The use of methyl ricinoleate in lactone production by Yarrowia lipolytica: Aspects of bioprocess operation that influence the overall performance

Abstract

Yarrowia lipolytica was used to produce γ-decalactone by the degradation of methyl ricinoleate (MR). A new method for inoculating the biotransformation medium was tested, which avoided the laborious step of washing cells from the growth medium. The consequent cell hydrophobicity increase led to an enhancement of aroma production. In a study of MR concentration in shake flasks, the highest productivity (15 mg L^?1 h^?1) was achieved using 30 g MR L^?1. Lipase and protease activities were induced but no correlation between lipase induction and aroma production was found. The effects of different aeration and agitation rates were studied in bioreactor assays. Productivity was improved to 87 mg L^?1 h^?1, and another compound, 3-hydroxy-γ-decalactone, was detected in large amounts. Dehydration of this lactone produced two decenolides with aroma characteristics. The direct influence of oxygen on the production of both lactones was demonstrated.     

l-Serine production by a methylotroph and its related enzymes

The production process of l-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of l-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32–34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers. Correspondence to: Y. Izumi     

Physiological responses,tolerance efficiency,and phytoextraction potential of Hylotelephium spectabile (Boreau) H. Ohba under Cd stress in hydroponic condition

Abstract

A sand hydroponic experiment with different concentrations of 0, 5, 10, 20, 40?mg L^?1 Cd was used to study the growth and physiological response of Hylotelephium spectabile (Boreau) H. Ohba. and its phytoextraction potential for Cd. The results showed that total plant biomass under 5?mg L^?1 Cd treatment was slightly affected. The content of malondialdehyde (MDA) in leaf exposed to Cd was higher, and the POD and CAT activity exhibited a positive response to the low level of Cd addition (5?mg·L^?1). The photosynthesis pigments were slightly inhibited, and the ultrastructure of chloroplast remained intact after treatment with 10?mg L^?1 Cd. The maximum leaf Cd content (603?mg·kg^?1) was found in 5?mg L^?1 Cd treatment, then decreased with the Cd level increased. The maximum Cd content in the shoots far exceeds the threshold level (100?mg kg^?1) for a Cd-hyperaccumulator plant with the value of translocation factor (TF_shoot/root) for Cd reaching up to 5.62. In conclusion, H. spectabile showed normal growth and physiological response and high shoot Cd accumulation under 5?mg L^?1 Cd stress, which made it to be a good candidate for phytoextraction of low-level Cd polluted environment.     

Metabolic engineering and flux analysis of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for L-serine production

l-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of l-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of l-serine to pyruvate and glycine, releasing the feedback inhibition by l-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH^r). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in l-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular l-serine pool reached (14.22±1.41) μmol g_CDM ⁻¹, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH^r improved the l-serine biosynthesis pathway. In addition, the flux from l-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that l-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C₁ units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for l-serine production in C. glutamicum.     

Biodegradation of 4-chlorobiphenyl by using induced cells and cell extract of Burkholderia xenovorans

This study aimed to evaluate the efficiency of Burkholderia xenovorans LB400 cells and their cell extract to remediate 4-chlorobiphenyl (4-CB). The bacterium previously induced with 4-CB was able to degrade up to 98% of initial 50 mg L^?1 of 4-CB from mineral medium within 96 h of incubation. The degradation of 4-CB occurred through the formation of meta-cleavage product 2-hydroxy-6-oxo-6phenylhexa-2,4-dienoic acid (HOPDA), as revealed through enzymatic assay of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD). A derivative of 1,2-benzenedicarboxylic acid was observed as one of the major intermediate metabolites of 4-CB degradation. Time course production of 2,3-DHBD during growth corresponds with the degradation pattern of 4-CB by the bacterium. In vitro degradation of 4-CB using cell extract of B. xenovorans showed complete degradation of initial 25 mg L^?1 of 4-CB within 6 h of incubation. To the best of the authors' knowledge, this is the first report in which in vitro degradation of 4-CB using cell extract of Burkholderia xenovorans is presented.     

