Trace Metals in Soot and PM2.5 from Heavy-Fuel-Oil Combustion in a Marine Engine.

Heavy fuel oil (HFO) particulate matter (PM) emitted by marine engines is known to contain toxic heavy metals, including vanadium (V) and nickel (Ni). The toxicity of such metals will depend on the their chemical state, size distribution, and mixing state. Using online soot-particle aerosol mass spectrometry (SP-AMS), we quantified the mass of five metals (V, Ni, Fe, Na, and Ba) in HFO-PM soot particles produced by a marine diesel research engine. The in-soot metal concentrations were compared to in-PM2.5 measurements by inductively coupled plasma-optical emission spectroscopy (ICP-OES). We found that <3% of total PM2.5 metals was associated with soot particles, which may still be sufficient to influence in-cylinder soot burnout rates. Since these metals were most likely present as oxides, whereas studies on lower-temperature boilers report a predominance of sulfates, this result implies that the toxicity of HFO PM depends on its combustion conditions. Finally, we observed a 4-to-25-fold enhancement in the ratio V:Ni in soot particles versus PM2.5, indicating an enrichment of V in soot due to its lower nucleation/condensation temperature. As this enrichment mechanism is not dependent on soot formation, V is expected to be generally enriched within smaller HFO-PM particles from marine engines, enhancing its toxicity.

An approach for manganese biomonitoring using a manganese carrier switch in serum from transferrin to citrate at slightly elevated manganese concentration.

After high-dose-short-term exposure (usually from occupational exposure) and even more under low-dose long term exposure (mainly environmental) manganese (Mn) biomonitoring is still problematic since these exposure scenarios are not necessarily reflected by a significant increase of total Mn in blood or serum. Usually, Mn concentrations of exposed and unexposed persons overlap and individual differentiation is often not possible. In this paper Mn speciation on a large sample size (n=180) was used in order to be able to differentiate between highly Mn-exposed or low or unexposed individuals at low total Mn concentration in serum (Mn(S)). The whole sample set consisted of three subsets from Munich, Emilia Romagna region in Italy and from Sweden. It turned out that also at low total Mn(S) concentrations a change in major Mn carriers in serum takes place from Mn-transferrin (Mn-Tf(S)) towards Mn-citrate (Mn-Cit(S)) with high statistical significance (p<0.000002). This carrier switch from Mn-Tf(S) to Mn-Cit(S) was observed between Mn(S) concentrations of 1.5µg/L to ca. 1.7µg/L. Parallel to this carrier change, for sample donors from Munich where serum and cerebrospinal fluid were available, the concentration of Mn beyond neural barriers - analysed as Mn in cerebrospinal fluid (Mn(C)) - positively correlates to Mn-Cit(S) when Mn(S) concentration was above 1.7µg/L. The correlation between Mn-Cit(S) and Mn(C) reflects the facilitated Mn transport through neural barrier by means of Mn-citrate. Regional differences in switch points from Mn-Tf(S) to Mn-Cit(S) were observed for the three sample subsets. It is currently unknown whether these differences are due to differences in location, occupation, health status or other aspects. Based on our results, Mn-Cit(S) determination was considered as a potential means for estimating the Mn load in brain and CSF, i.e., it could be used as a biomarker for Mn beyond neural barrier. For a simpler Mn-Cit(S) determination than size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS), ultrafiltration (UF) of serum samples was tested for suitability, the latter possibly being a preferred choice for routine occupational medicine laboratories. Our results revealed that UF could be an alternative if methodical prerequisites and limitations are carefully considered. These prerequisites were determined to be a thorough cleaning procedure at a minimum Mn(S) concentration >1.5µg/L, as at lower concentrations a wide scattering of the measured concentrations in comparison to the standardized SEC-ICP-MS results were observed.

The role of chemical speciation, chemical fractionation and calcium disruption in manganese-induced developmental toxicity in zebrafish (Danio rerio) embryos.

Manganese (Mn) is an essential nutrient that can be toxic in excess concentrations, especially during early development stages. The mechanisms of Mn toxicity is still unclear, and little information is available regarding the role of Mn speciation and fractionation in toxicology. We aimed to investigate the toxic effects of several chemical forms of Mn in embryos of Danio rerio exposed during different development stages, between 2 and 122h post fertilization. We found a stage-specific increase of lethality associated with hatching and removal of the chorion. Mn(II), ([Mn(H2O)6](2+)) appeared to be the most toxic species to embryos exposed for 48h, and Mn(II) citrate was most toxic to embryos exposed for 72 and/or 120h. Manganese toxicity was associated with calcium disruption, manganese speciation and metal fractionation, including bioaccumulation in tissue, granule fractions, organelles and denaturated proteins.

