Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain.

Butyric acid is a valuable building-block for the production of chemicals and materials and nowadays it is produced exclusively from petroleum. The aim of this study was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces butyric acid at a high yield and selectivity from lignocellulosic biomasses. Pretreated (by wet explosion) and enzymatically hydrolysed wheat straw (PHWS), rich in C6 and C5 sugars (71.6 and 55.4 g l(-1) of glucose and xylose respectively), was used as substrate. After one year of serial selections, an adapted strain of C. tyrobutyricum was developed. The adapted strain was able to grow in 80% (v v(-1) ) PHWS without addition of yeast extract compared with an initial tolerance to less than 10% PHWS and was able to ferment both glucose and xylose. It is noticeable that the adapted C. tyrobutyricum strain was characterized by a high yield and selectivity to butyric acid. Specifically, the butyric acid yield at 60-80% PHWS lie between 0.37 and 0.46 g g(-1) of sugar, while the selectivity for butyric acid was as high as 0.9-1.0 g g(-1) of acid. Moreover, the strain exhibited a robust response in regards to growth and product profile at pH 6 and 7.

Continuous Fermentation of Wheat Straw Hydrolysate by Clostridium tyrobutyricum with In-Situ Acids Removal.

The present study focused on fermentative butyric acid production by Clostridium tyrobutyricum from pre-treated and hydrolysed wheat straw (PHWS) based on continuous operation mode and in situ acids extraction by reverse electro enhanced dialysis (REED). Different dilutions of PHWS in a synthetic medium (60-100 % v/v) were tested. It was found that continuous fermentation of PHWS greatly enhanced the sugar consumption rates and butyric acid productivity compared to batch tests, while application of REED enhanced them even further. Specifically, applying combined continuous operation mode and REED system for the fermentation of 70 % PHWS resulted in 19- and 53-fold higher glucose (1.37 g L-1 h-1) and xylose (0.80 g L-1 h-1) consumption rates, respectively, compared to those obtained by batch processing. Fermentation of 100 % PHWS continued unhindered with just urea and K2HPO4 added with butyric acid production rate, yield and selectivity being 1.30 g L-1 h-1, 0.45 g g-1 sugars and 0.88 g g-1 acids, respectively. These results were also confirmed in a 20 L pilot plant bioreactor system.

Rapid and reliable method for identification of associated endonuclease cleavage and recognition sites.

UNLABELLED: One barrier to cross during genetic engineering is the restriction-modification system found in many bacteria. In this study, we developed a fast and reliable method for mapping the recognition and cleavage site of the restriction endonucleases. Clostridium pasteurianum, a model organism for the study of nitrogen fixation, has been found to harbour at least two restriction-modification systems including the restriction endonucleases CpaPI, which is an isoschizomer of MboI and CpaAI. Dam-methylated DNA was used to isolate the activity of CpaAI. Exposing freshly prepared cell lysate to known nucleotide fragments and directly sequencing the pool of digested nucleotide fragments enabled identification of the cleavage sites in the fragments. By aligning the sequences adjacent to the cleavage site, it was possible to identify the recognition sequence. Using this method, we successfully located all CpaAI recognition and cleavage sites within the template sequence. By modifying DNA with both Dam and CpG methylases (M.SssI) and thereby preventing digestion by CpaPI and CpaAI, no further endonuclease activity was detected. SIGNIFICANCE AND IMPACT OF THE STUDY: Restriction-modification systems are important barriers to successful genetic modification in many bacterial species. In this study, we demonstrate an efficient and general applicable method for identifying endonuclease recognition and cleavage sites. For the study and the trails, the model organism for nitrogen fixation Clostridium pasteurianum was used. The method was proven to be reliable, and by modifying DNA at the identified sites, it is possible to prevent digestion.

Comparison of two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion with one-stage thermophilic (55 degrees C) digestion of cattle manure.

