Anaerobic digestion of stillage fractions - estimation of the potential for energy recovery in bioethanol plants.

Stillage processing can require more than one third of the thermal energy demand of a dry-grind bioethanol production plant. Therefore, for every stillage fraction occurring in stillage processing the potential of energy recovery by anaerobic digestion (AD) was estimated. In the case of whole stillage up to 128% of the thermal energy demand in the process can be provided, so even an energetically self-sufficient bioethanol production process is possible. For wet cake the recovery potential of thermal energy is 57%, for thin stillage 41%, for syrup 40% and for the evaporation condensate 2.5%. Specific issues for establishing AD of stillage fractions are evaluated in detail; these are high nitrogen concentrations, digestate treatment and trace element supply. If animal feed is co-produced at the bioethanol plant and digestate fractions are to be reused as process water, a sufficient quality is necessary. Most interesting stillage fractions as substrates for AD are whole stillage, thin stillage and the evaporation condensate. For these fractions process details are presented.

Doubling the organic loading rate in the co-digestion of energy crops and manure--a full scale case study.

In the present study the increase of the organic loading rate from 2.11 to 4.25 kg VS m(-3) d(-1) in a two stage, agricultural biogas plant was investigated. The process enhancement resulted in the doubling of the plant capacity from 500 kW to 1000 kW retaining the same digester volume. Efficiency criteria showed good performance throughout the study. At the end of the monitoring, biogas yield was on the same level as before the enhancement, while volume related biogas productivity almost doubled from 1.50 to 2.91 Nm(3) m(-3) d(-1). However, as a consequence of the higher transfer of poorly degraded organic material into the effluent, the residual methane potential of the effluent multiplied by the factor 10. The results of this study show, that most agricultural biogas plants in Austria have a great potential for a significant capacity increase. However, to avoid atmospheric emissions, the effluent storage of high loaded processes has to be integrated into the gas-tight system of the digesters.

New data on temperature optimum and temperature changes in energy crop digesters.

As a result of self-heating in anaerobic digesters when using energy crops in the feedstock, the influence of temperature on the digestion process came back into focus. The aim of this study was to investigate the impact of such temperature increases on process stability. Furthermore, different strategies for the transition from mesophilic to thermophilic conditions and the resulting methane yields at different temperature levels were evaluated. Two main effects were identified with different bio-slurries from agricultural biogas plants: (1) a failure of methane production connected to changes in the microbial community; and (2), a slow but continuous accumulation of propionic acid, though without an immediate effect on methane production. All strategies for increasing the operating temperature showed negative effects on digester performance, some with serious economic consequences for the operator. It was shown that methane yields at different temperature levels in the mesophilic and sub-thermophilic ranges are similar.

The influence of energy crop substrates on the mass-flow analysis and the residual methane potential at a rural anaerobic digestion plant.

In a full scale anaerobic digestion plant exclusively operating on solid energy crops the mass-flows were analysed for two different substrate compositions over 583 d. The mono-fermentation of maize whole crop silage was compared to a mixture of maize and grass + clover silage. The two stage system required the input of dilution liquid guarantee digestion and agitation in the high loaded first stage (OLR: 5.50 kg VS.m(-3).d(-1)). Grass + clover demanded the double mass of process dilution liquid, which reduced SRT from 65 d to 34 d for each stage and leaded to an increased generation of Solid Digestion Residues by separation. Experiments showed that 70% of the Residual Methane Potential are caused by the 7% mass fraction of SDR. For maize and maize + grass + clover RMPs of 6.34% and 11.80% were observed, respectively. RMP can also be expressed as additional substrate input required for full granted operation. Thus, the mass stream analysis is used to determine mitigation strategies for RMP.

Comparing centralised and decentralised anaerobic digestion of stillage from a large-scale bioethanol plant to animal feed production.