Modeling of growth kinetics for an isolated marine bacterium,Oceanimonas sp. BPMS22 during the production of a trypsin inhibitor

Abstract

Protease inhibitors significantly control physiologically relevant protease activities. Protease inhibitors from marine microbial sources are unique due to their rough living environmental conditions. In the present study, a protein protease inhibitor (PI) was produced from marine Oceanimonas sp. BPMS22. Seven different media were screened for the growth of the bacterium and production of PI. Different carbon and nitrogen sources were screened and optimized for the specific protease inhibitor activity. Three different growth models were checked for the best fit of the bacterial growth. A modified Gompertz model was selected as the best model for the growth of Oceanimonas sp. BPMS22 with the maximum specific growth rate of 0.165?hr^?1 and doubling time of 4.2?hr. The production of PI takes place during the non-growing phase of the bacterial growth. A kinetic model for the production of PI during non-growing phase was used for studying various process parameters. From the model, the maximum trypsin inhibitor formation rate of 0.3802?IU per mg of biomass per hour was observed at 49.91?hr.     

Growth Kinetics of Microbacterium lacticum and Nitrate-Dependent Degradation of Ethylbenzene under Anaerobic Conditions

A pure strain of Microbacterium lacticum DJ-1 capable of anaer-obic biodegradation of ethylbenzene was isolated from soil contaminated with gasoline. Growth of the strain and biodegradation of ethylbenzene in batch cultures led to stoichiometric reduction of nitrate. M. lacticum DJ-1 could degrade 100 mg L^?1 of ethylbenzene completely, with a maximum degradation rate of 15.02 ± 1.14 mg L^?1 day^?1. Increasing the initial concentration of ethy-lbenzene resulted in decreased degradative ability. The cell-specific growth rates on ethylbenzene conformed to the Haldane–Andrew model in the substrate level range of 10–150 mg L^?1. Kinetic parameters were determined by nonlinear regression on specific growth rates and various initial substrate concentrat-ions, and the values of the maximum specific growth rate, half saturation constant, and inhibition constant were 0.71 day^?1, 34.3 mg L^?1, and 183.5 mg L^?1, respectively. This is the first report of ethylbenzene biodegradation by a bacterium of Microbacterium lacticum under nitrate-reducing conditions.     

Biodegradation of the organochlorine insecticide, endosulfan, and the toxic metabolite, endosulfan sulfate, by Klebsiella oxytoca KE-8

Biodegradation of endosulfan, a chlorinated cyclodiene insecticide, is generally accompanied by production of the more toxic and more persistent metabolite, endosulfan sulfate. Since our reported endosulfan degrader, Klebsiella pneumoniae KE-1, failed to degrade endosulfan sulfate, we tried to isolate an endosulfan sulfate degrader from endosulfan-polluted soils. Through repetitive enrichment and successive subculture using mineral salt medium containing endosulfan or endosulfan sulfate as the sole source of carbon and energy, we isolated a bacterium capable of degrading endosulfan sulfate as well as endosulfan. The bacterium KE-8 was identified as Klebsiella oxytoca from the results of 16S rDNA sequence analysis. In biodegradation assays with KE-8 using mineral salt medium containing endosulfan (150 mg l^–1) or endosulfan sulfate (173 mg l^–1), the biomass was rapidly increased to an optical density at 550 nm of 1.9 in 4 days and the degradation constants for - and -endosulfan, and endosulfan sulfate were 0.3084, 0.2983 and 0.2465 day^–1, respectively. Analysis of the metabolites further suggested that K. oxytoca KE-8 has high potential as a biocatalyst for bioremediation of endosulfan and/or endosulfan sulfate.     

Improved accumulation of betulin and betulinic acid in cell suspension culture of Betula pendula roth by abiotic and biotic elicitors

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5?mg L^?1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200?mg L^?1 of chitosan was found to be 5.9?×?(6.5?mg g^?1 DW) higher over control cells. Treating the cells with 2?mg L^?1 of CCC, after 7?days, led to 149.3× enhancement of B content (19.4?mg g^?1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.     