A two-stage 68 degrees C/55 degrees C anaerobic degradation process for treatment of cattle manure was studied. In batch experiments, an increase of the specific methane yield, ranging from 24% to 56%, was obtained when cattle manure and its fractions (fibers and liquid) were pretreated at 68 degrees C for periods of 36, 108, and 168 h, and subsequently digested at 55 degrees C. In a lab-scale experiment, the performance of a two-stage reactor system, consisting of a digester operating at 68 degrees C with a hydraulic retention time (HRT) of 3 days, connected to a 55 degrees C reactor with 12-day HRT, was compared with a conventional single-stage reactor running at 55 degrees C with 15-days HRT. When an organic loading of 3 g volatile solids (VS) per liter per day was applied, the two-stage setup had a 6% to 8% higher specific methane yield and a 9% more effective VS-removal than the conventional single-stage reactor. The 68 degrees C reactor generated 7% to 9% of the total amount of methane of the two-stage system and maintained a volatile fatty acids (VFA) concentration of 4.0 to 4.4 g acetate per liter. Population size and activity of aceticlastic methanogens, syntrophic bacteria, and hydrolytic/fermentative bacteria were significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. The density levels of methanogens utilizing H2/CO2 or formate were, however, in the same range for all reactors, although the degradation of these substrates was significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. Temporal temperature gradient electrophoresis profiles (TTGE) of the 68 degrees C reactor demonstrated a stable bacterial community along with a less divergent community of archaeal species.

Metabolic interactions between methanogenic consortia and anaerobic respiring bacteria.

Most types of anaerobic respiration are able to outcompete methanogenic consortia for common substrates if the respective electron acceptors are present in sufficient amounts. Furthermore, several products or intermediate compounds formed by anaerobic respiring bacteria are toxic to methanogenic consortia. Despite the potentially adverse effects, only few inorganic electron acceptors potentially utilizable for anaerobic respiration have been investigated with respect to negative interactions in anaerobic digesters. In this chapter we review competitive and inhibitory interactions between anaerobic respiring populations and methanogenic consortia in bioreactors. Due to the few studies in anaerobic digesters, many of our discussions are based upon studies of defined cultures or natural ecosystems.

Molecular ecology of anaerobic reactor systems.

Anaerobic reactor systems are essential for the treatment of solid and liquid wastes and constitute a core facility in many waste treatment plants. Although much is known about the basic metabolism in different types of anaerobic reactors, little is known about the microbes responsible for these processes. Only a few percent of Bacteria and Archaea have so far been isolated, and almost nothing is known about the dynamics and interactions between these and other microorganisms. This lack of knowledge is most clearly exemplified by the sometimes unpredictable and unexplainable failures and malfunctions of anaerobic digesters occasionally experienced, leading to sub-optimal methane production and wastewater treatment. Using a variety of molecular techniques, we are able to determine which microorganisms are active, where they are active, and when they are active, but we still need to determine why and what they are doing. As genetic manipulations of anaerobes have been shown in only a few species permitting in-situ gene expression studies, the only way to elucidate the function of different microbes is to correlate the metabolic capabilities of isolated microbes in pure culture to the abundance of each microbe in anaerobic reactor systems by rRNA probing. This chapter focuses on various molecular techniques employed and problems encountered when elucidating the microbial ecology of anaerobic reactor systems. Methods such as quantitative dot blot/fluorescence in-situ probing using various specific nucleic acid probes are discussed and exemplified by studies of anaerobic granular sludge, biofilm and digester systems.

State of the art and future perspectives of thermophilic anaerobic digestion.