A comparison of stillage treatment options for large-scale bioethanol plants was based on the data of an existing plant producing approximately 200,000 t/yr of bioethanol and 1,400,000 t/yr of stillage. Animal feed production--the state-of-the-art technology at the plant--was compared to anaerobic digestion. The latter was simulated in two different scenarios: digestion in small-scale biogas plants in the surrounding area versus digestion in a large-scale biogas plant at the bioethanol production site. Emphasis was placed on a holistic simulation balancing chemical parameters and calculating logistic algorithms to compare the efficiency of the stillage treatment solutions. For central anaerobic digestion different digestate handling solutions were considered because of the large amount of digestate. For land application a minimum of 36,000 ha of available agricultural area would be needed and 600,000 m(3) of storage volume. Secondly membrane purification of the digestate was investigated consisting of decanter, microfiltration, and reverse osmosis. As a third option aerobic wastewater treatment of the digestate was discussed. The final outcome was an economic evaluation of the three mentioned stillage treatment options, as a guide to stillage management for operators of large-scale bioethanol plants.

The impact of increasing energy crop addition on process performance and residual methane potential in anaerobic digestion.

In a full-scale agricultural biogas plant, the changes in process performance connected with the increasing energy crop addition were monitored. The substrates applied were pig manure, solid energy crops and agricultural residues. During the study, the organic loading rate and the volume-related biogas productivity were doubled to 4.2 kg VS/(m(.3)d) and 2.83 Nm(3)/(m(3).d) respectively, by means of increasing the energy crop ratio in the feedstock to 96.5% (volatile solids). This resulted in an increase of the electrical capacity on a level twice as high as before. At the same time, methane yield and organic degradation rate decreased slightly to 0.35 Nm(3)/kg VS(added) and 87.4%, respectively. The strongest impact observed was on the transfer of partly degraded organic material into the digestate storage and with this, an increase of the residual methane potential of the digestate. A maximum theoretical methane load in the digestate of 14.4% related to total methane production of the biogas plant was observed. This maximum level could be reduced to 5.5%.

Self-heating of anaerobic digesters using energy crops.

With the increasing application of energy crops in agricultural biogas plants and increasing digester volumes, the phenomenon of self-heating in anaerobic digesters appeared in some cases. Until now this development was just known from aerobic systems. To obtain an idea of the thermodynamics inside an anaerobic digester, a detailed analysis of all heat fluxes in a full-scale agricultural biogas plant was carried out. Several experiments were realised to quantify the influences of different internal and external energy sources. To estimate the impact of self-heating in anaerobic systems, data of other full-scale agricultural biogas plants in Austria were collected. Alternatives to the cooling of the digesters are discussed based on individual experiences of several plants. A connection between carbohydrate-rich substrates, especially with high starch contents, and the self-heating could be shown. From the results it can be assumed that the anaerobic digestion of most energy crops is exothermic, which is in contrast to the current thermodynamic belief.

Prion protein: detection in 'spiked' anaerobic sludge and degradation experiments under anaerobic conditions.

The behavior of the transmissible spongiform encephalopathies (TSE) causing agent denominated "prion protein" in anaerobic sludge (biogas reactor) was assessed with incubation tests. A widely applied screening method for BSE in cattle on the basis of the Western blotting protocol was adapted to detect the Proteinase K resistant, scrapie-form prion protein (PrPSC). As PrPsc source homogenized TSE infected brain tissue of animals late in the clinical phase of disease was taken (301V/VM mouse-BSE; bovine BSE and 22A/SV mouse-scrapie). The incubation under mesophilic conditions did not show any significant reduction of the PrPsc titer. Under thermophilic conditions contradictory results were obtained. The reduction time of PrPsc in water was equal to or longer than the PrPsc reduction time in anaerobic sludge. In comparison, with sterilized (121 degrees C, steam pressure) or poisoned (sodium azide, 1% w/v) sludge used as incubation matrix a much shorter time resulted until no prion protein could be detected.

Optimised anaerobic treatment of house-sorted biodegradable waste and slaughterhouse waste in a high loaded half technical scale digester.