Phytoremediative potential of salt-tolerant grass species for cadmium and lead under contaminated nutrient solution

Abstract

Phytoremediation of heavy metal contaminated soils represents a promising technique and salt-tolerant hyperaccumulators for multiple metals are the need of time. Therefore, phytoremediation potential of four salt-tolerant grass species [Dhab (Desmostachya bipinnata), Kallar (Leptochloa fusca), Para (Brachiaria mutica) and Sporobolus (Sporobolus arabicus Boiss)] was evaluated for cadmium (Cd) and lead (Pb) in a hydroponic study. The plants were harvested after a growth period of 3 months in a nutrient solution containing different levels of Cd (0, 5, and 25?mg?L^?1) and Pb (0, 25, and 125?mg L^?1). Results indicated that Dhab grass showed the highest root and shoot dry matter yield followed by Para, Kallar and Sporobolus grass irrespective of metal or its level under which they were grown. All the grass species showed considerable Cd-accumulating potential with an accumulation of >150?mg kg^?1of shoot dry matter at a higher level of Cd-contamination (25?mg?L^?1). While in case of shoot Pb-accumulation only Para grass performed well and accumulated Pb >1000?mg kg^?1 of shoot dry matter at the higher level of Pb-contamination (125?mg?L^?1). Moreover, Para and Dhab grasses performed better for shoot Cd-uptake, while only Para grass showed promising shoot Pb uptake potential. In conclusion, these grass species could be penitentially used for phytoremediation of salt-affected Cd and Pb contaminated soils.     

Biodegradation of a Phthalate Plasticizer, Di-Isononyl Phthalate (DINP), by Sphingobium chungbukense

In this study, di-isononyl phthalate (DINP) was efficiently degraded by Sphingobium chungbukense KCTC 2955. The optimal conditions for DINP (100 mg L⁻¹) degradation by S. chungbukense in a mineral salts medium were found to be pH 7.0, 30°C, and stirring at 200 rpm. The maximum specific rate of DINP degradation was found to be concentration dependent, with a maximum of 4.12 mg DINP L⁻¹ h⁻¹. DINP was transformed rapidly by S. chungbukense, with the formation of monoisononyl phthalate (MIP) and phthalic acid, which subsequently degraded further. These results highlight the potential of this bacterium for removing DINP-contaminated waste in the environment. Jae-Min Park and Miri Jeon contributed equally to this work.     

Isolation and Characterization of Phenol-Degrading Soil Bacterium Xanthobacter flavus

A soil bacterium isolated from a contaminated site degraded phenol when provided as the sole carbon and energy source in the medium. The bacterium was identified as Xanthobacter flavus MTCC 9130. This microbial strain was able to tolerate phenol up to 1000 mg L^?1 concentration. The lag phase increased with the increase in phenol concentration. The optimum growth temperature was 37°C. The organism efficiently utilized phenol and could degrade it completely within 120 h when initial concentration was less than 600 mg L^?1. Degradation of phenol was through ortho pathway, enzyme assay through cell-free extract exhibited the presence of catechol 1,2-dioxygenase. The specific activity was 0.146 μ mol min^?1 mg^?1 protein. However, higher concentrations of phenol in the medium had a negative effect on the growth of the bacterium. Hence this ability of Xanthobacter flavus can be effectively used for bioremediation studies of phenol-contaminated sites.     

Production of carotenoids by Brevibacterium linens: variation among strains, kinetic aspects and HPLC profiles

This work describes carotenoid pigment production by the red bacterium Brevibacterium linens covering strain diversity, kinetic and analytical aspects. Pigment production of 23 B. linens strains ranged from 0.05 to 0.60 mg pigments L⁻¹ culture, with specific productivity from 0.2 to 0.6 mg pigments per g dry biomass. The pigment production time curve showed a sigmoid shape, that matched cell growth. HPLC analysis revealed three groups of peaks, possibly non-hydroxylated, mono- and di-hydroxylated carotenoids. Polar molecules were mainly represented. Journal of Industrial Microbiology & Biotechnology (2000) 24, 64–70. Received 19 April 1999/ Accepted in revised form 25 September 1999     

Diesel Biodegradation Capacities and Biosurfactant Production in Saline-Alkaline Conditions by Delftia sp NL1, Isolated from an Algerian Oilfield

