3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads.

Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek(®) Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.

Biomek Cell Workstation: A Flexible System for Automated 3D Cell Cultivation.

The shift from 2D cultures to 3D cultures enables improvement in cell culture research due to better mimicking of in vivo cell behavior and environmental conditions. Different cell lines and applications require altered 3D constructs. The automation of the manufacturing and screening processes can advance the charge stability, quality, repeatability, and precision. In this study we integrated the automated production of three 3D cell constructs (alginate beads, spheroid cultures, pellet cultures) using the Biomek Cell Workstation and compared them with the traditional manual methods and their consequent bioscreening processes (proliferation, toxicity; days 14 and 35) using a high-throughput screening system. Moreover, the possible influence of antibiotics (penicillin/streptomycin) on the production and screening processes was investigated. The cytotoxicity of automatically produced 3D cell cultures (with and without antibiotics) was mainly decreased. The proliferation showed mainly similar or increased results for the automatically produced 3D constructs. We concluded that the traditional manual methods can be replaced by the automated processes. Furthermore, the formation, cultivation, and screenings can be performed without antibiotics to prevent possible effects.

Effect of varying salinity, temperature, ammonia and nitrous acid concentrations on nitrification of saline wastewater in fixed-bed reactors.

Nitrification under changing salinities (0-9%), temperatures (6-50°C), ammonia (0-5 g NL(-1)) and nitrite concentrations (0-0.4 g NL(-1)) was investigated in fixed-bed reactors. For all conditions ammonia oxidation rates (AOR) were lower than nitrite oxidation rates (NOR). AORs and NORs increased from 12.5 to 40°C and were very low at 6°C and almost zero at 50°C. No recovery of nitrification was obtained after incubation at 50°C, whereas nitrification was restorable after incubation at 6°C. Ammonia concentrations of 5 g NL(-1) or nitrite concentrations up to 0.125 g NL(-1) decreased AOR to almost zero. AORs and NORs recovered if ammonia or nitrite was removed. At concentrations of 1 and 5 g NL(-1) ammonia AOR and NOR were inhibited by 50%, whereas 27 mg N/L nitrite inhibited AOR by 50%.

Biodegradation of high phenol containing synthetic wastewater by an aerobic fixed bed reactor.

The continuous aerobic degradation of phenol, mixed with readily degradable synthetic wastewater was studied over a period of 400 days at 25+/-5 degrees C temperature in a fixed bed biofilm reactor using 'Liapor' clay beads as packing material. The phenol concentration added to the reactor ranged from 0.19 to 5.17g/l and was achieved by a gradual increase of phenol in wastewater, thus adapting the microbial flora to high contaminant concentrations. A maximal removal rate of 2.92g phenol/(ld) at a hydraulic retention time (HRT) of 0.95 days and a total organic loading rate (OLR) of 15.3g COD/(ld) with a phenol concentration of 4.9g/l was observed. However, this was not a stable rate at such high phenol loading. At the end of reactor operation on day 405, the phenol removal rate was 2.3g/(ld) at a influent phenol concentration of 4.9g/l. There were no phenol intermediates present in the reactor, as evident from corresponding COD, phenol removal and the absence of fatty acids. Omission of organic nitrogen compounds or of urea in influent feed was not favourable for optimal phenol removal. The phenol degradation profile that was studied in shake flasks indicated that the presence of a acetate which represent as an intermediate of phenol degradation retarded the phenol degradation. The highest phenol degradation rate observed in batch assays was 3.54g/(ld).

Chitin purification from shrimp wastes by microbial deproteination and decalcification.

Chitin was purified from Penaeus monodon and Crangon crangon shells using a two-stage fermentation process with anaerobic deproteination followed by decalcification through homofermentative lactic acid fermentation. Deproteinating enrichment cultures from sewage sludge and ground meat (GM) were used with a proteolytic activity of 59 and 61 mg N l(-1) h(-1) with dried and 26 and 35 mg N l(-1) h(-1) with wet P. monodon shells. With 100 g wet cells of proteolytic bacteria per liter, protein removal was obtained in 42 h. An anaerobic spore-forming bacterium HP1 was isolated from enrichment GM. Its proteolytic activity was 76 U ml(-1) compared to 44 U ml(-1) of the consortium. Glucose was fermented with Lactobacillus casei MRS1 to lactic acid. At a pH of 3.6, calcium carbonate of the shells was solubilised. After deproteination and decalcification of P. monodon or C. crangon shells, the protein content was 5.8% or 6.7%, and the calcium content was 0.3% or 0.4%, respectively. The viscosity of the chitin from P. monodon and C. crangon was 45 and 135 mPa s, respectively, whereas purchased crab shell chitin (practical grade) had a viscosity of 21 mPa s, indicating a higher quality of biologically purified chitin.