Anaerobic co-digestion of organic wastes from households, slaughterhouses and meat processing industries was optimised in a half technical scale plant. The plant was operated for 130 days using two different substrates under organic loading rates of 10 and 12 kgCOD.m(-3).d(-1). Since the substrates were rich in fat and protein components (TKN: 12 g.kg(-1) the treatment was challenging. The process was monitored on-line and in the laboratory. It was demonstrated that an intensive and stable co-digestion of partly hydrolysed organic waste and protein rich slaughterhouse waste can be achieved in the balance of inconsistent pH and buffering NH4-N. In the first experimental period the reduction of the substrate COD was almost complete in an overall stable process (COD reduction >82%). In the second period methane productivity increased, but certain intermediate products accumulated constantly. Process design options for a second digestion phase for advanced degradation were investigated. Potential causes for slow and reduced propionic and valeric acid degradation were assessed. Recommendations for full-scale process implementation can be made from the experimental results reported. The highly loaded and stable codigestion of these substrates may be a good technical and economic treatment alternative.

Evidence for a putative senescence gene locus within the chromosomal region 10p14-p15.

High-risk human papillomavirus (HPV) types 16 and 18 are involved in the multistep process of cervical cancer. Transfection of normal keratinocytes with high-risk HPV-DNA generally gives rise to immortal cultures. This may be explained by the loss of senescence genes as a consequence of HPV-induced genetic instability. On the basis of the dominance of cellular senescence over immortality, fusion of normal keratinocytes with HPV-immortalized cells results in complementation of these putative gene defects. In a previous study, we showed that underrepresentation of chromosome 10 is a characteristic phenomenon during the early phase of immortalization. Here we show that introduction of a normal copy of chromosome 10 into HPV16-immortalized cells (HPKII) by Microcell-mediated chromosome transfer resulted in senescence of a significant number of hybrids. By using several derivatives of chromosome 10 for further fusion experiments, the chromosomal region responsible for senescence could be assigned to 10p14-p15. The potential significance of loss of gene function in this region is underlined by the high frequency (38.7%) of loss of heterozygosity in cervical cancers including early stage tumors.

Myocardial Gi alpha-protein levels in patients with hypertensive cardiac hypertrophy, ischemic heart disease and cardiogenic shock.

OBJECTIVE: Increased inhibitory G-protein alpha-subunits (Gi alpha) have been reported to be related to adenylyl cyclase desensitization in the failing human heart. In order to investigate whether this cellular alteration occurs already at the stage of hypertensive cardiac hypertrophy or in catecholamine-refractory cardiogenic shock, Gi alpha levels were studied in myocardial samples from patients with hypertensive cardiac hypertrophy, coronary heart disease without heart failure and from patients with cardiogenic shock on high-dose catecholamine therapy as well as from patients without evidence of heart disease. METHODS: Gi alpha was quantified with pertussis-toxin-catalyzed 32P-ADP-ribosylation and with a radioimmunoassay in myocardial samples from patients within 16 h of death. The radioimmunoassay was constructed with recombinant G-protein alpha-subunits (rGi alpha 1) from transformed E. coli harbouring the full-length cDNA of Gi alpha 1, iodinated peptide 125I-KENLKDCGLF and immunoprecipitating antiserum (MB 1) raised against the synthetic peptide (KENLKDCGLF) in rabbits. RESULTS: Pertussis toxin substrates and immunochemical Gi alpha remained stable up to 80 h following storage at room temperature in myocardium obtained during cardiac transplantation. Gs alpha, adenylyl cyclase, beta-adrenoceptors and inhibitory receptors were not stable and could not be determined. Increases in myocardial Gi alpha of 65-82% of both pertussis toxin substrates and immunologically quantified Gi alpha were observed in hypertensive cardiac hypertrophy. Catecholamine therapy in patients who died of catecholamine-refractory shock increased myocardial Gi alpha by 225% compared to myocardium from patients with coronary heart disease without heart failure and without catecholamine therapy or compared to control myocardium. CONCLUSION: These findings provide evidence than an increase in myocardial Gi alpha-proteins could be of relevance in pathological conditions other than chronic heart failure. Since an increase in Gi alpha levels already occurs in hypertensive cardiac hypertrophy, it could play a role in contributing to the development of contractile dysfunction and heart failure in later stages of the syndrome. Finally, an increase in Gi alpha could be one mechanism contributing to catecholamine refractoriness in shock. This could provide a target for pharmacological treatment in this condition.