Calculation of internal dose from ingested soil-derived uranium in humans: Application of a new method.

The aim of the present study was to determine the internal dose in humans after the ingestion of soil highly contaminated with uranium. Therefore, an in vitro solubility assay was performed to estimate the bioaccessibility of uranium for two types of soil. Based on the results, the corresponding bioavailabilities were assessed by using a recently published method. Finally, these bioavailability data were used together with the biokinetic model of uranium to assess the internal doses for a hypothetical but realistic scenario characterized by a daily ingestion of 10 mg of soil over 1 year. The investigated soil samples were from two former uranium mining sites of Germany with (238)U concentrations of about 460 and 550 mg/kg. For these soils, the bioavailabilities of (238)U were quantified as 0.18 and 0.28 % (geometric mean) with 2.5th percentiles of 0.02 and 0.03 % and 97.5th percentiles of 1.48 and 2.34 %, respectively. The corresponding calculated annual committed effective doses for the assumed scenario were 0.4 and 0.6 µSv (GM) with 2.5th percentiles of 0.2 and 0.3 µSv and 97.5th percentiles of 1.6 and 3.0 µSv, respectively. These annual committed effective doses are similar to those from natural uranium intake by food and drinking water, which is estimated to be 0.5 µSv. Based on the present experimental data and the selected ingestion scenario, the investigated soils-although highly contaminated with uranium-are not expected to pose any major health risk to humans related to radiation.

Particle emissions from a marine engine: chemical composition and aromatic emission profiles under various operating conditions.

The chemical composition of particulate matter (PM) emissions from a medium-speed four-stroke marine engine, operated on both heavy fuel oil (HFO) and distillate fuel (DF), was studied under various operating conditions. PM emission factors for organic matter, elemental carbon (soot), inorganic species and a variety of organic compounds were determined. In addition, the molecular composition of aromatic organic matter was analyzed using a novel coupling of a thermal-optical carbon analyzer with a resonance-enhanced multiphoton ionization (REMPI) mass spectrometer. The polycyclic aromatic hydrocarbons (PAHs) were predominantly present in an alkylated form, and the composition of the aromatic organic matter in emissions clearly resembled that of fuel. The emissions of species known to be hazardous to health (PAH, Oxy-PAH, N-PAH, transition metals) were significantly higher from HFO than from DF operation, at all engine loads. In contrast, DF usage generated higher elemental carbon emissions than HFO at typical load points (50% and 75%) for marine operation. Thus, according to this study, the sulfur emission regulations that force the usage of low-sulfur distillate fuels will also substantially decrease the emissions of currently unregulated hazardous species. However, the emissions of soot may even increase if the fuel injection system is optimized for HFO operation.

PIXE as a complement to ICP-OES trace metal analysis in Sudanese medicinal plants.

This paper compares trace element concentrations (Ca, K, Sr, Fe, Mn, Zn, Ni, Cu, Co and Cr) in 27 Sudanese medical plants determined in parallel by PIXE and ICP-OES to get information on which technique is preferable at different matrices and element concentrations. PIXE correlates well to ICP-OES for Sr, Mn, Ca, K, Zn and Fe determinations. ICP-OES seems to be the superior technique over PIXE when measuring low concentrated elements (chromium, cobalt, nickel and copper) in the medicinal plants.

Levels of Cr, Co, Ni and Mo in erythrocytes, serum and urine after hip resurfacing arthroplasty.