Propionic acid accumulation and degradation during restart of a full-scale anaerobic biowaste digester.

The methane formation rate of 300 m(3) of sludge from a full scale biowaste reactor, that was stored without feeding for six weeks during a maintenance period, was about 60% of the methanogenic activity before maintenance. The 300 m(3) sludge was then pumped back into the biowaste reactor. On the third day, after refilling of the stored biowaste suspension, anaerobic conditions were obtained and feeding was started by addition of 36.1 m(3) of fresh biowaste suspension (=11.3 tons biowaste). The pH dropped from originally pH 7.7 to pH 7.3 and later on to pH 6.8, which was considered the minimum allowed pH for methanogenesis to recover. Maximum concentrations of acetate (1.78 gl(-1)), n-butyrate (0.57 gl(-1)) and n-valerate (0.44 gl(-1)) accumulated during the following days with feeding of 11.8 tons on day 5 and twice 6.5 tons on days 7 and 9, respectively. Thereafter, acetate, n-butyrate and n-valerate were degraded completely, whereas the concentration of propionate was still increasing. Propionic acid was the dominant fatty acid during the restart period and reached its maximum concentration of 6.2 gl(-1) 17 days after start of feeding. This high level of propionate was degraded completely in about 5 days with maximum degradation rates of 2.14 gl(-1)d(-1), and the pH of the anaerobic sludge increased from 7.1 to 7.4. During restart, the methane content of the biogas increased successively to 65%. Samples that were taken at different time intervals during the restart phase of the methane reactor showed different fatty acid degradation capabilities. After 10 days, when acetate and n-butyrate still accumulated in the methane reactor the maximum acetate degradation rate was 1.52 gl(-1)d(-1) and the n-butyrate degradation rate was 0.51 gl(-1)d(-1). Oxidation of n-valerate caused an increase of propionate, which was degraded after a lag phase of 6 days with a maximum rate of 0.6 gl(-1)d(-1). In the samples taken after 16 and 23 days, the propionate degradation rate increased to 1.42 gl(-1)d(-1) and 1.55 gl(-1)d(-1), respectively, and the lag phase for propionate degradation was reduced or had disappeared completely. The maximum propionate degradation rate was measured in the methane reactor in the fourth week after restart. The synthrophic propionate oxidizing bacteria were apparently the most suffering bacteria during sludge storage. If the propionate oxidizing bacteria could be kept active and the propionate degrading activity of the biowaste suspension of 6.16 gl(-1)d(-1) before the maintenance period could be maintained, then accumulation of 6.2 gl(-1) propionate in the methane reactor after restart could be avoided and full activity reached even earlier.

Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers.

More than 750 isolates of faecal coliforms (>200 strains), enterococci (>200 strains) and pseudomonads (>340 strains) from three wastewater treatment plants (WTPs) and from four groundwater wells in the vicinity of leaking sewers were tested for resistance against 14 antibiotics. Most, or at least some, strains of the three bacterial groups, isolated from raw or treated sewage of the three WTPs, were resistant against penicillin G, ampicillin, vancomycin, erythromycin, triple sulfa and trimethoprim/sulfamethoxazole (SXT). Only a few strains of pseudomonads or faecal coliforms were resistant against some of the other tested antibiotics. The antibiotic resistances of pseudomonads, faecal coliforms and enterococci from groundwater varied to a higher extent. In contrast to the faecal coliforms and enterococci, most pseudomonads from all groundwater samples, including those from non-polluted groundwater, were additionally resistant against chloramphenicol and SXT. Pseudomonads from sewage and groundwater had more multiple antibiotic resistances than the faecal coliforms or the enterococci, and many pseudomonads from groundwater were resistant to more antibiotics than those from sewage. The pseudomonads from non-polluted groundwater were the most resistant isolates of all. The few surviving faecal coliforms in groundwater seemed to gain multiple antibiotic resistances, whereas the enterococci lost antibiotic resistances. Pseudomonads, and presumably, other autochthonous soil or groundwater bacteria, such as antibiotic-producing Actinomyces sp., seem to contribute significantly to the gene pool for acquisition of resistances against antibiotics in these environments.

