Construction of Effective Minimal Active Microbial Consortia for Lignocellulose Degradation.

Enriched microbial communities, obtained from environmental samples through selective processes, can effectively contribute to lignocellulose degradation. Unfortunately, fully controlled industrial degradation processes are difficult to reach given the intrinsically dynamic nature and complexity of the microbial communities, composed of a large number of culturable and unculturable species. The use of less complex but equally effective microbial consortia could improve their applications by allowing for more controlled industrial processes. Here, we combined ecological theory and enrichment principles to develop an effective lignocellulose-degrading minimal active microbial Consortia (MAMC). Following an enrichment of soil bacteria capable of degrading lignocellulose material from sugarcane origin, we applied a reductive-screening approach based on molecular phenotyping, identification, and metabolic characterization to obtain a selection of 18 lignocellulose-degrading strains representing four metabolic functional groups. We then generated 65 compositional replicates of MAMC containing five species each, which vary in the number of functional groups, metabolic potential, and degradation capacity. The characterization of the MAMC according to their degradation capacities and functional diversity measurements revealed that functional diversity positively correlated with the degradation of the most complex lignocellulosic fraction (lignin), indicating the importance of metabolic complementarity, whereas cellulose and hemicellulose degradation were either negatively or not affected by functional diversity. The screening method described here successfully led to the selection of effective MAMC, whose degradation potential reached up 96.5% of the degradation rates when all 18 species were present. A total of seven assembled synthetic communities were identified as the most effective MAMC. A consortium containing Stenotrophomonas maltophilia, Paenibacillus sp., Microbacterium sp., Chryseobacterium taiwanense, and Brevundimonas sp. was found to be the most effective degrading synthetic community.

Isolation and identification of biocellulose-producing bacterial strains from Malaysian acidic fruits.

UNLABELLED: Biocellulose (BC) is pure extracellular cellulose produced by several species of micro-organisms that has numerous applications in the food, biomedical and paper industries. However, the existing biocellulose-producing bacterial strain with high yield was limited. The aim of this study was to isolate and identify the potential biocellulose-producing bacterial isolates from Malaysian acidic fruits. One hundred and ninety-three bacterial isolates were obtained from 19 local acidic fruits collected in Malaysia and screened for their ability to produce BC. A total of 15 potential bacterial isolates were then cultured in standard Hestrin-Schramm (HS) medium statically at 30°C for 2 weeks to determine the BC production. The most potent bacterial isolates were identified using 16S rRNA gene sequence analysis, morphological and biochemical characteristics. Three new and potent biocellulose-producing bacterial strains were isolated from soursop fruit and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. Stenotrophomonas maltophilia WAUPM42 was the most potent biocellulose-producing bacterial strain that produced the highest amount of BC 0·58 g l(-1) in standard HS medium. Whereas, the isolates P. vagans WAUPM45 and B. fluminensis WAUPM53 showed 0·50 and 0·52 g l(-1) of BC production, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Biocellulose (BC) is pure extracellular cellulose that is formed by many micro-organisms in the presence of carbon source and acidic condition. It can replace plant-based cellulose in multifarious applications due to its unique characteristics. In this study, three potential biocellulose-producing bacterial strains were obtained from Malaysian acidic fruits and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. This study reports for the first time the new biocellulose-producing bacterial strains isolated from Malaysian acidic fruits.

Isolation of bacterial strains able to metabolize lignin and lignin-related compounds.

UNLABELLED: In this study, we identified five strains isolated from soil and sediments able to degrade kraft lignin, aromatic dyes and lignin derivatives. Using 16S rRNA gene sequencing, the isolates were identified as Serratia sp. JHT01, Serratia liquefacien PT01, Pseudomonas chlororaphis PT02, Stenotrophomonas maltophilia PT03 and Mesorhizobium sp. PT04. All the isolates showed significant growth on lignin with no water-extractable compounds. Synthetic aromatic dyes were used to assess the presence of oxidative enzymes. All the isolates were able to use the thiazine dye Methylene blue and the anthraquinone dye Remazol Brilliant Blue R as the sole carbon source. Guaiacol, veratryl alcohol and biphenyl were also mineralized by all the strains isolated. These results suggest they could be used for the treatment of aromatic pollutants and for the degradation of the lignocellulosic biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: The valorization of waste lignin and lignocellulosic biomass by biocatalysis opens up new possibilities for the production of value-added substituted aromatics, biofuel and for the treatment of aromatic pollutants. Bacteria with ligninolytic potential could be a source of novel enzymes for controlled lignin depolymerization. In this work, five soil bacteria were isolated and studied. Every isolate showed significant growth on lignin and was able to degrade several lignin monomers and ligninolytic indicator dyes. They could thus be a source of novel ligninolytic enzymes as well as candidates for a bacterial consortium for the delignification of lignocellulosic biomass.

