Zymomonas Research Articles

Characterization of Two Zymomonas mobilis Wild Strains and Analysis of Populations Dynamics during Their Leavening of Bread-like Doughs.

Two Zymomonas mobilis wild strains (UMB478 and 479) isolated from water kefir were characterized for their biomass production levels and leavening performance when used as the inoculum of a real bread-like dough formulation. The obtained baked product would be consumable by people with adverse responses to Saccharomyces cerevisiae. In liquid cultures, the two strains reached similar biomass concentration (0.7 g CDW/L). UMB479 showed an interesting resistance to NaCl (MBC 30 g/L), that may be useful in the bakery sector. When inoculated in doughs, UMB479 produced the maximum dough volume (650 mL) after 5 h, glucose was almost consumed and 1 g/100 g of ethanol produced, +200% respective to UMB478. Using S. cerevisiae for comparison purposes, the dough doubled its volume fast, in only 2 h, but reached a final level of 575 mL, lower than that achieved by Z. mobilis. The analysis of bacterial and fungal population dynamics during dough leavening was performed through the Automated Ribosomal Intergenic Spacer Analysis (ARISA); doughs leavened by UMB479 showed an interesting decrease in fungal richness after leavening. S. cerevisiae, instead, created a more complex fungal community, similar before and after leavening. Results will pave the way for the use of Z. mobilis UMB479 in commercial yeast-free leavened products.

Comparison of Bioethanol Production Using Saccharomyces cerevisiae and Zymomonas mobilis in Fermented Jackfruit Peel Treated with Blend Crude Cellulose Enzymes

Bioethanol is an alternative energy source that is increasingly needed along with the depletion of petroleum stocks. Bioethanol can be produced by utilizing various wastes, including jackfruit peel and a mixture of crude cellulose enzymes from Trichoderma reesei and Aspergillus niger as well as fermentation using Saccharomyces cerevisiae and Zymomonas mobilis. This study aimed to compare the production of bioethanol using S. cerevisiae and Z. mobilis in fermented jackfruit peel with a mixture of crude cellulose enzymes from T. reesei and A. niger. The experimental design used a completely randomized design with the ratio of crude cellulase enzymes from T. reesei and A. niger as independent variables (0:0), (1:0), (0:1), (1:1), (1:2), (2:1), (1:3), and (3:1) as well as sugar and ethanol content as dependent variables. The sugar content was determined using the DNS method, while the ethanol content was determined using an alcoholmeter. Data analysis used one way ANOVA assisted by SPSS 16. The results showed that the highest sugar content (14.21 percent) was obtained in the ratio of crude cellulase enzymes T. reesei and A. niger (1:3), while the highest ethanol content (3, 16 percent) at a ratio of 1: 2 and fermented using Z. mobilis.

Effect of Pretreatment and Composition of Trichoderma Viride and Zymomonas Mobilis Consortium on Bioethanol Production from Leaf Litter

Bioethanol is an alternative biofuel that is widely produced in bulk to replace and complement petroleum-based fuels. Bioethanol is produced through the alcoholic fermentation of sucrose or simple sugars. One of the potential source for bioethanol production that has not been studied is leaf litter. Various studies have shown that T. viride and Z. mobilis can be used for bioethanol production, but there has been no report on the production of bioethanol from leaf litter using a consortium of T. viride and Z. mobilis using the SSF method. This study aims to examine the effect of pretreatment and composition of the consortium of T. viride and Z. mobilis on the production of bioethanol from leaf litter. Leaf litter was dried and mashed, then pretreatment was carried out with 32% NH3 solution. The bioethanol production process using the SSF method uses a consortium of T. viride and Z. mobilis with a composition of 5%: 5%, 10%: 5%, 5%: 10% and 10%: 10% (w/v). SSF was carried out for 72 hours at a temperature of 35oC and pH 5. The bioethanol was purified by distillation and the ethanol content was tested with GCMS. Higher levels of bioethanol were found in leaf litter with pretreatment than without pretreatment. The highest ethanol content (0.6721%) was obtained from SSF with consortium composition of T. viride : Z. mobilis (10% : 10%) from leaf litter with pretreatment. Pretreatment and composition of the consortium T. viride and Z. mobilis affect the production of bioethanol from leaf litter.