Increase of myocardial inhibitory G-proteins in catecholamine-refractory septic shock or in septic multiorgan failure.

PURPOSE: The aim of the study was to investigate the mechanisms of myocardial catecholamine refractoriness in septic shock. METHODS: The inhibitory guanine nucleotide-binding proteins (Gi alpha) were studied with pertussis toxin labeling and radioimmunologically in myocardium from patients who died while in catecholamine-refractory septic shock and from patients who died of noncardiac disease. RESULTS: An increase by 62% (immunological Gi alpha) and 221% (pertussis toxin substrate) of myocardial Gi alpha was observed in patients with catecholamine-refractory shock compared with controls. The increases of Gi alpha were greater than those found in chronic heart failure reported earlier. CONCLUSIONS: An increase in the expression of Gi alpha could also be important in conditions other than chronic heart failure, eg, septic shock. An increase of Gi alpha could play a pathophysiologically relevant role in catecholamine refractoriness in septic shock and could provide a target for pharmacologic treatment in this condition.

C-terminal modifications of pertussis toxin-sensitive G-protein alpha-subunits differentially affect immunoreactivity. Evidence against endogenous ADP-ribosylation in human heart, lung, thrombocytes and adipose tissue.

Immunochemical detection of pertussis toxin-sensitive guanine-nucleotide binding proteins has been suggested to represent the most direct approach to quantitate the protein than pertussis toxin-catalysed [32P]ADP-ribosylation. The latter technique is potentially hampered by pre-existing covalent modification of the C-terminus. However, limited data exist as to whether and in what way modifications of the C-terminus affect immunoreactivity of Gi alpha (alpha-subunit of the inhibitory G-protein of adenylyl cyclase). Membranes from human myocardium, thrombocytes, adipose tissue and lung were treated with pertussis toxin or N-ethylmaleimide. Both, conditions prevented high affinity agonist binding to m-cholinoceptors and inhibited [32P]ADP-ribosylation by pertussis toxin consistent with the notion that the modifications took place at the C-terminus. Pertussis toxin treatment increased immunoreactivity to different antisera raised against the C-terminal decapeptide of transducin alpha (KENLKDCGLF, DS 1-4, AS). N-Ethylmaleimide reduced immunoreactivity towards all antisera studied. Pertussis toxin reduced the mobility of Gi alpha on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) depending on the presence of the toxin and sensitivity to inhibition of ADP-ribosylation by nicotinamide. In native membranes from none of the tissues studied, immunoreactive material comigrating with pertussis toxin-modified form of Gi alpha was detected. It is concluded that modification of the C-terminus by pertussis toxin or N-ethylmaleimide resulting in the same functional consequence, i.e. prevention of high affinity agonist receptor binding, is capable of producing opposite changes of immunoreactivity. Pertussis toxin treatment reduces the electrophoretic mobility on SDS-PAGE. Separation of the native and pertussis toxin-modified form of Gi alpha on SDS-PAGE demonstrates that endogenously ADP-ribosylated Gi alpha is lacking in membranes from human myocardium, thrombocytes, lung and adipose tissue.

Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts.

Virus integration into the host genome is a characteristic step during cervical carcinogenesis. Experimental data provide evidence that integration could result in increased levels of oncogene (E6/E7) transcripts. This is the first study in which the level of viral transcripts is correlated to the physical state of the viral genome in cervical intraepithelial neoplasia (CIN) and cervical carcinomas (CxCa). Using the APOT-assay integrate-derived transcripts only were detected in 3/28 (11%) CIN and in 28/55 (51%) carcinomas, respectively. The remaining biopsies contained either episome-derived transcripts only or both mRNA species. SybrGreen real time reverse transcriptase-PCR assays were used to quantify viral gene expression for (i) all transcripts initiated from p97, (ii) full-length E6, (iii) E6*I and (iv) E5 transcripts. E6/E7 transcript levels showed a broad distribution but similar median values irrespective of histopathological grading and physical state of the viral genome. Biopsies with integrate-derived transcripts only generally lacked E5-specific mRNA. Our data do not support the hypothesis that HPV integration invariably results in high levels of oncogene transcripts. Instead, constitutive expression of oncogene transcripts rather than the level of expression appears to be decisive for transformation and the maintenance of the malignant phenotype.