[Biofilm on a metal surface as a factor of microbial corrosion].

Main attention was given in the present review to the research methods, phases of biofilm's forming, exopolymer compounds of bacteria as main biofilm forming factor. A microbial corrosion as a result of interaction between the biofilm and metal surface was considered. The interaction was displayed in biomineralization. The future trends of biofilms study were bound with research of their architecture. That architecture was determined by the structure and function of biofilms compounds: biopolymers and biominerals.

Characterization of an efficient estrogen-degrading bacterium Stenotrophomonas maltophilia SJTH1 in saline-, alkaline-, heavy metal-contained environments or solid soil and identification of four 17ß-estradiol-oxidizing dehydrogenases.

The efficient bioremediation of estrogen contamination in complex environments is of great concern. Here the strain Stenotrophomonas maltophilia SJTH1 was found with great and stable estrogen-degradation efficiency even under stress environments. The strain could utilize 17ß-estradiol (E2) as a carbon source and degrade 90% of 10 mg/L E2 in a week; estrone (E1) was the first degrading intermediate of E2. Notably, diverse pH conditions (3.0-11.0) and supplements of 4% salinity, 6.25 mg/L of heavy metal (Cd2+ or Cu2+), or 1 CMC of surfactant (Tween 80/ Triton X-100) had little effect on its cell growth and estrogen degradation. The addition of low concentrations of copper and Tween 80 even promoted its E2 degradation. Bioaugmentation of strain SJTH1 into solid clay soil achieved over 80% removal of E2 contamination (10 mg/kg) within two weeks. Further, the whole genome sequence of S. maltophilia SJTH1 was obtained, and a series of potential genes participating in stress-tolerance and estrogen-degradation were predicted. Four dehydrogenases similar to 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) were found to be induced by E2, and the four heterogenous-expressed enzymes could oxidize E2 into E1 efficiently. This work could promote bioremediation appliance potential with microorganisms and biodegradation mechanism study of estrogens in complex real environments.

Combination of chromogenic differential medium and estA-specific PCR for isolation and detection of phytopathogenic Xanthomonas spp.

A xanthomonad differential medium (designated Xan-D medium) was developed, on which streaks and colonies of xanthomonads, including 13 species of the genus Xanthomonas, turned wet-shining yellow-green and were surrounded with a smaller milky zone and a bigger clear zone in 3 to 4 days. The characteristics could easily be differentiated from those of yellow nonxanthomonads and other bacteria. The mechanism of color change and formation of a milky zone on the medium are mainly due to the Tween 80 hydrolytic capacity of xanthomonads. The gene, estA, responsible for Tween 80 hydrolysis was cloned and expressed in Escherichia coli, which acquired a capacity to hydrolyze Tween 80 and could turn green and form a milky zone on the Xan-D medium. The nucleotide sequence of estA is highly conserved in the xanthomonads, and the sequence was used to design a specific PCR primer set. The PCR amplification using the primer set amplified a 777-bp specific DNA fragment for all xanthomonad strains tested. The Xan-D medium was used to isolate and differentiate Xanthomonas campestris pv. campestris from naturally infected cabbages with black rot symptoms for a rapid diagnosis. All isolated X. campestris pv. campestris strains developed characteristic colonies and were positive in the PCR with the estA primer set. The Xan-D medium was further amended with antibiotics and successfully used for the detection of viable X. campestris pv. campestris cells from plant seeds. Although some yellow nonxanthomonads and other saprophytic bacteria from plant seeds could still grow on the medium, they did not interfere with the color development of X. campestris pv. campestris. However, Stenotrophomonas maltophilia, which is closely related to xanthomonads, existing in a seed lot could also develop yellow-green color but had different colony morphology and was negative in the PCR with the estA primer set. Accordingly, the combination of the Xan-D medium with the estA-specific PCR is a useful and reliable method for the isolation and detection of viable xanthomonad cells from plant materials.

Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility.