Transcriptomic Data Sets for Zymomonas mobilis 2032 during Fermentation of Ammonia Fiber Expansion (AFEX)-Pretreated Corn Stover and Switchgrass Hydrolysates.

ABSTRACTThe transcriptomes of Zymomonas mobilis 2032 were captured during the fermentation of ammonia fiber expansion (AFEX)-pretreated corn stover and switchgrass hydrolysates containing different concentrations of glucose and xylose. RNA samples were collected when Z. mobilis was fermenting glucose or xylose. Here, we present transcriptome sequencing (RNA-Seq) data obtained during separate phases of glucose or xylose consumption.

Experimental investigation on simultaneous production of bioethanol and biodiesel from macro-algae

The present work compares the biofuel production efficiency of Padina tetrastromatica and Sargassum swartzii macro-algae biomass by fermentation with Saccharomyces cerevisiae and Zymomonas Mobilis. The optimal pretreatment factors were identified by studying the effect of temperature, pH, and enzyme concentration on the conversion of macroalgae biomass to fermentable reducing sugars. The Low concentration of enzyme did not help conversion of biomass to reducing sugars even after 15 h of treatment with enzyme, but when the enzyme concentration was increased, the desired conversion was completed within 4 h of enzyme treatment. Both Padina tetrastromatica and Sargassum swartzii had an ethanol yield of above 80% irrespective of the difference in the species used for the fermentation. The highest possible ethanol yield (95.12%) was achieved for Padina tetrastromatica biomass when it was fermented with Zymomonas Mobilis. From the above results, it is clear that both Padina tetrastromatica and Sargassum swartzii have the potential to be used as reliable feedstock for biofuel production. The spent biomass after the fermentation process was used for the lipid extraction process and the extracted lipid was used for biodiesel production. The percentage of biodiesel yield obtained by means of the Soxhlet extraction process from Padina tetrastromatica and Sargassum swartzii macro-algae with the conventional NaOH catalyst was found to be 84% and 89% respectively. MgO.La2O3 was synthesized by means of the co-precipitation method. With the introduction of MgO.La2O3 nano-catalyst biodiesel yield was improved up to 88% and 92% for Padina tetrastromatica and Sargassum swartzii macro-algae, respectively.

High-sodium maltobionate production by immobilized Zymomonas mobilis cells in polyurethane

The purpose of this study was the production of maltobionic acid, in the form of sodium maltobionate, by Z. mobilis cells immobilized in polyurethane. The in situ immobilized system (0.125-0.35mm) was composed of 7g polyol, 3.5g isocyanate, 0.02g silicone, and 7g Z. mobilis cell, at the concentration of 210g/L. The bioconversion of maltose to sodium maltobionate was performed with different cell concentrations (7.0-9.0 gimobilized/Lreaction_medium), temperature (30.54-47.46°C), pH (5.55-7.25), and substrate concentration (0.7-1.3mol/L). The stability of the immobilized system was evaluated for 24h bioconversion cycles and storage of 6months. The maximum concentration of sodium maltobionate was 648.61mmol/L in 34.34h process (8.5 gdry_cell/Lreaction_medium) at 39°C and pH 6.30. The immobilized system showed stability for 19 successive operational cycles of 24h bioconversion and 6months of storage, at 4°C or 22°C.

Effect of bio-electrochemical system on the metabolic changes of Zymomonas mobilis

A bio-electrochemical system can promote the interaction between microorganism and electrode and consequently change cellular metabolism. To investigate the metabolic performance of Zymomonas mobilis in the bio-electrochemical system, we applied an H-type bio-electrochemical reactor to control Z. mobilis fermentation under 3 V. Compared with the control group without applied voltage, the glycerol in the anode chamber increased by 24%, while the glucose consumption in the cathode chamber increased by 16%, and the ethanol and succinic acid concentration increased by 13% and 8%, respectively. Transcriptomic analysis revealed that the pathways related to organic acid metabolism, redox balance, and electron transfer played roles in metabolic changes. Three significantly differentially expressed genes, ZMO1060 (superoxide dismutase), ZMO0401 (diguanylate cyclase), and ZMO1819 (nitrogen fixation protein), were selected to verify their functions in the bio-electrochemical system. Overexpression of ZMO1060 and ZMO1819 improved the electrochemical activity of Z. mobilis. This study provides insights into the microbial metabolism regulated by the bio-electrochemical system.