[Spectrum of HPV infections and HPV-associated diseases in the female and male]. / Spektrum von genitalen HPV-Infektionen und HPV-assoziierten Erkrankungen bei Frau und Mann.

Genital human papilloma viruses cause anogenital infections and are, in addition, associated with premalignant and malignant neoplasias. In HPV infections, clinical, subclinical and latent infections must be differentiated. HPV associated diseases comprise anogenital precancers and invasive cancers. HPV infections are the most common genital partner infections. In particular the diagnosis of subclinical and latent infections is only possible using molecular-biological techniques. For the diagnosis of HPV infections, colposcopy, cytology, and histology are of varying value. The general risk of infection seems almost identical for women and men. HPV infections and HPV-associated diseases comprise a similar morphologic spectrum in women and in men and are frequently of multifocal and/or multicentric character. This latter notion must be kept in mind when evaluating therapeutic approaches. At the moment, no clear statement can be made regarding the biological course of subclinical infections with the so-called "oncogenic" HPV types such as HPV 16 and 18.

Pork intake and human papillomavirus-related disease.

Clinical and ecological evidence supporting an association between human papillomavirus (HPV)-related tumors and dietary factors are presented. Abstinence from high intake of fried pork (600-1,000 g/day) was associated with regression of an urethral condyloma in a healthy 19-year-old man treated with interferon gamma. International correlations suggest that pork intake is positively associated with incidence of cervical cancer, a disease also related to HPV. Pork meat or dietary factors associated with pork meat consumption may be involved in the development of HPV-related diseases.

Prevalence of human papillomavirus genomes in tissues from the lower genital tract as detected by molecular in situ hybridization.

Incidence cases of human papillomavirus (HPV)-related disease of the lower anogenital tract were prospectively collected and submitted to RNA-DNA in situ hybridization, which allows a direct correlation between morphology and the presence of viral DNA. The overall HPV detection rate in condylomata and intraepithelial or invasive neoplasia varied for the different genital areas: cervix (64%), vagina (56%), vestibule (59%), vulva (50%), and penis (42%). HPVs 6/11 were associated with condylomata, whereas HPV 16 was associated with intraepithelial and invasive neoplasia. Not a single case of intraepithelial or invasive neoplasia contained HPV 6/11, which supports the notion of the oncogenic potential of HPV 16. Condylomata were positive for HPV 16 only when adjacent to HPV 16-positive cervical intraepithelial neoplasias (CINs). The amount of viral DNA (copy number) was low in invasive cancers, intermediate in intraepithelial neoplasia, and conspicuously high in condylomata. In biopsies (n = 142) taken from lesions suggestive of subclinical papillomavirus infection on the basis of minor colposcopic and histologic abnormalities, HPV DNA was not detectable. Histologically normal tissue (n = 210) adjacent to condyloma, CIN, or cancer, and thus potential carrier of latent HPV infection, also proved negative for HPV DNA by in situ hybridization. The inability to detect viral DNA by in situ hybridization in tissues diagnosed as subclinically or latently infected may have been due to limited sensitivity of the hybridization method applied in this study.

Subclinical human papillomavirus infections in male sexual partners of female carriers.