Degradation of alkyllead compounds to inorganic lead in contaminated soil.

In glass columns with sandy soil from a former antiknocking agents factory hydrophobic tetraalkyllead was transformed in oxygen-saturated water to inorganic lead. Up to 324 mg l(-1) trialkyllead, but only very little dialkyllead accumulated. After 740 days 49.1+/-6.7% of the organic lead was converted to inorganic lead. Conversion of hydrocarbons was 39.6+/-5.1%. To reduce toxicity of high trialkyllead concentrations the water of soil columns was replaced by tap water after 450d. Trialkyllead in the new water increased again to more than 150 mg l(-1). If the alkyllead-containing water from these columns was diluted to concentrations of alkyllead compounds that were found in the groundwater after air injection (total alkyllead<10 mg l(-1)) and used as a source of alkyllead compounds in columns with non-contaminated sandy soil, elimination of tetra-, tri- and dialkyllead compounds followed first-order kinetics. In the soil 85.8-93.6% of the alkyllead dissappeared in only 170 days with 51% being converted to inorganic lead. This makes in situ remediation reasonable.

Scale-up of anaerobic digestion of the biowaste fraction from domestic wastes.

In the City of Karlsruhe/Germany anaerobic digestion of 7200 ta(-1) of separately collected biowaste has proven its feasibility at an organic loading rate (OLR) of up to 8.5 kg CODm(-3)d(-1). An extension of biowaste collection over the whole city area would increase the amount of biowaste to 12,000 ta(-1), leading to an OLR of the existing anaerobic reactor of up to 15 kg CODm(-3)d(-1). To test, whether the increased amount of biowaste could be stabilized in the existing plant, biowaste suspensions were digested in a laboratory reactor at a maximum OLR, that exceeded the future OLR of the full-scale plant. The laboratory reactor was started with effluent of the full-scale biowaste digester. Like in full-scale, biowaste suspension from the hydropulper was added in a fed-batch mode. The elimination of organic material (measured as COD, chemical oxygen demand) and the volumetric gas production were linearly increasing with the OLR from 4.3 to 19 kg CODm(-3)d(-1). Thus, safe operation of the full-scale plant at an OLR of 15 kg CODm(-3)d(-1) should be possible, leaving still some reserve capacity. To determine the metabolic reserves for fatty acid degradation during digestion at an OLR of 10 kg CODm(-3)d(-1), digester effluent was supplemented with either 40 mmoll(-1) acetate, propionate, i-butyrate or n-butyrate. Results of these batch assays indicated a rapid degradation of all fatty acids and fatty acid conversion rates, that would allow a stable anaerobic fermentation at 15 kg CODm(-3)d(-1)OLR. On the basis of the laboratory results the OLR of the full-scale methane reactor was increased to 15 kg CODm(-3)d(-1). After 7 months, results of full-scale digestion were still consistent with the previously obtained laboratory results.

Solid and liquid residues as raw materials for biotechnology.

In the past few decades huge amounts of solid and paste-like wastes of domestic and industrial origin have been deposited on sanitary landfills worldwide. Only a small proportion was incinerated, where incineration plants were available. Since primary resources, such as ores for metal production or crude oil for the production of gasoline, diesel, solvents and plastics, or coal and natural gas as sources for energy or chemicals are not available in unlimited quantities, and because the deposition of residues, wastes and worn-out commodities on sanitary landfills causes pollution of the atmosphere, the soil and the groundwater due to hazardous gaseous emissions and toxic leachates, wastes from households and from industry must be avoided or minimized at an early stage. Whenever waste material can be recycled it must be re-introduced into production processes and the non-recyclable fractions should be used as a fuel for energy recovery. After incineration, the highly toxic dust fractions of ashes and slags resulting from burning the wastes should be deposited on sanitary landfills, while the granulated mineral slag fractions could be used as a substitute for the sand in cement as a construction material. Here we review various processes for the treatment of organic fractions of differently composed wastes to upgrade them to more valuable, re-usable products or at least to recover their energy content. Upgrading processes of organic wastes include composting, biogas fermentation, production of organic acids and solvents, and biopolymer or biosurfactants production. We also include biological purification procedures for the most important components of wastes, such as chitin from the shells of Crustaceae. Typical examples from pilot-scale or full-scale studies are discussed for each process.

Bioremediation of soil contaminated with alkyllead compounds.