A temporal view of the water kefir microbiota and flavour attributes

Water kefir is a fermented beverage often produced in a household setting through the inoculation of a microbial community (water kefir grains) into a sugar rich solution. To date, a detailed understanding of how this community evolves during the fermentation, and the resulting impact on the final beverage flavour has not been elucidated. To address this metagenomics, flavour analysis and sensory science were used to resolve the temporal evolution of a model water kefir. We observed that the diversity of the water kefir studied is relatively unique, being dominated throughout the fermentation by the gluconic acid producer Zymomonas mobilis. We describe the metagenome-assembled genome of two likely new Curvibacter species, that is mostly found in the liquid fraction. Finally, we show that, in addition to Z. mobilis, members of the Bifidobacterium, Acetobacter and Saccharomyces species as well as former members of the Lactobacillus genus, were the main contributors to flavour. Industrial relevanceWater kefir has become an increasingly attractive alternative natural beverage for consumers, while also serving as an interesting model to enhance our understanding of microbial communities consisting of eukaryotes and prokaryotes. By integrating metagenomics, chemical profiling and sensory evaluation, we illustrate and clarify the dynamic nature of water kefir fermentations. In addition to elucidating its detailed temporal diversity, we also highlight the potential flavour and sensory contributions of the different microbial members of the community. These combined findings represent a significant milestone in the understanding of water kefir microbiology while also paving the way for the development of defined starter cultures that can enable the manufacture of a beverage which reproduces the main attributes of a traditionally brewed water kefir.

PRODUKSI BIOETANOL DARI LIMBAH BATANG KELAPA SAWIT (Elaeis guineensis) MENGGUNAKAN Zymomonas mobilis DENGAN PERLAKUAN CRUDE ENZIM Trichoderma reesei DAN Aspergillus niger

<p><em>Bioethanol is an alternative energy that is starting to be needed, along with the depletion of petroleum reserves. Bioethanol can be obtained from various wastes, including palm oil trunk waste which contains 86.03% cellulose. The purpose of this study was to determine the production of bioethanol from oil palm trunk waste by treatment with crude enzymes Trichoderma reesei and Aspergillus niger. This research is an experimental study using a completely randomized design, namely the ratio of crude enzymes from Trichoderma reesei and Aspergillus niger (0:0), (1:0), (0:1), (1:1), (2:1), (1:2), (3:1), (1:3) on sugar and bioethanol content of fermented oil palm stem waste using Zymomonas mobilis. Sugar content was measured using the DNS method, bioethanol content was measured using an alcoholmeter. Data were analyzed using one way ANOVA. The results showed that the crude enzymes T. reesei and A. niger could increase the sugar content, namely the ratio of crude enzymes T. reesei and A. niger 3:1 (11.89 g/mL). The highest bioethanol content was in the ratio of crude enzymes T. reesei and A. niger (2:1), which was 2.34%.</em><em></em></p>

Kinase expression enhances phenolic aldehydes conversion and ethanol fermentability of Zymomonas mobilis.