We tested 40 clinical Stenotrophomonas maltophilia strains to investigate the possible correlation between adherence to and formation of biofilm on polystyrene, and cell surface properties such as hydrophobicity and motility. Most of the strains were able to adhere and form biofilm, although striking differences were observed. Eleven (27.5%) of the strains were hydrophobic, with hydrophobicity greatly increasing as S. maltophilia attached to the substratum. A positive correlation was observed between hydrophobicity and levels of both adhesion and biofilm formation. Most of the isolates showed swimming and twitching motility. A highly significant negative correlation was observed between swimming motility and level of hydrophobicity. Hydrophobicity is thus a significant determinant of adhesion and biofilm formation on polystyrene surfaces in S. maltophilia.

Stenotrophomonas maltophilia as a part of normal oral bacterial flora in captive snakes and its susceptibility to antibiotics.

Only little is known about normal oral bacterial flora in captive snakes containing Stenotrophomonas maltophilia. This microbe has been reported as a causative agent of numerous infections in reptiles. Therefore, the goal of the study was to detect its presence in the mouths of a significant number of healthy captive snakes and determining its susceptibility to antibiotics at 30 and 37 degrees C. The isolates were obtained in 1999-2005 from mouth swabs of 115 snakes of 12 genera and 22 species-most often Elaphe guttata (24 individuals; 20.9%). Susceptibility to 24 antibiotics was tested by the microdilution method. The microbe was demonstrated in 34 (29.6%) individuals. Overall, 47 strains of S. maltophilia were acquired. Evaluation using PFGE profiles and antibiograms resulted in confirmation of one strain of S. maltophilia in 23 (20.0%) individuals, two strains in nine (7.8%) and three in two (1.8%) snakes. All tested antibiotics were more effective at 37 degrees C, with the partial exception of cotrimoxazole and cefoperazone/sulbactam. At a temperature of 37 degrees C, the lowest frequency of resistance to levofloxacin (no resistant strains), cotrimoxazole and ofloxacin (97.9% of susceptible strains) was recorded. At 30 degrees C, the most active agents were cotrimoxazole (97.9% of susceptible strains), levofloxacin (91.5%) and ofloxacin (85.1%). In conclusion, S. maltophilia is present in the mouths of about one third of healthy captive snakes, showing good susceptibility to cotrimoxazole, some fluoroquinolones and aminoglycosides. The antibiotics (particularly aminoglycosides) are more effective at 37 degrees C.

[Effect of the biofilm biopolymers on the microbial corrosion rate of the low-carbon steel].

The relationship between exopolymer's specific production, relative carbohydrate and protein content in the biofilm exopolymers of the pure and mixed Thiobacillus thioparus and Stenotrophomonas maltophilia cultures and their corrosion activity was studied. Change of growth model of investigated cultures from plankton to biofilm led to an increase of specific exopolymer's production. In the biofilm formed by T. thioparus and S. maltophilia biofilm on the low-carbon steel surface one could observe an increase of relative protein content in the exopolymer complex in comparison with those in the pure culture. The development of such biofilms stimulatied the 7-fold corrosion activity.

Involvement of a quinoprotein (PQQ-containing) alcohol dehydrogenase in the degradation of polypropylene glycols by the bacterium Stenotrophomonas maltophilia.

Previous work has shown that when the bacterium Stenotrophomonas maltophilia is grown on polypropylene glycol, different dye-linked polypropylene glycol dehydrogenase (PPG-DH) activities are induced during growth. Here the purification and characterization of the dehydrogenase activity induced in the stationary phase, and present in the periplasmic space, is described. The homogeneous enzyme preparation obtained consists of a homodimeric protein with a molecular mass of about 123 kDa and an isoelectric point of 5.9. The cofactor of the enzyme appeared to be pyrroloquinoline quinone (PQQ), no heme c was present, and holo-enzyme contained two PQQ molecules per enzyme molecule. In these respects, PPG-DH described here is similar to already known quinoprotein alcohol dehydrogenases, but in other respects, it is different. Therefore, it is suggested that PPG-DH could be a new type of quinoprotein alcohol dehydrogenase. Based on its strong preference for polyols, PPG-DH seems well fitted to carry out the first step in the degradation of PPGs, synthetic polymers containing a variety of hydroxyl groups.

Differences in the permeability of high-flux dialyzer membranes for bacterial pyrogens.

