Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g⁻¹ with productivities of 3.2–5.0 g l⁻¹ h⁻¹. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l⁻¹ h⁻¹ by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l⁻¹ furfural and 10 g l⁻¹ acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.     

A pilot study on the regeneration of ferrous chelate complex in NOx scrubber solution by a biofilm electrode reactor

A chemical absorption-biological reduction integrated process has been proposed for the removal of nitrogen oxides (NO_x) from flue gases. In this study, we report a new approach using biofilm electrode reactor (BER) to regenerate Fe(II)EDTA via simultaneously reducing Fe(II)EDTA-NO and Fe(III)EDTA in NO_x scrubber solution. Biofilm formed on the surface of the cathode was confirmed by Environmental Scan Electro-Microscope. Experimental results demonstrated that it was effective and feasible to utilize the BER to promote the reduction of Fe(II)EDTA-NO and Fe(III)EDTA simultaneously. The reduction efficiency of Fe(II)EDTA-NO and Fe(III)EDTA was up to 85% and 78%, respectively when the BER was continuously operated with electricity current at 30 mA. The absence of electricity induced an immediate decrease in reduction efficiency, indicating that the bio-regeneration of ferrous chelate complex was electrochemically accelerated. The present approach is considered advantageous for the enhanced bio-reduction in the NO_x scrubber solution.     

Resynchronization with unknown pregnancy status using progestin-based timed artificial insemination protocol in beef cattle

Two experiments were designed to evaluate the use of resynchronization (RESYNCH) protocols using a progestin-based timed artificial insemination (TAI) protocol in beef cattle. In experiment 1, 475 cyclic Nelore heifers were resynchronized 22 days after the first TAI using two different inducers of new follicular wave emergence (estradiol benzoate [EB; n = 241] or GnRH [n = 234]) with the insertion of a norgestomet ear implant. At ear implant removal (7 days later), a pregnancy test was performed, and nonpregnant heifers received a dose of prostaglandin plus 0.5 mg of estradiol cypionate, with a timed insemination 48 hours later. The pregnancy rate after the first TAI was similar (P = 0.97) between treatments (EB [41.9%] vs. GnRH [41.5%]). However, EB-treated heifers (49.3%) had a greater (P = 0.04) pregnancy per AI (P/AI) after the resynchronization than the GnRH-treated heifers (37.2%). In experiment 2, the pregnancy loss in 664 zebu females (344 nonlactating cows and 320 cyclic heifers) between 30 and 60 days after resynchronization was evaluated. Females were randomly assigned to one of two groups (RESYNCH 22 days after the first TAI [n = 317] or submitted only to natural mating [NM; n = 347]). Females from the NM group were maintained with bulls from 15 to 30 days after the first TAI. The RESYNC-treated females were resynchronized 22 days after the first TAI using 1 mg of EB on the first day of the resynchronization, similar to experiment 1. No difference was found in P/AI (NM [57.1%] vs. RESYNC [61.5%]; P = 0.32) or pregnancy loss (NM [2.0%] vs. RESYNC [4.1%]; P = 0.21) after the first TAI. Moreover, the overall P/AI after the RESYNCH protocol was 47.5%. Thus, the administration of 1 mg of EB on day 22 after the first TAI, when the pregnancy status was undetermined, promotes a higher P/AI in the resynchronized TAI than the use of GnRH. Also, the administration of 1 mg of EB 22 days after the TAI did not affect the preestablished pregnancy.     

Gyro-scope: an individual-based computer model to forecast gyrodactylid infections on fish hosts

Individual-based computer models (IBM) feature prominently in current theoretical ecology but have only been applied in a small number of parasitological studies. Here we designed an IBM to simulate the infection dynamics of gyrodactylid parasites and immune defence of na?ve hosts (i.e. fish previously not exposed to these parasites). We compared the results of the model with empirical data from guppies (Poecilia reticulata) infected with Gyrodactylus parasites. The laboratory experiments on guppies showed that larger fish acquired a heavier parasite load at the peak of the infection. The survival probability declined with increased body size and no fish survived a parasite load of 80 or more worms in this experiment (i.e. lethal load). The model was a good predictor of the Gyrodactylus infection dynamics of guppies and the model output was congruent with previously published data on Gyrodactylus salaris infections of salmon (Salmo salar). Computer simulations indicated that the infections persisted longer on larger hosts and that the parasite load increased exponentially with the body size of the host. Simulations furthermore predicted that the parasite load of fish with a standard length in excess of 17mm (i.e. the size of adult guppies) reached a lethal load. This suggests that in the conditions of the experiment, the immune defence of na?ve guppies can offer moderate protection against gyrodactylid infections to juveniles, but not to na?ve adult guppies. The model is a useful tool to forecast the development of gyrodactylid infections on single hosts and make predictions about optimal life history strategies of parasites.     