Kinases modulate the various physiological activities of microbial fermenting strains including the conversion of lignocellulose-derived phenolic aldehydes (4-hydroxyaldehyde, vanillin, and syringaldehyde). Here, we comprehensively investigated the gene transcriptional profiling of the kinases under the stress of phenolic aldehydes for ethanologenic Zymomonas mobilis using DNA microarray. Among 47 kinase genes, three genes of ZMO0003 (adenylylsulfate kinase), ZMO1162 (histidine kinase), and ZMO1391 (diacylglycerol kinase), were differentially expressed against 4-hydroxybenzaldehyde and vanillin, in which the overexpression of ZMO1162 promoted the phenolic aldehydes conversion and ethanol fermentability. The perturbance originated from plasmid-based expression of ZMO1162 gene contributed to a unique expression profiling of genome-encoding genes under all three phenolic aldehydes stress. Differentially expressed ribosome genes were predicted as one of the main contributors to phenolic aldehydes conversion and thus finally enhanced ethanol fermentability for Z. mobilis ZM4. The results provided an insight into the kinases on regulation of phenolic aldehydes conversion and ethanol fermentability for Z. mobilis ZM4, as well as the target object for rational design of robust biorefinery strains.

Acceleration of Bio-Ethanol Production from Waste Vegetables and Fruits by Using Zymomonas mobilis Genetically Modified

The genetic modification pathways have been considered the shortest and most efficient for the production of bioethanol, in particular, that knock-in Alcohol dehydrogenase (ADH1) originally from Saccharomyces cerevisiae into the overexpression plasmid (pET28a), in order to continuous stimulate and accelerate the fermentation after inserted directly into the Zymomonas mobilis bacteria. At the same time, there are huge volumes of waste vegetables and fruit which are produced every day from restaurants and shops, and need to eliminate them with modern scientific methods, in order to the clean the environment and produced biofuel to reduce at least the use of fossil fuels. The percentage of production bioethanol was 52 % / 172 hours, which is considered a qualitative leap in the percentage of production during that period. In contrast, HPLC technique has determined the purity of bioethanol was 89.07% compared to 99% absolute ethanol which is possible to increase the purity according to the biochemical method. This study illustrated that may be commercially bioethanol produced in the future by the development of technology-based on genetic modification of bacteria.

Invertase enzyme

The enzyme known as invertase catalysis the conversion of sucrose into glucose and fructose (EC 3.2.1.26). The best source of invertase is yeast. Internal and exterior in vertases are the two varieties found in S. cerevisiae. Invertase catalyses the hydrolysis of sucrose into fructose and glucose. Pharmaceuticals, analytical measuring probes, and yeast species detection ELISA kits all include this invertase. Immobilization of the invertase naturally is what is meant by this. Different levels of glycoprotein polymerization, glycosylation, and phosphorylation make bacteria invertase unique. A range of gram-positive and gram-negative organisms, including Zymomonas mobilis, and Bacillus strains, produce invertase. Comparing fungal growth to the time course of invertase synthesis by fungi, which ranged from 24 to 192 hours, was analysed. Twenty-four-hour intervals were used to measure the invertase activity. The biological characteristics that were looked at in this study included the amount of invertase-producing bacteria and soil invertase activity. Invertase is used to replace traditional acid hydrolysis.

Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli.

A promoter plays a crucial role in controlling the expression of the target gene in cells, thus being one of the key biological parts for synthetic biology practices. Although significant efforts have been made to identify and characterize promoters with different strengths in various microorganisms, the compatibility of promoters within different hosts still lacks investigation. In this study, we chose the native Pgap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting promoter compatibility between Escherichia coli and Z. mobilis. Pgap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial cell factories. Using EGFP as a reporter, a Z. mobilis-derived Pgap mutant library was constructed and sorted in E. coli, with candidate promoters exhibiting high fluorescence intensity collected. A total of 53 variants were finally selected and sequenced by Sanger sequencing. The sequencing results grouped these variants into 12 different Pgap variant types, among which seven types presented higher promoter strength than native Pgap in E. coli. The next-generation sequencing technique was then employed to identify key mutations within the Pgap promoter region that affect the promoter compatibility. Finally, six important sites were identified and confirmed to help increase Pgap strength in E. coli while keeping similar strength of native Pgap in Z. mobilis. Compared to native Pgap, synthetic promoters combining these sites had enhanced strength; especially, Pgap-6M combining all six sites exhibited 20-fold greater strength than native Pgap in E. coli. This study thus not only determined six important sites affecting promoter compatibility but also confirmed a series of Pgap promoter variants with strong promoter activity in both E. coli and Z. mobilis. In addition, a strategy was established in this study to investigate and determine nucleotide sequences in promoter regions affecting promoter compatibility, which can be applied in other microorganisms to help reveal universal factors affecting promoter compatibility and design promoters with desired strengths among different microbial cell factories.