BACKGROUND: Hip resurfacing arthroplasty is known to increase the metal ion concentration in the serum, urine and whole blood, with potentially adverse effects on the organism. However, only few data are available about the metal ion concentrations in erythrocytes, although they are directly exposed to the higher concentrations of the serum. METHODS: The ion levels of chromium, cobalt, nickel and molybdenum in erythrocytes of 25 patients with a hip resurfacing implant were analysed with high resolution ICP-sf-MS (inductively-coupled-plasma-sector-field-mass-spectrometry). The results were compared to the ion levels in the serum and urine of the patients, and also to the ion levels of 27 control persons without an implant. RESULTS: Compared to the control group, ion levels in the erythrocytes of the hip resurfacing group were markedly increased for cobalt (0.10 vs. 3.26 microg/kg) and slightly for chromium (6.04 vs. 7.38 microg/kg). In contrast, ion levels in the serum of the hip resurfacing group were increased for cobalt (0.21 vs. 1.92 microg/l), chromium (1.48 vs. 5.64 microg/l), nickel (1.53 vs. 4.25 microg/l) and molybdenum (2.17 vs. 3.78 microg/l). CONCLUSION: Ion concentrations of cobalt and chromium are also increased in erythrocytes after hip resurfacing arthroplasty. Further research is required to evaluate the impact of the elevated ion levels on the erythrocytes, and to evaluate if metal ions also accumulate in other tissues of the body.

Manganese speciation in paired serum and CSF samples using SEC-DRC-ICP-MS and CE-ICP-DRC-MS.

Occupational manganese (Mn) overexposure leads to accumulation in the brain and has been shown to cause progressive, permanent, neuro-degenerative damage with syndromes similar to idiopathic Parkinsonism. Mn is transported by an active mechanism across neural barriers (NB) finally into the brain; but to date, modes of Mn neurotoxic action are poorly understood. This paper investigates the relevant Mn-carrier species which are responsible for widely uncontrolled transport across NB. Mn speciation in paired serum/cerebrospinal fluid (CSF) samples was performed by size exclusion chromatography-inductively coupled plasma-dynamic reaction cell-mass spectrometry (SEC-ICP-DRC-MS) and capillary zone electrophoresis coupled to ICP-DRC-MS in a 2D approach for clear identification. For additional species verification, electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry was used after SEC-ICP-DRC-MS (second 2D approach). The Mn species from the different sample types were interrelated and correlation coefficients were calculated. In serum protein-bound Mn species like Mn-transferrin/albumin (Mn-Tf/HSA) were dominant, which had the main influence on total Mn in serum if Mn(total) was <1.5 µg/L. Above serum Mn(total) concentration of 1.6 µg/L the serum Mn(total) concentration was correlated with increasing Mn-citrate (Mn-Cit) concentration. In parallel Mn(total) and Mn species in CSF were determined. It turned out that Mn(total) from CSF was about half of Mn(total) in serum; Mn-Tf/HSA was only about 10% compared to serum. It turned out that above 1.6 µg/L Mn(total) in serum Mn-Cit was not only the leading Mn species in serum but also was the main influencing factor of both Mn(total) and Mn-Cit concentration in CSF. These results were further investigated using two statistical models (orthogonal partial least squares discriminant analysis, canonical discriminant analysis). Both models discriminated the samples in two groups where CSF samples were either correlated to Mn(total) and Mn-Cit (samples with serum Mn(total) > 1,550 ng/L) or correlated to Mn-Tf/HSA (samples with serum Mn(total) < 1,550 ng/L). We conclude that elevated Mn-Cit(serum) could be a valuable marker for increased total Mn in CSF (and brain), i.e., it could be a marker for elevated risk of Mn-dependent neurological disorders such as manganism in occupational health.

Enhanced extract preparation of native manganese and iron species from brain and liver tissue.

To date, no reference method for the extraction of labile Mn species from biological tissues is published which provides sufficient extraction efficiency combined with monitoring speciation. Here, an extraction method is reported using cryogenic conditions (+N) under inert gas atmosphere. Fresh brain and liver tissues were used, then stored either 1 day (+N) or 1 month in N(2liq) (+N 1 m) to evaluate degradation effects during long-term storage. Both attempts were compared to a previous extraction method (-N) using neither N(2liq) nor storage ability. Mn and Fe concentrations in extracts and pellets were determined with inductively coupled plasma (ICP)-atomic emission spectroscopy (AES) and compared to acid digests of the same sample. Element ratios of extracts/digest indicated the extraction efficiency, which was increased from 17% (-N) to 26% (+N) for Mn in brain or from 28% (-N) to 44% (+N) in liver extracts. For Fe species, the increase was only from 40% (-N) to 44% (+N) in brain but from 64% (-N) to 74% (+N) in liver. Size exclusion chromatography (SEC)-ICP-mass spectrometry (MS) was employed to screen for Mn and Fe species pattern in extracts. In brain, surplus extracted Mn (+N, +N 1 m) was assigned to organic Mn species, mainly from the 0.7-4 kDa fraction, while in the liver, it was seen in the 70-80 kDa fraction. Fe speciation was similar for -N and +N methods in brain extracts. In liver, higher amounts of Fe species were extracted from the 140-160 kDa fraction. Storage at -196 °C for 1 month did neither affect Mn speciation in brain nor in liver extracts. Fe species pattern showed a negligible shift (≤5%) from 140-160 to 70-80 kDa fraction in liver extracts stored 1 month in N(2liq).