AIMS: The increasing use of high-flux membranes for hemodialysis has raised concerns that patients dialyzed with these membranes may be at higher risk of being exposed to cytokine-inducing bacterial substances in the dialysate than patients dialyzed with low-flux membranes. We investigated the permeability of various high-flux membranes for both purified E. coli lipopolysaccharide (LPS) as well as for LPS derived from Stenotrophomonas (Sten.) maltophilia. MATERIALS AND METHODS: An in vitro dialysis circuit with saline in the blood compartment of 3 dialyzers containing different membranes (polysulfone, helixone and Diapes) was employed. The dialysate was challenged with increasing doses of sterile filtrates derived from Sten. maltophilia cultures or with purified LPS from E. coli. Samples from the blood compartment were tested for cytokine induction (IL-1beta, IL-6 and TNF) in mononuclear cells as well as for LPS by limulus amebocyte lysate test (LAL). RESULTS: IL-6 induction above sterile controls (< 0.02 ng/ml IL-6) was observed by samples from the blood side of DIAPES dialyzers (1.2 +/- 0.7 ng/ml IL-6) after challenging the dialysate with 4.1 +/- 3.6 U/ml E. coli LPS (9.9 +/- 4.5 ng/ml IL-6). In contrast, at the same challenge dose no significant IL-6 induction above sterile controls was observed by blood side samples of polysulfone (0.15 +/- 0.07 ng/ml) and helixone (0.09 +/- 0.05 ng/ml) dialyzers. Increasing the amount of E. coli LPS in the dialysate further augmented IL-6 induction by blood side samples of Diapes but not of polysulfone and helixone dialyzers. Similar results were obtained for IL-1beta and TNF. After challenging the dialysate with E. coli LPS as well as with cultures of Sten. maltophilia, significantly more LAL reactivity was observed in the blood compartment of Diapes compared to polysulfone and helixone. CONCLUSIONS: There are considerable differences between high-flux membranes regarding their permeability for cytokine-inducing substances from E. coli as well as for LPS derived from E. coli and Sten. maltophilia. Dialyzers that leak CIS under aqueous conditions in vivo should not be used unless the dialysate has passed through an ultrafilter.

Tracking of the viability of Stenotrophomonas maltophilia bacteria population in polyvinylalcohol nanofiber webs by positron annihilation lifetime spectroscopy.

Polyvinylalcohol (PVA) fiber web containing embedded bacteria was prepared by electrospinning technique. From the point of the complex functionality of such potential delivery systems, it will be of impact how bacteria can survive in such artificial biotopes. The present study suggests a possible fast method for the tracking of the viability of the embedded bacteria based on the changes of the supramolecular structure of the polymeric delivery system caused by the metabolic product of the bacteria. Positron annihilation lifetime spectroscopy (PALS) was applied to track the free volume changes of the system in the course of storage. The PALS method sensitively detected the free volume changes, thus the viability of the bacteria in the polymeric fiber web.

[Monosaccharide composition of exopolymer complex in Thiobacillus thioparus and Stenotrophomonas maltophilia].

The bacteria of Thiobacillus thioparus and Stenotrophomonas maltophilia are capable to form a thick biofilm complicated as to its structure on the surface of low-carbon steel. This biofilm formation on any surface occurs under the adhesion of the cells of the plankton growth model with the help of synthesis of muciferous exopolymers with adhesive properties. Hence the monosaccharide composition of the exopolymer complex in the form of polyol acetates was studied by chromate-mass-spectrum method. A significant difference in the composition of exopolymer monosaccharides with the presence of steel model in the medium and without it was established; a change in the monosaccharide composition of mono- and binary culture in the conditions of plankton and biofilm growth was also observed.

The use of silica gel prepared by sol-gel method and polyurethane foam as microbial carriers in the continuous degradation of phenol.

A mixed microbial culture was immobilized by entrapment into silica gel (SG) and entrapment/ adsorption on polyurethane foam (PU) and ceramic foam. The phenol degradation performance of the SG biocatalyst was studied in a packed-bed reactor (PBR), packed-bed reactor with ceramic foam (PBRC) and fluidized-bed reactor (FBR). In continuous experiments the maximum degradation rate of phenol (q(s)max) decreased in the order: PBRC (598 mg l(-1) h(-1)) > PBR (PU, 471 mg l(-1)h(-1)) > PBR(SG, 394 mg l(-1) h(-1)) > FBR (PU, 161 mg l(-1) h(-1)) > FBR (SG, 91 mg l(-1) h(-1)). The long-term use of the SG biocatalyst in continuous phenol degradation resulted in the formation of a 100-200 microm thick layer with a high cell density on the surface of the gel particles. The abrasion of the surface layer in the FBR contributed to the poor degradation performance of this reactor configuration. Coating the ceramic foam with a layer of cells immobilized in colloidal SiO2 enhanced the phenol degradation efficiency during the first 3 days of the PBRC operation, in comparison with untreated ceramic packing.