Mathematical model with time‐delay and delayed controller for a bioreactor

In this paper, a fractional Lotka–Volterra mathematical model for a bioreactor is proposed and used to fit the data provided by a bioprocess known as continuous fermentation of Zymomonas mobilis. The model contemplates a time‐delay due to the dead‐time (non‐trivial) that the microbe needed to metabolize the substrate. A Hopf bifurcation analysis is performed to characterize the inherent self oscillatory experimental bioprocess response. As consequence, stability conditions for the equilibrium point together with conditions for limit cycles using the delay as bifurcation parameter are obtained. Under the assumptions that the use of observers, estimators, or extra laboratory measurements are avoided to prevent the rise of computational or monetary costs, for the purpose of control, we will only consider the measurement of the biomass. A simple controller that can be employed is the proportional action controller , which is shown to fail to stabilize the obtained model under the proposed analysis. Another suitable choice is the use of a delayed controller which successfully stabilizes the model even when it is unstable. The delay in the feedback control is due to the dead‐time necessary to obtain the measurement of the biomass in the bioreactor by dry weight. Finally, the proposed theoretical results are corroborated through numerical simulations.

Improved Hydrogen Peroxide Stress Resistance of Zymomonas mobilis NADH Dehydrogenase (ndh) and Alcohol Dehydrogenase (adhB) Mutants

Unintended shifts in stress resistance of microbial strains with engineered central metabolism may impact their growth and production performance under oxidative, lignocellulosic, solvent, and other stress conditions, and as such, must be taken into account in bioprocess design. In the present work, we studied oxidative stress resistance in mutant strains of the facultatively anaerobic, ethanologenic bacterium Zymomonas mobilis with modified respiratory (inactivated NADH dehydrogenase Ndh, by disruption of ndh) and ethanologenic (inactivated iron-containing alcohol dehydrogenase isoenzyme ADH II, by disruption of adhB) catabolism, using exogenously added H2O2 in the concentration range of 2–6 mM as the oxidative stressor. Both mutations improved H2O2 resistance and enhanced catalase activity by a factor of 2–5, while the overexpression of Ndh had an opposite effect. Strains with a catalase-negative background were unable to grow already at 1 mM hydrogen peroxide, and their H2O2 resistance did not depend on AdhB or Ndh expression levels. Hence, the improved resistance of the ndh and adhB mutants to H2O2 resulted from their elevated catalase activity. The interrelation between these mutations, the catabolic redox balance, catalase activity, and oxidative stress defense in Z. mobilis is discussed.

Odd one out? Functional tuning of Zymomonas mobilis pyruvate kinase is narrower than its allosteric, human counterpart.

Various protein properties are often illuminated using sequence comparisons of protein homologs. For example, in analyses of the pyruvate kinase multiple sequence alignment, the set of positions that changed during speciation ("phylogenetic" positions) were enriched for "rheostat" positions in human liver pyruvate kinase (hLPYK). (Rheostat positions are those which, when substituted with various amino acids, yield a range of functional outcomes). However, the correlation was moderate, which could result from multiple biophysical constraints acting on the same position during evolution and/or various sources of noise. To further examine this correlation, we here tested Zymomonas mobilis PYK (ZmPYK), which has <65% sequence identity to any other PYK sequence. Twenty-six ZmPYK positions were selected based on their phylogenetic scores, substituted with multiple amino acids, and assessed for changes in Kapp-PEP . Although we expected to identify multiple, strong rheostat positions, only one moderate rheostat position was detected. Instead, nearly half of the 271 ZmPYK variants were inactive and most others showed near wild-type function. Indeed, for the active ZmPYK variants, the total range of Kapp,PEP values ("tunability") was 40-fold less than that observed for hLPYK variants. The combined functional studies and sequence comparisons suggest that ZmPYK has evolved functional and/or structural attributes that differ from the rest of the family. We hypothesize that including such "orphan" sequences in MSA analyses obscures the correlations used to predict rheostat positions. Finally, results raise the intriguing biophysical question as to how the same protein fold can support rheostat positions in one homolog but not another.