Abstract

In this study, a diesel oil-degrading bacterium was isolated from an oilfield water injection (water-bearing formations, 1,205?m depth) in Algeria. The bacterial strain, designated NL1, was cultivated on diesel oil as sole carbon and energy sources. Molecular analyses of the 16S rRNA gene sequence (KY397882) placed NL1 strain closely related to distinct cultivated species of the Delftia genus. Optimal diesel oil biodegradation by Delftia sp NL1 strain occurred at pH 11, 40?°C, 2?M NaCl and initial hydrocarbon concentration of 5% (v/v) as sole carbon source. GC-MS analyses evidenced that strain Delftia sp NL1 was able to degrade more than 66.76% of diesel oil within only 7?days. On the other hand, and in the same conditions, biosurfactant production by Delftia sp NL1 was also evaluated evidencing high emulsifying capacity (E₂₄ = 81%), ability to lower the surface tension of growing media (with the value of 25.7?mN m^?1), and production of glycolipids (8.7?g L^?1) as biosurfactants. This research presents indigenous strain Delftia sp NL1 for diesel degradation and synthesis of biosurfactant in extreme conditions. In this sense, strain NL1 is a good candidate for possible in situ oil recovery and in wastewater treatment in refineries and oil terminals in petroleum industry.     

Experimental evaluation of using calcein and alizarin red S for immersion marking of bighead carp Aristichthys nobilis (Richardson, 1845) to assess growth and identification of marks in otoliths,scales and fin rays

In order to evaluate the effects of immersion marking with calcein (CAL) and alizarin red S (ARS) on growth and mortality of juvenile bighead carp Aristichthys nobilis, and assess mark quality in otoliths, scales, and fin rays, CAL from 50 to 200 mg L^?1 and ARS from 150 to 300 mg L^?1 concentrations were used. With the exception of non‐lateral line scales from 50 mg L^?1 CAL treatments, immersion for 24 h produced detectable marks in sagittae, lateral line and non‐lateral line scales, and fin rays (dorsal, pectoral, ventral, anal, and caudal) at 100 days post‐marking. Detectable fluorescent marks in sagittae were readily observed at concentrations of 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. Marks were poorly visible in all non‐lateral line scales from both CAL‐ and ARS‐treated groups. Fluorescent marks were readily detected in lateral line scales at 100–200 mg L^?1 CAL or 150–300 mg L^?1 ARS, and in fin rays at 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. In particular, optimal marks were observed at the highest concentrations investigated in sagittae (300 mg L^?1 ARS), lateral line scales (150–200 mg L^?1 CAL or 250–300 mg L^?1 ARS), and fin rays (200 mg L^?1 CAL or 250–300 mg L^?1 ARS). However, fluorescent marks visible to the naked eye were not produced by any of the CAL or ARS treatments in sagittae, scales, or fin rays during this experiment. In addition, there was no significant difference on survival and growth of marked fish compared to controls throughout the experiment (P > 0.05).     

Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

Optimization of growth conditions and fatty acid analysis for three freshwater diatom isolates

Diatoms are a group of highly abundant and diverse aquatic algae species. They contain high lipid content along with many bioactive compounds that can be exploited for biotechnological applications. Despite these attractive attributes, diatoms are underrepresented in production projects due to difficulties in their cultivation. To optimize the growth of three freshwater diatom isolates, Cyclotella sp., Synedra sp. and Navicula sp., an orthogonal assay on N, P, Si and Fe, as well as temperature and pH, was designed using traditional single‐factor tests. We also studied the effect of using nanosilica as an alternate Si source on growth and found that the diatom isolates studied achieved their highest growth rates under different combinations of nutrient and environmental conditions. Silica had the greatest influence on growth, followed by phosphate and iron. The optimized growth conditions for Synedra sp. were N: 30 mg L^?1, P: 3 mg L^?1, Si: 14.8 mg L^?1, Fe: 0.448 mg L^?1, temperature 25°C and pH 8. For Navicula sp.: N: 20 mg L^?1, P: 2.5 mg L^?1, Si: 19.7 mg L^?1, Fe: 0.112 mg L^?1, temperature 30°C and pH 7.5–8. For Cyclotella sp.: N: 20 mg L^?1, P: 2.5 mg L^?1, Si: 19.7 mg L^?1, Fe: 0.448 mg L^?1, temperature 30°C and pH 7.5–8. Nano silica negatively affected growth in Navicula sp. and Cyclotella sp., but no such effect was observed in Synedra sp. Fatty acid profiling showed C16:0, C16:1(n ? 7), C18:0 and C20:5(n ? 3) as major fatty acids, with no significant differences in fatty acid methyl ester profiles between traditional and modified media. This work gives us a new insight into the growth requirements of freshwater diatom species, which are less studied than marine species.     