Infection with an acanthocephalan manipulates an amphipod's reaction to a fish predator's odours

Many parasites with complex life cycles increase the chances of reaching a final host by adapting strategies to manipulate their intermediate host's appearance, condition or behaviour. The acanthocephalan parasite Pomphorhynchus laevis uses freshwater amphipods as intermediate hosts before reaching sexual maturity in predatory fish. We performed a series of choice experiments with infected and uninfected Gammarus pulex in order to distinguish between the effects of visual and olfactory predator cues on parasite-induced changes in host behaviour. When both visual and olfactory cues, as well as only olfactory cues were offered, infected and uninfected G. pulex showed significantly different preferences for the predator or the non-predator side. Uninfected individuals significantly avoided predator odours while infected individuals significantly preferred the side with predator odours. When only visual contact with a predator was allowed, infected and uninfected gammarids behaved similarly and had no significant preference. Thus, we believe we show for the first time that P. laevis increases its chance to reach a final host by olfactory-triggered manipulation of the anti-predator behaviour of its intermediate host.     

Effect of interval from induction of puberty to initiation of a timed AI protocol on pregnancy rate in Nellore heifers

Prepubertal Bos indicus heifers (n = 774) were submitted to an E₂/P₄-based timed artificial insemination (TAI) protocol at three different intervals after induction of their pubertal ovulation by insertion of an intravaginal progesterone (P₄) device for 12 days. Heifers were randomly assigned to start the TAI protocol at 10 (group 10; n = 253), 12 (group 12; n = 265), or 14 (group 14; n = 256) days after the P₄ device was removed. The TAI protocol consisted of the following: insertion of intravaginal device containing P₄ (Controlled internal drug release [CIDR]; previously used twice for 9 days each) + estradiol benzoate (2 mg) on Day 0, CIDR withdrawal + estradiol cypionate (0.5 mg) and PGF2α (12.5 mg) on Day 9, and TAI on Day 11. A subgroup of heifers (n = 472) was evaluated by ultrasound on Days 9 and 11 to evaluate the ovaries and to determine P₄ concentrations on Day 9. On Day 9, more (P < 0.05) CLs were present, and follicular diameter was smaller (P < 0.05) for group 10 than for groups 12 and 14 (38.4%, 29.3%, and 23.3% with CL and 9.4 ± 0.1, 9.9 ± 0.1, and 9.8 ± 0.1 mm diameter, respectively), but P₄ concentrations did not differ (P > 0.1) between treatments (2.4 ± 0.06 ng/mL). Follicular diameter at TAI (11.08 ± 0.09 mm) and ovulation rate (88.4%) did not differ between treatments (P > 0.1). However, conception and pregnancy rates for all heifers were greater (P < 0.05) in group 12 (50.4% and 45.5%, respectively) than in group 10 (38.2% and 33.7%, respectively), with group 14 intermediate to other treatments (45.6% and 40.6%, respectively). The final pregnancy rate did not differ between treatments (80.9%). In conclusion, a 12-day interval from the end of the puberty induction protocol to the start of the TAI protocol resulted in greater conception and pregnancy rates in prepubertal Nellore heifers.     