The state of the art of thermophilic digestion is discussed. Thermophilic digestion is a well established technology in Europe for treatment of mixtures of waste in common large scale biogas plants or for treatment of the organic fraction of municipal solid waste. Due to a large number of failures over time with thermophilic digestion of sewage sludge this process has lost its appeal in the USA. New demands on sanitation of biosolids before land use will, however, bring the attention back to the use of elevated temperatures during sludge stabilization. In the paper we show how the use of a start-up strategy based on the actual activity of key microbes can be used to ensure proper and fast transfer of mesophilic digesters into thermophilic operation. Extreme thermophilic temperatures of 65 degrees C or more may be necessary in the future to meet the demands for full sanitation of the waste material before final disposal. We show data of anaerobic digestion at extreme thermophilic temperatures.

Expression and purification of dynamin II domains and initial studies on structure and function.

Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of dynamin II (dynamin(500-870)) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His)(6) peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose. On the contrary, the PRD and dynamin(500-870) had to be tagged with a (His)(6) peptide at the N-terminus to bind to nickel-chelating agarose. Additional tagging with the S-peptide, which forms a stable complex with immobilized S-protein, allowed removal of strongly interacting E. coli proteins. Circular dichroic spectra indicate a structured recombinant PHD with a secondary structure content similar to that of the known PHD from dynamin I. The N-terminally tagged, recombinant PRD is unfolded but nevertheless binds specifically to the SH3 domain of amphiphysin II as well as to proteins extracted from rat brain. The described methods are suitable to isolate functionally active domains of dynamin II in sufficient amount and purity for further studies.

Protein kinase C alpha-dependent phosphorylation of Golgi proteins.

Golgi-enriched membranes were phosphorylated in order to understand the mechanism for protein kinase C (PKC) regulation of exocytic vesicle formation at the trans-Golgi network. Two of the main PKC substrates were identified as MARCKS and Mac-MARCKS by two-dimensional electrophoresis (2-DE) and mass spectrometric sequencing. Annexin IV and profilin I, two other Golgi-associated proteins--although known as in vitro PKC substrates--were not phosphorylated in the Golgi-bound state.

Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network.

Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of dynamin II to the Golgi membrane. The impact of dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of dynamin II which concomitantly displaces dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.

Autonomic nervous system-controlled cardiac pacing: a comparison between intracardiac impedance signal and muscle sympathetic nerve activity.

A recently introduced rate responsive cardiac pacing system is based on information derived from the intracardiac impedance signal containing information on the inotropic state of the ventricle. This study compared the inotropic state index (ISI) with muscle sympathetic activity (MSA), both being modulated by the autonomic nervous system. Nine patients (66 +/- 3 years, mean +/- SEM) with Inos2DR pacemakers were included. Each patient was studied at rest and during cold pressor test (CPT). Microneurography of the peroneal nerve was performed to measure MSA continuously, which was digitally stored along with continuous surface ECG and blood pressure. The intracardiac impedance signal was transmitted by the pacemaker and stored simultaneously. Linear correlation between ISI and MSA was calculated for the period of the CPT. During CPT, mean systolic blood pressure increased from 122 +/- 4 to 149 +/- 6 mmHg (P < 0.0001), diastolic blood pressure increased from 74 +/- 8 to 86 +/- 4 mmHg (P = 0.02), and intrinsic heart rate increased from 69 +/- 7 to 75 +/- 7 beats/mill (P = 0.019). ISI increased by 21 +/- 7% (P = 0.018), MSA by 26 +/- 6% (P = 0.004). ISI and MSA were positively correlated during the CPT in eight of nine patients (R2 = 0.86-0.99, P < 0.0001). Negative correlation was found in one patient (R2 = 0.94). This study demonstrates parallel increases of the ISI and MSA during CPT. ISI and MSA showed a close linear relationship during provoked changes of sympathetic activity. These results provide further evidence that the sympathetic nervous system is responsible for the observed ISI changes.