Enzymatic degradation of the antibiotic sulfamethazine by using crude extracts of different halophytic plants

Abstract

The biodegradation of the antibiotic sulfamethazine (SMT) by using different crude extracts of halophytes was investigated. For this purpose, crude water extracts of the halophytes Chenopodium quinoa, Sesuvium portulacastrum, and Tripolium pannonicum were prepared. Different amounts of SMT were added to the different extracts (final concentration of 1, 2, and 5?mg L^?1) and incubated at 37?°C. Crude extracts of T. pannonicum were further used to evaluate the degradation rate over time. In order to evaluate the influence of endophytic or naturally plant-associated microorganisms on the biodegradation of SMT, extracts from plants grown in sterile and non-sterile conditions were compared. SMT was analyzed by liquid chromatography coupled to positive ion electrospray mass spectrometry (ESI LC-MS). Based on the findings, crude extracts of T. pannonicum have a high potential to biodegrade SMT with a decrease up to 85.4% (4.27?±?0.10?mg L^?1) from an initial concentration of 5?mg L^?1. The lowest activity was obtained using extracts of C. quinoa with degradation of 4.5%. Extracts of plants cultivated under sterile and non-sterile conditions do not have any significant difference in SMT degradation. Therefore, microorganisms and their enzymatic activities do not seem to play a significant role during this process.     

Photoautotrophic cultivating options of freshwater green microalgal Chlorococcum humicola for biomass and carotenoid production

Photoautotrophic cultivation of Chlorococcum humicola was performed in batch and continuous modes in different cultivating system arrangements to compare biomass and carotenoids’ concentration and their productivities. Batch result from stirred tank and airlift photobioreactors indicated the positive effect of increasing light intensity on growth and carotenoid production, whereas the finding from continuous cultivation indicated that carotenoid enhancement preferred high light intensity and nitrogen-deficient environment. The highest biomass (1.31?±?0.04?g?L^?1) and carotenoid (4.59?±?0.06?mg?L^?1) concentration as well as the highest productivities, 0.46?g?L^?1 d^?1 for biomass and 1.61?mg?L^?1 d^?1 for carotenoids, were obtained when maintaining high light intensity of 10 klx, BG-11 medium and 2% (v/v) CO₂ simultaneously, while the highest carotenoid content (4.84?mg?g^?1) was associated with high light intensity and nitrogen-deficient environment, which was induced by feed-modified BG-11 growth medium containing nitrate 20 folds lower than the original medium. Finally, the cultivating system arranged into smaller stirred tank photobioreactors in series yielded approximately 2.5 folds increase in both biomass and carotenoid productivities relative to using single airlift photobioreactor with equivalent working volume and similar operating condition.     

A Novel Zeolite Coating for Protection of Concrete Sewers from Biological Sulfuric Acid Attack

Microbial growth inhibition and resistance to biological deterioration of concrete specimens coated with silver-loaded zeolite was evaluated by measuring the time course of bacterial growth, biological sulfur oxidation, and sulfate production using Acidithiobacillus thiooxidans as a corrosive agent. Live bacterial cells declined from an initial inoculum concentration of 1.1 × 10₄ cell ml_-1 to zero in 10 days, during which only 0.5–1% of the initial sulfur concentration of 10 g l_-1 was biologically oxidized, corresponding to sulfate production rates of 35–42 mg SO ₄ ^{2 ?} g ^{? 1} S ^{? 1} . Leaching coefficients of calcium and silicon in the specimens coated with silver-loaded zeolite of 1.6 × 10 ^{? 4} to 4.6 × 10 ^{? 2} cm ² d ^{? 1} respectively, were only 0.8% and 1% of the uncoated specimens.