Mucor indicus: Biology and industrial application perspectives: A review

Mucor indicus, one of the most important strains of zygomycetes fungi, has been the subject of several studies since a couple of hundred years ago. This fungus, regarded as a non-pathogenic dimorphic microorganism, is used for production of several beers and foods. Morphology of the fungus can be manipulated and well controlled by changing a number of parameters. Furthermore, M. indicus can grow on a variety of substrates including lignocellulosic hydrolysates which are mixtures of hexoses, pentoses, and different severe fermentation inhibitors. Indeed, high yield ethanol production is among the most important features of this strain. Presence of considerable amounts of chitosan in the cell wall is another important aspect of the fungus. Besides production of ethanol and chitosan, the biomass of this fungus has shown a great potential to be used as a rich nutritional source, e.g. fish feed. The fungus is also among the oleaginous fungi and produces high amounts of polyunsaturated fatty acids particularly γ-linolenic acid. Furthermore, the biomass autolysate has a high potential for yeast extract replacement in fermentation by the fungus. Additionally, the strain has shown promising results in heavy metal removal from wastewaters. This review discusses different aspects of biology and industrial application perspectives of M. indicus. Furthermore, open areas for the future basic and applied levels of research are also presented.     

PKA from Mucor circinelloides: model to study the role of linker I in the interaction between R and C subunits

Protein kinase A from the fungus Mucor circinelloides shows high affinity interaction between regulatory (R) and catalytic (C) subunits. Its R subunit shows a differential presence of several acidic residues in linker I region, in the amino terminus. Mutants R1, lacking the N-terminal region, and R2, lacking the acidic cluster, were used to analyze its effect on the interaction with the C subunit, assessed through inhibition of catalytic activity and cAMP activation of reconstituted holoenzyme. A similar decrease in the interaction was obtained when using R1 and R2 with the homologous C subunit; however when using heterologous bovine C, only R1 had a decreased interaction. The results show the general importance of linker I region in the R-C interaction in protein kinases A and point to the importance of the acidic cluster present in the N-terminus of M. circinelloides R subunit in the high affinity interaction between R and C in this holoenzyme.     

Metal biosorption by two cyanobacterial mats in relation to pH, biomass concentration, pretreatment and reuse

The pH-dependent metal sorption by Oscillatoria- and Phormidium-dominated mats was effectively expressed by the Hill function. The estimated Hill functions can fruitfully predict the amount of metal sorbed at a particular initial pH. Pretreatment of biomass with 0.1 mmol L⁻¹ HCl was more effective than pretreatment with CaCl₂, HNO₃, NaOH, and SDS in enhancing metal sorption ability of the biomass. Desorption of metal ions in the presence of 100 mmol L⁻¹ HCl from metal-loaded mat biomass was completed within 1 h. After six cycles of metal sorption/desorption, sorption decreased by 6-15%. Only 6% and 11% of the biomass derived from the Oscillatoria sp.- and Phormidium sp.-dominated mats was lost during the cycling. The cyanobacterial mats seem to have better potential than several biomass types for use in metal sorption from wastewaters as they are ubiquitous, self-immobilized, and have good reusability.     

Arthrospira maxima prevents the acute fatty liver induced by the administration of simvastatin,ethanol and a hypercholesterolemic diet to mice

An evident fatty liver, corroborated morphologically and chemically, was produced in CD-1 mice after five daily doses of simvastatin 75 mg/Kg body weight, a hypercholesterolemic diet and 20 percent ethanol in the drinking water. After treating the animals, they presented serum triacylglycerols levels five times higher than the control mice, total lipids, cholesterol and triacylglycerols in the liver were 2, 2 and 1.5 times higher, respectively, than in control animals. When Arthrospira maxima was given with diet two weeks prior the onset of fatty liver induction, there was a decrement of liver total lipids (40%), liver triacylglycerols (50%) and serum triacylglycerols (50%) compared to the animals with the same treatment but without Arthrospira maxima. In addition to the mentioned protective effect, the administration of this algae, produced a significant increase (45%) in serum high density lipoproteins. The mechanism for this protective effect was not established in these experiments.     

Seed development in Malpighiaceae species with an emphasis on the relationships between nutritive tissues

Malpighiaceae ovules have a well-developed nucellus; previous observations indicate that during seed development, the endosperm does not proliferate, thus, remaining scarce. This study aimed at identifying the nutritive tissues during seed development in Malpighiaceae, focusing especially on the endosperm. We analysed the seed development of Janusia mediterranea, J. occhionii, Mascagnia cordifolia, and Tetrapterys chamaecerasifolia, which were collected and processed by traditional methods for light microscopy. Ovules are subcampylotropous, crassinucellate and unitegmic in Janusia and bitegmic in M. cordifolia and T. chamaecerasifolia. The nucellus is well developed and protrudes through the micropyle, touching the funicular obturator. During development, a pachychalaza is formed, and the integuments coalesce in bitegmic species. Through a series of nucellar cell divisions, the perisperm is formed. In Janusia species, the endosperm is not produced. In M. cordifolia and T. chamaecerasifolia, the endosperm is nuclear, but it is scarce and ephemeral. The mature seed is exalbuminous, and the perisperm is consumed, and thus, the mature embryo is total. The absence of endosperm in Janusia is newly observed for the family and indicates functional transfer for the abundant perisperm.     