Kinetics of sulfate and hydrogen uptake by the thermophilic sulfate-reducing bacteria thermodesulfobacterium sp. Strain JSP and thermodesulfovibrio sp. Strain R1Ha3

Half-saturation constants (Km), maximum uptake rates (Vmax), and threshold concentrations for sulfate and hydrogen were determined for two thermophilic sulfate-reducing bacteria (SRB) in an incubation system without headspace. Km values determined for the thermophilic SRB were similar to the constants described for mesophilic SRB isolated from environments with low sulfate concentrations.

Site-specific modification of 4.5S RNA apical domain by complementary oligodeoxynucleotides carrying an alkylating group.

Site-specific alkylation of RNA by reactive oligodeoxynucleotides provides structural information and represents the first step towards the design of RNA derivatives to be used for functional studies. Specific alkylation of 4.5S RNA at G53, the first base of the apical tetraloop, was achieved by incubation with oligodeoxynucleotide ON2, complementary to nucleotides 38-53, which carries a p-(N-2-chloroethyl-N-methylamino)benzylamidophosphate group at the 5' end. Alkylation efficiency was increased by a factor of 6, without alteration of specificity, in the presence of a helper oligodeoxynucleotide, ON1, complementary to nucleotides 58-71 of the opposite strand of the RNA helix. A second reactive oligodeoxynucleotide, ON1-3'-R, was obtained by attaching the alkylating group to the 3' end of ON1. ON1-3'-R was able to modify G58. In the presence of ON2 as a helper oligodeoxynucleotide, the specificity of ON1-3'-R changes and efficient alkylation of nucleotides G54, A56 and G57 of the apical region of 4.5S RNA was observed.

Protein kinase C bound to the Golgi apparatus supports the formation of constitutive transport vesicles.

Constitutive secretion of heparan sulphate proteoglycans (HSPGs) was stimulated in human hepatoma HepG2 cells by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC). To delineate more closely the site of PKC action, the packaging in vitro of 35SO4-labelled HSPGs into transport vesicles was investigated. Formation of transport vesicles at the trans-Golgi network was stimulated by PMA and inhibited by calphostin C or Ro 31-8220 by using a post-nuclear supernatant. Treatment of either isolated Golgi-enriched membranes or cytosolic proteins with calphostin C provided evidence that membrane-bound PKC forms strongly supported vesicle formation, whereas cytosolic PKC forms showed a marginal effect. The PKC isoforms PKC-alpha and PKC-zeta were attached to highly purified Golgi membranes, as shown by Western blotting. Both isoforms were localized by confocal immunofluorescence microscopy in the Golgi area of HepG2 cells. Immunoelectron microscopy of ultrathin cryosections of HepG2 cells showed that PKC-zeta predominantly attaches to the trans-Golgi region, whereas PKC-alpha binds to the cis- and trans-Golgi area.

Hybridization of two oligodeoxynucleotides to both strands of an RNA hairpin structure increases the efficiency of RNA-DNA duplex formation.

Hybridization of two oligodeoxyribonucleotides (ON1 and ON2), complementary to opposite strands of the apical domain of Escherichia coli 4.5S RNA, was studied. ON1, complementary to bases 58-71, was not able to form a stable RNA-DNA hybrid whereas ON2, complementary to bases 38-53, was. Addition of both oligonucleotides at the same time resulted in the formation of a ternary complex permitting hybridization of ON1 and increasing hybridization of ON2. Under this condition, binary complexes of ON1 or ON2 with 4.5S RNA were not observed. The data demonstrate that hybridization of oligonucleotides to both strands of an RNA hairpin structure increases the efficiency of hybridization of either oligonucleotide.

Carcinoembryonic antigen expression, antibody localisation and immunophotodetection of human colon cancer liver metastases in nude mice: a model for radioimmunotherapy.