Bioavailability of zinc from NutriSet zinc tablets compared with aqueous zinc sulfate.

BACKGROUND/OBJECTIVES: The apparent widespread extent of zinc (Zn) deficiency in developing countries and the efficacy of oral Zn supplements as an adjunct to oral rehydration therapy make oral Zn supplementation an increasingly important modality in clinical medicine and public health. In this study we aimed to compare the relative bioavailability of oral doses of 30 mg of Zn in two dosing forms. SUBJECTS/METHODS: In total, 10 healthy male volunteers ingested oral Zn doses with 200 ml plain water at about 0830 hours in the fasting state on two occasions, once as 30 mg of Zn in an aqueous solution of reagent grade zinc sulfate (ZnSO(4)) and another time as 1.5 NutriSet Zn tablets (Nutriset, Malaunay, France); on a third occasion, only plain water was consumed. Venous blood specimens were collected at baseline, 60, 120, 180 and 240 min after ingestion and the plasma Zn was measured for each sample. RESULTS: The relative bioavailability of oral Zn from a commonly used, tableted (NutriSet) form is only about half of that of a reference dose of aqueous ZnSO(4) as indicated by the area under the curve of serial plasma Zn excursion and maximal change in circulating Zn. CONCLUSIONS: Reduced or absent functional outcomes in Zn intervention trials may derive, in part, from a lower than anticipated intestinal uptake of the Zn in the tableted form.

Levels of toxic metals in multisectoral samples from Winam Gulf of Lake Victoria.

In this study, the distribution and sources of inputs of trace metals including Cd, Cu, Zn and Pb from various sources as well as Fe which is widely used in the construction industry, into Winam Gulf of Lake Victoria were investigated. The sampling sites were located up streams and down streams of four rivers (Sio, Nyamasaria, Nyando and Sondu-Miriu), in four beaches along the lake (Port Victoria, Kisumu Car Wash, Dunga and Hippo point beaches) and in three estates (Nyamasaria, Migosi and Nyawita) in Kisumu city, covering potential agrochemical and industrial sources and drinking water points, respectively. The concentrations (in microg/L) of trace metals analysed in the lake and river waters ranged from <1.79 (Cd), <3.83 (Pb), <1.53-3.86 (Cu), 4.37-11.6 (Zn), 11.8-2,440 (Fe). The sediment concentrations (in microg/kg x 10(3)) ranged from 0.19 to 1.91 (Cd), 6.86-138 (Pb), 18-100 (Cu), 36.2-443 (Zn) and 960-73,200 (Fe), with highest concentrations of all metals being recorded at Kisumu Car Wash area. The study confirmed that the concentrations of the metals accumulate downstream in the rivers both in water and sediment and these rivers are major sources of the heavy metal load into Winam Gulf of Lake Victoria.

Observation of changes in urinary excretion of thorium in humans following ingestion of a therapeutic soil.

The study investigated the changes in urinary thorium excretion by humans following ingestion of a therapeutic soil, which contains about 10 ppm of thorium. This well-known healing earth in Germany has been considered as an alternative medicine for diarrhoea and gastric hyper-acidity. Six adult volunteers ingested this therapeutic soil in varying quantities for 1-15 days at levels approximating those described in the package insert of the medicine (10-60 g of soil per day). The subjects ingested about 0.1-0.6 mg of thorium daily, which is 100-600 times higher than the normal daily intake of about 1 microg thorium in Germany. All 24-h urine samples collected from the subjects during pre-ingestion, ingestion and post-ingestion periods of the soil were analyzed for (232)Th using inductively coupled plasma mass spectrometry (ICP-MS). The measured excretion values varied in a wide range. Apparently, the high thorium amounts administered did not increase the (232)Th excretion in urine as expected, suggesting that this soil ingestion will not result in a considerably higher and harmful uptake of thorium into the human body.