Intracellular monitoring of superoxide dismutase expression in an Escherichia coli fed-batch cultivation using on-line disruption with at-line surface plasmon resonance detection

An on-line cell disruption system for at-line monitoring of the intracellular concentration of recombinant human superoxide dismutase (rhSOD) in a genetically modified Escherichia coli strain, HMS174(DE3) (pET11a/rhSOD), in bioreactor cultivations is described. The sampled bacteria were disrupted on-line by rapid mixing with a nonionic detergent. The recombinant protein content of the lysed bacterial sample was quantitated by a subsequent surface plasmon resonance biosensor with a specific monoclonal antibody. Extraction efficiency of the monitoring system was optimized with respect to the flow rate ratio of the cell suspension and the detergent at relevant cell densities with the aim to attain rapid monitoring. Monitoring was demonstrated for a shake flask culture and a glucose-limited fed-batch cultivation. The results are compared with a traditional enzyme-linked immunosorbent assay method showing a correlation coefficient of R2 = 0.97. Extraction efficiency of rhSOD reached 95-99% at a total processing time of 1.8-2.6 min and a contact time of 0.8-1.4 min. The possibility of extending the monitoring system to other intracellular proteins is discussed.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both).Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism.Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.     

Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.     

ARI-1, an RBR family ubiquitin-ligase, functions with UBC-18 to regulate pharyngeal development in C. elegans

The LIN-35 retinoblastoma protein homolog and the ubiquitin-conjugating enzyme UBC-18 function redundantly to control an early step of pharyngeal morphogenesis in C. elegans. In order to identify ubiquitin-ligases acting downstream of UBC-18, we carried out a two-hybrid screen using UBC-18 as the bait molecule. Our screen identified three putative ubiquitin-ligases, one of which, ARI-1, showed genetic interactions leading to defective pharyngeal development that were identical to that previously observed for UBC-18. ARI-1 is a member of the RBR family of ubiquitin-ligases and contains a C-terminal motif that places it within the highly conserved Ariadne subfamily of RBR ligases. Our analyses indicate that ARI-1 is the principal Ariadne family member in C. elegans that is involved in the control of pharyngeal development with UBC-18. Using GFP reporters, we find that ARI-1 is expressed dynamically in a wide range of tissues including muscles and neurons during embryonic and postembryonic development. We also provide evidence that dsRNA species containing 14 or fewer base pairs of contiguous identity with closely related mRNAs are sufficient to mediate off-target silencing in C. elegans.     

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone,equine chorionic gonadotropin,or temporary calf removal

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n = 36) and anestrous (n = 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P = 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows.     

A combined cultivation and cultivation-independent approach shows high bacterial diversity in water-miscible metalworking fluids

Ten metalworking fluids (MWF) and seven water preparation basis samples (WPB) were taken from five industrial plants in Germany. Total cells (TCC) and colony forming units (CFU) were counted, strains were isolated and their 16S rRNA gene was sequenced. Additionally, DNA was extracted directly from the samples, and clone libraries of 16S rRNA genes were built and gene sequenced. TCC ranged from 7.6×10(4) TCC/mL MWF to 1.6×10(8) TCC/mL MWF, and from 4.6×10(2) TCC/mL WPB to 7.8×10(7) TCC/mL WPB. The CFU showed similar but often lower results. A total of 70 isolates and 732 clones were 16S rRNA gene sequenced and all isolates, as well as 183 of the nearly full length 16S rRNA of these clones, were gene sequenced. A total of 98 different genera were detected in all 17 samples. The number of genera within each sample varied highly, with 1-22 genera per sample. The dominant genera in MWF were Leucobacter, Desemzia, Sphingomonas and Wautersiella. From these, only Sphingomonas was detected in WPB as well. This study showed that MWF can harbour a high bacterial diversity, which differs significantly from the bacterial flora of the corresponding WPB.