometric ratio at the same temperature.[8] The overall structure of Cs7NbIn3As5 is quite simple and unremarkable,[9] an ionic assembly of isolated anions of [NbIn3As5] immersed in a TMsea∫ of cesium cations that screen them from each other (inter-anion dmin 5.009(4) ä). What is remarkable is the structure and bonding of the anion (Figure 1). Its geometry can be viewed in a few different ways. The more obvious approach is to recognize the cubane shape made of one Nb, three In, and four As atoms, [NbIn3As4], and its TMhandle∫ of a fifth arsenic atom attached to the niobium

The yeast Rhodotorula glutinis contains an enantioselective epoxide hydrolase. Previous work showed that the enzyme is a membrane-associated enzyme that can be solubilised from the membranes by a detergent treatment. Now, the effect of detergents on reaction rate and particularly enantioselectivity was investigated. Three types of detergents were tested: non-ionic, anionic and zwitterionic. Non-ionic detergents stimulated the specific activity of the enzyme. Enantioselectivity of the enzyme was strongly affected by several detergents. Thesit and sucrosemonolaurate had the most pronounced effects and enantiomeric ratios were strongly enhanced. The effects are most likely due to the ability of detergents to stabilise membrane-proteins by forming micelles and thus mimicking the membrane structure.

The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance.

The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general. For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained. Pseudomonas putida strains containing the todC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida F1, which degrades toluene via 3-methylcatechol, or into mutant P. putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette. As a result, up to 14 mM (1.74 g l–1) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 µmol min–1 g–1cell dry weight, which is four times higher than other catechol production systems. It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the production process. This is an important step for obtaining designer biocatalysts.

Twenty-one microorganisms were screened for their ability to convert nitroaromatics into 3-nitrocatechol as a result of the action of an oxygenase. Cultures containing toluene dioxygenases and phenol monooxygenases accumulated 3-nitrocatechol during incubation with nitrobenzene and nitrophenol, respectively. Nocardia S3 was selected and studied in more detail. Toluene-pregrown cultures were able to degrade nitrobenzene with a concomitant formation of 3-nitrocatechol. The rates of nitrobenzene utilization decreased throughout the biotransformation period and finally the accumulation ceased. The gradual deterioration of the biotransformation rates was not a consequence of depletion of the NADH pool, but was due to the accumulation of 3-nitrocatechol. The inhibition of nitrobenzene biotransformation by 3-nitrocatechol greatly impacts 3-nitrocatechol production processes.

Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMod-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a partial sequence of the interrupted genes was determined. Comparison of the deduced amino acid sequences with a protein database revealed the following interrupted putative gene products: organic solvent efflux proteins SrpA and SrpB, the flagellar structural proteins FlgK, FlaG, FliI, FliC, and FliH, the transcriptional activator FleQ, the alternative RNA polymerase sigma factor RpoN, and the flagellum-specific RNA polymerase sigma factor FliA (RpoF). The transposon mutants, except for the organic solvent efflux mutants, were nonmotile as determined by a swarm assay and the formation of the flagellum was totally impaired. Expression studies with a srp promoter probe showed a decreased expression of the SrpABC efflux pump in the nonmotile mutants.

Batch and soil column experiments were performed to investigate the potential of anaerobic oxidation processes for natural and stimulated degradation of chlorinated and non-chlorinated hydrocarbons. Model pollutants which included 1,2-dichloroethane (DCA), 1,1,1-trichloroethane (TCA), vinylchloride (VC), cis-1,2-dichloroethene (CIS), trans-1,2-dichloroethene (TRANS), trichloroethene (TCE), monochlororbenzene (MCB), 2-chloroethanol (CE) and octane were supplied as electron donor under iron (III), manganese (IV) or nitrate reducing conditions. Evidence was obtained that DCA, VC, MCB, CE and octane can be oxidised under anoxic conditions. Particularly nitrate-reducing conditions may have great potential for natural or stimulated remediation of sites contaminated with these particular hydrocarbons.

The novel insertion sequence ISS12plays a key role in the tolerance of Pseudomonas putida S12 to sudden toluene stress. Under normal culturing conditions theP. putida S12 genome contained seven copies of ISS12. However, a P. putida S12 population growing to high cell density after sudden addition of a separate phase of toluene carried eight copies. The survival frequency of cells in this variant P. putida S12 population was 1000 times higher than in “normal” P. putida S12 populations. Analysis of the nucleotide sequence flanking the extra ISS12 insertion revealed integration into the srpS gene. srpSforms a gene cluster with srpR and both are putative regulators of the solvent resistance pump SrpABC. SrpABC makes a major contribution to solvent tolerance in P. putida S12 and is induced by toluene. The basal level of srp promoter activity in the P. putida S12 variant was seven times higher than in wild-type P. putida S12. Introduction of the intact srpRS gene cluster in the variant resulted in a dramatic decrease of survival frequency after a toluene shock. These findings strongly suggest that interruption of srpS by ISS12 up-regulates expression of the solvent pump, enabling the bacterium to tolerate sudden exposure to lethal concentrations of toxic solvents. We propose that P. putida S12 employs ISS12 as a mutator element to generate diverse mutations to swiftly adapt when confronted with severe adverse conditions.

Several lactic acid bacteria produce exopolysaccharides (EPS), either attached to the cell wall or excreted into the environment as slime material. EPS produced by Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) and Streptococcus thermophilus play an important role in improving the texture and stability of yogurt and preventing syneresis (Cerning, 1990; Nakajima et al. 1990). The amount and composition of the EPS produced by lactic acid bacteria are dependent on a number of factors, such as temperature, initial pH, carbon source and the availability of minerals, vitamins and other medium components.In previous work it was shown that the production and sugar composition of the EPS from Lb. bulgaricus NCFB2772 are affected by the carbohydrate source (Grobben et al. 1995, 1996). In a simplified defined medium, from which several vitamins and trace elements were omitted, EPS production by Lb. bulgaricus significantly increased, although growth of the strain was reduced (Grobben et al. 1998).

Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h-1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.

Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1 R ,2 S )- and then the (1 S ,2 R )-isomer was observed. The hydrolysis of (4 R )- and (4 S )-limonene-1,2-epoxide resulted in, respectively, (1 S ,2 S ,4 R )- and (1 R ,2 R ,4 S )-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities ( E ratios between 34 and 3).