Measurements of daily urinary uranium excretion in German peacekeeping personnel and residents of the Kosovo region to assess potential intakes of depleted uranium (DU).

Following the end of the Kosovo conflict, in June 1999, a study was instigated to evaluate whether there was a cause for concern of health risk from depleted uranium (DU) to German peacekeeping personnel serving in the Balkans. In addition, the investigations were extended to residents of Kosovo and southern Serbia, who lived in areas where DU ammunitions were deployed. In order to assess a possible DU intake, both the urinary uranium excretion of volunteer residents and water samples were collected and analysed using inductively coupled plasma-mass spectrometry (ICP-MS). More than 1300 urine samples from peacekeeping personnel and unexposed controls of different genders and age were analysed to determine uranium excretion parameters. The urine measurements for 113 unexposed subjects revealed a daily uranium excretion rate with a geometric mean of 13.9 ng/d (geometric standard deviation (GSD)=2.17). The analysis of 1228 urine samples from the peacekeeping personnel resulted in a geometric mean of 12.8 ng/d (GSD=2.60). It follows that both unexposed controls and peacekeeping personnel excreted similar amounts of uranium. Inter-subject variation in uranium excretion was high and no significant age-specific differences were found. The second part of the study monitored 24 h urine samples provided by selected residents of Kosovo and adjacent regions of Serbia compared to controls from Munich, Germany. Total uranium and isotope ratios were measured in order to determine DU content. (235)U/(238)U ratios were within +/-0.3% of the natural value, and (236)U/(238)U was less than 2 x 10(-7), indicating no significant DU in any of the urine samples provided, despite total uranium excretion being relatively high in some cases. Measurements of ground and tap water samples from regions where DU munitions were deployed did not show any contamination with DU, except in one sample. It is concluded that both peacekeeping personnel and residents serving or living in the Balkans, respectively, were not exposed to significant amounts of DU.

Distribution pattern of inhaled ultrafine gold particles in the rat lung.

The role of alveolar macrophages in the fate of ultrafine particles in the lung was investigated. Male Wistar-Kyoto rats were exposed to ultrafine gold particles, generated by a spark generator, for 6 h at a concentration of 88 microg/m3 (4 x 10(6)/cm3, 16 nm modal mobility diameter). Up to 7 days, the animals were serially sacrificed, and lavaged cells and lung tissues were examined by transmission electron microscopy. The gold concentration/content in the lung, lavage fluid, and blood was estimated by inductively coupled plasma-mass spectrometry. Gold particles used were spherical and electron dense with diameters of 5-8 nm. The particles were individual or slightly agglomerated. By inductively coupled plasma-mass spectrometry analysis of the lung, 1945 +/- 57 ng (mean +/- SD) and 1512 +/- 184 ng of gold were detected on day 0 and on day 7, respectively, indicating that a large portion of the deposited gold particles was retained in the lung tissue. In the lavage fluid, 573 +/- 67 ng and 96 +/- 29 ng were found on day 0 and day 7, respectively, which means that 29% and 6% of the retained gold particles were lavageable on these days. A low but significant increase of gold (0.03 to 0.06% of lung concentration) was found in the blood. Small vesicles containing gold particles were found in the cytoplasm of alveolar macrophages. In the alveolar septum, the gold particles were enclosed in vesicles observed in the cytoplasm of alveolar type I epithelial cells. These results indicate that inhaled ultrafine gold particles in alveolar macrophages and type I epithelial cells are processed by endocytotic pathways, though the uptake of the gold particles by alveolar macrophages is limited. To a low degree, systemic particle translocation took place.

Speciation of Pt(II) and Pt(IV) in spiked extracts from road dust using on-line liquid chromatography-inductively coupled plasma mass spectrometry.