Sandy soil, which was highly contaminated with alkyllead compounds, was taken from bore cores from a site of a former tetraalkyllead producing company. It was analyzed for its capacity to chemically and/or biologically degrade alkyllead contaminants. For this purpose, soil samples were supplied with oxygen or oxygen + minerals at different water saturation. For long-term elution, contaminated soil was packed into glass columns of 1.5m length and 10cm diameter. Oxygen-saturated water was recirculated in an upflow mode. Within a time span of 260 days tetraethyllead was completely eluted from the sandy soil and was apparently converted to triethyllead by chemical or microbiological reaction. The triethyllead concentration in the circulating water accounted for 60-80% of the maximal amount, that could be formed from tetraethyllead by a single dealkylation. This indicated that between 20-40% of the triethyllead were apparently further degraded. Only very little diethyllead accumulated in the water. The triethyllead concentration in the circulating water was highly toxic for non-adapted microorganisms. However, if a readily degradable carbon source was added, fast growth of indigenous soil bacteria was observed, but only little alkyllead degradation occurred.

Degradation of latex and of natural rubber by Streptomyces strain La 7.

Streptomyces strain La 7 was isolated from the banquete of a city high way in Karlsruhe. According to partial 16S rRNA gene sequencing it was identical with Streptomyces albogriseolus and Streptomyces viridodiastaticus. DNA-DNA-similarity studies revealed 80.3-82.4% similarity between each of two of the three strains. Although phylogenetically closely related, Streptomyces strain La 7 differed from the two reference strains by morphological as well as physiological features and might represent a new species aside of S. albogriseolus and S. viridodiastaticus. The new Streptomyces strain La 7 was grown in a medium containing a latex emulsion or squares of natural rubber gloves as the only carbon source. On agar plates with a latex overlay agar, translucent halo formation around the colonies was observed. The unvulcanized latex was metabolized and the carbon from the isoprene units was apparently used for cell growth. In shake cultures with unlimited oxygen supply, during 60 days of incubation, 140 mg of the 175 mg totally emulgated latex were degraded exponentially. In sterile control flasks about 3% of the initial amount of latex could not be recovered after incubation on a shaker, presumably due to photochemical transformation. During static incubation of sterile medium, the latex formed a sticky layer at the surface of the medium and on the glass walls and recovery of the material was more difficult. Estimation of the protein content of cells from total nitrogen resulted in about 50% of the degraded latex being incorporated into cells, if a standard cell composition was assumed. Direct protein analysis according to Bradford (1976) gave much lower estimates, presumably due to a low content of aromatic amino acids. Stripes of natural rubber were degraded by Streptomyces strain La 7 during 70 days to an extent of about 30%. Scanning electron microscopy demonstrated, that hyphes of Streptomyces strain La 7 colonized and penetrated the latex surface with a concomitant deterioration of the latex material.

Effect of ammonia on the anaerobic degradation of protein by a mesophilic and thermophilic biowaste population.

The influence of ammonia on the anaerobic degradation of peptone by mesophilic and thermophilic populations of biowaste was investigated. For peptone concentrations from 5 g l-1 to 20 g l-1 the mesophilic population revealed a higher rate of deamination than the thermophilic population, e.g. 552 mg l-1 day-1 compared to 320 mg l-1 day-1 at 10 g l-1 peptone. The final degree of deamination of the thermophilic population was, however, higher: 102 compared to 87 mg NH3/g peptone in the mesophilic cultures. If 0.5-6.5 g l-1 ammonia was added to the mesophilic biowaste cultures, deamination of peptone, degradation of its chemical oxygen demand (COD) and formation of biogas were increasingly inhibited, but no hydrogen was formed. The thermophilic biowaste cultures were most active if around 1 g ammonia l-1 was present. Deamination, COD degradation and biogas production decreased at lower and higher ammonia concentrations and hydrogen was formed in addition to methane. Studies of the inhibition by ammonia of peptone deamination, COD degradation and methane formation revealed a Ki (50%) for NH3 of 92, 95 and 88 mg l-1 at 37 degrees C and 251, 274 and 297 mg l-1 at 55 degrees C respectively. This indicated that the thermophilic flora tolerated significantly more NH3 than the mesophilic flora. In the mesophilic reactor effluent 4.6 x 10(8) peptone-degrading colony-forming units (cfu)/ml were culturable, whereas in the thermophilic reactor effluent growth of only 5.6 x 10(7) cfu/ml was observed.