The male sexual partners of 156 women with human papillomavirus infection of the cervix uteri were examined. In 120 men (77 per cent) penile lesions were found on examination of the penis via a colposcope (peniscopy) the most common of which were flat acetowhite lesions (53 per cent). Predilection sites of lesions were the urethral meatus and the corona glandis (23 and 19 per cent of the lesions, respectively). Using filter in situ hybridization human papillomavirus-deoxyribonucleic acid was found in penile smears of 61 men (39 per cent). The cancer-associated viral types (human papillomavirus 16 and 18) were identified in 75 per cent of the human papillomavirus positive men. Viral types of sexual partners were identical in 87 per cent. The correlation between infections with human papillomavirus 16 and 18, and the severity of the cervical lesion was significant in corresponding sexual partners. Our results support the hypothesis that male sexual partners represent a risk factor by acting as a reservoir for genital infections with papillomaviruses. The majority of human papillomavirus infections are of subclinical character. They require sensitive diagnostic techniques, such as peniscopy and hybridization for their identification. Detection and treatment of subclinical human papillomavirus infection in men may be important for the prevention of genital cancer in women.

Different detectability of high-risk HPV in smears from incident and prevalent high-grade squamous intraepithelial lesions of the cervix.

Human papillomavirus (HPV) status in cervical smears from cervical intraepithelial neoplasia (CIN) 2/3 diagnosed in 36 of 892 women with a history of normal cytology and colposcopy (incident CIN 2/3) was compared with CIN 2/3 in 40 patients with a history of abnormal cytology (prevalent CIN 2/3). In all patients cervical smears for HPV testing and cytology and two cervigrams were taken. The scrapes were collected in hybrid capture assay solution and analyzed with the hybrid capture and general primer/type-specific primer polymerase chain reaction system (GP/TS-PCR) after DNA extraction. Patients with incident and prevalent CIN 2/3 were similar with respect to age. By GP/TS-PCR carried out under suboptimal conditions due to DNA extraction, HPV DNA was detected in 69.4% (25 of 36) of smears from incident CIN 2/3 compared to 95% (38 of 40) in prevalent CIN 2/3 (P = 0.003). Using hybrid capture, smears of incident CIN 2/3 were HPV positive in 50% (18 of 36) compared to 80% (32 of 40) in prevalent CIN 2/3 (P = 0.006). High-risk HPVs were significantly less common in smears from incident CIN 2/3 compared with prevalent CIN 2/3: 36.1% vs 72.5% by GP/TS-PCR (P = 0.001) and 47.2% vs 80% by hybrid capture assay (P = 0.003), respectively. Virus load in HPV-positive smears of prevalent CIN 2/3 was significantly higher than of incident CIN 2/3 using semiquantitative PCR (P = 0.0005). Thus, high-risk HPV types were detected less frequently and in lower concentration in smears from incident CIN 2/3 than in smears from prevalent CIN 2/3.

Screening for cervical intraepithelial neoplasia grade 2/3: validity of cytologic study, cervicography, and human papillomavirus detection.

OBJECTIVE: A commercial test for human papillomavirus detection (hybrid capture assay) was examined for its potential value to augment the sensitivity of cytologic study or cervicography for screening for cervical intraepithelial neoplasia grade 2/3. STUDY DESIGN: In a cohort of 967 women with a mean age of 37.1 years who underwent routine cytologic screening, cytologic examination, cervicography, and testing for high-risk human papillomavirus by the hybrid capture assay were compared for their ability to detect cervical intraepithelial neoplasia grade 2/3. Cervical punch biopsy specimens were taken in 20.5% (198/967) patients because they had an atypical or positive cervigram or abnormal cytologic results in the primary screening smears. The data were analyzed by two-tailed chi(2) and Fisher's exact test. RESULTS: Thirty-eight patients were diagnosed with cervical intraepithelial neoplasia grade 2/3 (prevalence 3.9%) by histologic study. Cytologic study identified 29%, cervicography 45%, and testing for high-risk human papillomavirus 50% of cervical intraepithelial neoplasia grade 2/3. When combined, detection of high-risk human papillomavirus or cervicography augmented sensitivity of cytologic study to 58% (p = 0.01) with positive predictive values of 23% and 17%, respectively. Results of the different techniques should be interpreted in relationship to each other and not as absolute values because collection of specimens for cytologic study was done with cotton swabs, which may be suboptimal for screening but is general practice in Germany. CONCLUSIONS: Screening for cervical intraepithelial neoplasia grade 2/3 can significantly be improved by human papillomavirus testing with the hybrid capture assay.