The emission of platinum from automobile catalytic exhaust converters is well known and the accumulation of Pt in road dust has been studied by the analysis of total Pt contents. However, there are few studies on the speciation of the emitted Pt-compounds in the environment. A separation method with HPLC-UV has been developed by Nachtigall et al. [Chromatogr. A 775 (1997) 197] with aqueous standards of Pt(II)- and Pt(IV)-chloro complexes. Due to the limited selectivity and sensitivity of the UV-detection this method is not suitable for Pt-speciation in road dust extracts. Therefore, in this study the method of Nachtigall et al. was modified to realize an on-line coupling to ICP-MS with the advantage of Pt-specific detection. Calibration was performed with aqueous standards and spiked road dust extracts. Calibration curves were linear with low residual standard deviation (1.3-3.1% for the aqueous standards) and approximately 10-times lower detection limits compared to the HPLC-UV-method. Moreover, the stability of the model species was investigated using isocratic as well as gradient separation. Isotopic ratios of 194Pt, 195Pt, 196Pt and 198Pt were determined for quality control. A new mathematical correction method for the HfO-interference on the 195Pt-signal was developed. Additionally, the elution pattern of further elements in the road dust extracts was monitored and total element contents were determined in unspiked road dust extracts.

Identification and quantification of metallothionein isoforms and superoxide dismutase in spiked liver extracts using HPLC-ESI-MS offline coupling and HPLC-ICP-MS online coupling.

A two-dimensional chromatographic method for the characterization of metallothionein isoforms (MT) and superoxide dismutase (SOD) in spiked liver extracts was developed for the optimization of extraction procedures from liver samples. Element-specific detection (ICP-MS) and molecule-specific detection (ESI-MS) were applied for maximum species information. A special focus was laid on the quantitative data evaluation (species stoichiometry, calibration with and without matrix, recovery), which is neglected in most MT/SOD publications with hyphenated techniques. Linearity, precision (residual standard deviation of calibration curves <10%), and detection limits (<0.6 mg L(-1) for MT isoforms and 13 mg L(-1) for SOD) prove the suitability of the method for quantification. An alternative quantification is proposed for the extension towards other lesser or even unknown trace element species, especially the native porcine MT and SOD.

[Mineral analysis of roentgenologically defined calcifications in patients with chronic calcifying tendinitis of the rotator cuff]. / Mineralogische Analyse röntgenologisch definierter Verkalkungen bei Patienten mit chronischer Tendinosis calcarea der Rotatorenmanschette.

AIM: Extracorporeal shockwave application (ESWA) is in clinical use to promote disintegration of symptomatic calcifications of the rotator cuff of the shoulder. However, disintegration of roentgenologically comparable calcifications of the rotator cuff is not always successfull. It is known from urologic stone lithotripsy that the susceptibility for disintegration of stone-like concrements depends on their mineral content. Therefore, in the present investigation the relative contents of calcium and phosphorus in rotator cuff calcifications were determined. METHOD: 39 surgically removed rotator cuff calcifications were analyzed by means of atomic emission spectrometry. RESULTS: The relative content of calcium of the rotator cuff calcifications was found to be 22.3 % +/- 5.7 % (mean +/- SD; 6.8 % - 32.4 %), that of phosphorus as 10.5 % +/- 2.4 % (2.7 % - 14.4 %). The data neither depend on the gender of the patients nor on their age at the time point of surgical removal of the rotator cuff calcifications. CONCLUSION: Roentgenologically comparable calcifications of the rotator cuff demonstrated distinct individual differences concerning the relative contents of calcium and phosphorus. The present results may serve as the first indication that the susceptibility of rotator cuff calcifications for disintegration may depend on their relative contents of calcium and phosphorus.

Effect of different extraction procedures on the yield and pattern of Se-species in bacterial samples.

Investigations are described to extract Se-species from a bacterial sample. The five extraction methods investigated were: hot water, protease, lysozyme, lysozyme-protease, and HCl hydrolysis. The extraction efficiency was determined by comparing the total amounts of selenium in the sample after pressure digestion with the amounts extracted by the different methods described. Efficiencies were found to be only 1% (hot water), ca. 8% (protease, HCl hydrolysis) or ca. 12% (lysozyme, lysozyme-protease). The Se-peak patterns were compared after investigating the extracts with strong anion exchange chromatography-inductively coupled plasma mass spectrometry (SAX-ICP-MS). Most promising were the lysozyme-assisted procedures, which showed the highest diversity of species. Here, in the protease-lysozyme approach, the protease seemed to break down species that had been extracted by lysozyme from the bacterial wall (murein sacculus). The other approaches seemed not to extract many species. Hot water extraction was completely unsuitable, extracting only low amounts of a single, unknown species.