Colorectal cancer frequently disseminates through the portal vein into the liver. In this study, outbred Swiss nude mice were adapted to facilitate the induction of liver metastases by a pre-grafting treatment with 6 Gy total body irradiation and i.v. injection of anti-asialo GM1 antibody. One day later, cultured LS 174T human colon cancer cells were injected into the surgically exposed spleen, which was resected 3 min later. In 48 of 65 mice, a few to several hundred liver metastases were macroscopically observed at dissection 3 to 4 weeks after transplantation. Ten of 10 mice, followed-up for survival, died with multiple large confluent liver metastases. By reducing the radiation dose to 4 or 0 Gy, or omitting the anti-asialo GM1 antibody injection, only 60%, 37% or 50% of mice, respectively, had visible metastases 3 weeks after transplantation. Carcinoembryonic antigen (CEA) measured in tumour extracts was in the mean 25.6 micrograms/g in liver metastases compared with 9.2 micrograms/g in s.c. tumours. Uptake of radiolabelled anti-CEA monoclonal antibody (MAb) in the metastases 12, 24 and 48 hr after injection gave a mean value of 39% of the injected dose per gram of tissue (ID/g). In comparison, MAb uptake in s.c. and intrasplenic tumours or lung metastases gave a mean percentage ID/g of 20, 18 and 15, respectively. Laser-induced fluorescence after injection of indocyanin-MAb conjugate allowed direct visual detection of small liver metastases, including some that were not visible under normal light. Preliminary results showed that mice, pre-treated with 4 Gy irradiation and the anti-asialo GM1 injection, were tolerant to radioimmunotherapy with a total dose of 500 muCi 131I labeled anti-CEA intact MAbs given in 3 injections.

Dynamin II binds to the trans-Golgi network.

Two dynamin II-specific antibodies were used to detect dynamin II attached to highly purified Golgi membranes of HepG2 cells. Within the Golgi apparatus dynamin II is predominantly localized to the trans-Golgi network, as shown by immunoelectron microscopy. Increased binding of dynamin II to the trans-Golgi network in the presence of GTP-gamma-S and a reduced binding upon stimulation of secretion by phorbolester indicates that dynamin II may have a function in post-Golgi vesicle transport.

Temperature regulation of anaerobic degradation of organic matter.

Anaerobic degradation of organic matter follows similar pathways in digesters and anaerobic freshwater sediments. The responsible microorganisms are linked in a complex food web, where short chain fatty acids and H2 are important intermediates. Degradation of short-chain fatty acids is endothermic under standard conditions and is only possible at low H2 partial pressures maintained by exothermic methanogenesis. The coupling between these endothermic and exothermic processes is delicate, and hence sensitive to environmental changes such as temperature variations. The effect of temperature on thermodynamics and on kinetics of these and other anaerobic degradation processes with emphasis on freshwater ecosystems is discussed.

Clinical pharmacokinetic studies of tetra(meta-hydroxyphenyl)chlorin in squamous cell carcinoma by fluorescence spectroscopy at 2 wavelengths.

To optimize photodynamic therapy (PDT) and photodetection of cancer with second-generation photosensitizers, knowledge of important variables such as the uptake of the dye and the dye contrast between normal and tumoral tissue after injection is necessary. The pharmacokinetics of a second-generation photosensitizer, tetra(meta-hydroxyphenyl)chlorin (mTHPC), is presented. To study this in a clinical context, an apparatus based on fluorescence spectroscopy and a noninvasive optical fiber probe has been used. The mTHPC fluorescence is induced at 2 excitation wavelengths (420 and 520 nm) with different penetration depth. The pharmacokinetics of mTHPC in patients with a squamous-cell carcinoma in the oral cavity show a signal selectivity as high as 16 about 3 hr after i.v. injection for the more advanced carcinomas. The magnitude of this selectivity appears to correlate with the staging of the cancer, the more invasive tumors showing the highest selectivity. Results obtained at 420 and 520 nm show little difference. These pharmacokinetics can be used directly for optimizing photodetection with mTHPC. However, complementary information on the localization of the drug by fluorescence microscopy, and a correlation of this data with tumor necrosis efficacy, are needed to optimize PDT timing.