High-level production of L-valine in Escherichia coli using multi-modular engineering

L-valine is a valuable amino acid in mammals that is used as the main component of feed additives. The low efficiency of the fermentation titer limits the industrial application of L-valine. Here, an L-valine-producing strain of Escherichia coli was obtained using a multi-modular strategy. Initially, a chassis strain was generated by mutagenesis and high-throughput screening. The L-valine biosynthetic pathway and transport module were modified to improve the L-valine titer. Subsequently, the transcription factors associated with L-valine biosynthesis were investigated. Overexpression of PdhR and inhibition of the expression of RpoS promoted L-valine synthesis. Finally, the NADPH supply was enhanced after the introduction of the heterologous Entner-Doudoroff (ED) pathway from Zymomonas mobilis. The strain VAL38 produced 92 g/L L-valine in a 5-L bioreactor with a yield of 0.34 g/g glucose. This strategy is provided as a reference for improving the production performance of cell factories for L-valine and its derivatives.

Tailoring fructooligosaccharides composition with engineered Zymomonas mobilis ZM4.

Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated β-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6g L-1 of FOS, with a productivity of 1.53 ± 0.03g L-1h-1 and a yield of 0.31 ± 0.03 gFOS gsucrose-1. This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. KEY POINTS: • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented.

Microbial conversion of waste baklava syrup to biofuels and bioproducts

In Turkey, approximately 17.5 million liters of waste baklava syrup (WBS) are generated annually. WBS contains a high amount of sugar; however, it does not find any application. In this study, microbial conversion of WBS to biofuels and bioproducts was investigated. The experiments were performed in shaking flask cultures. WBS was not subjected to any hydrolysis process and was directly used as substrate in the medium, since it was in liquid form. The total sugar content of WBS was determined as 870 g/L. At the optimal concentrations of WBS, 38.6 g/L citric acid by Aspergillus niger, 27.3 g/L levan by Zymomonas mobilis and 8.9 g/L lipids by Rhodotorula glutinis could be produced. The amounts of ethanol produced by S. cerevisiae and Z. mobilis were 129 and 75.2 mL/L, respectively. The maximum absorbances for yellow, red and orange pigments which were produced by Monascus purpureus were determined as 0.7768, 0.928 and 0.835, respectively. Evaluating WBS as a substrate in microbial fermentation studies can contribute to reducing both the medium cost and the environmental pollution problem. This is the first report about the use of WBS as a substrate in microbial studies.

THE POTENTIAL OF Azadirachta indica (NEEM TREE) LEAVES FOR BIOETHANOL PRODUCTION USING Zymomanas mobilis AS FERMENTING ORGANISMS

Bioethanol is a renewable energy produced from lignocellulosic biomass mainly by the fermentation process. It is used as an alternative to fossil fuel which doesn’t contribute to global warming. This research work was carried out to determining the potential of Neem tree leaves for bioethanol production. The process used in this study include pretreatment, hydrolysis fermentation and distillation. Hot water and Acid methods were used for the pretreatment. The hydrolysis of the substrate was carried out using Aspergillus niger for five days. Benedict test method was used to determine the reducing sugar concentration of the hydrolysate. The highest yield of 1500-2000 (mg/dl) was obtained equally for both the pre-treatment method. After hydrolysis, fermentation process was carried out for seven days using Zymomonas mobilis to ferment the hydrolysate. The fermented broth was separate using fractional distillation. Acid potassium dichromate was used to determine the concentration of the bioethanol produced. The results obtained shows that liquid hot water pretreatment produced the highest yield of 0.294±0.882 (mg/L) whereas alkaline method produced 0.282±.882 (mg/L) yield. Gas chromatography and mass spectroscopy analysis was also carried out to determine the presence of volatile compounds in the bioethanol produced. The highest volatile compound was Ethanol with 33.12% where Butyl Glycol was found to be least volatile compound with 6.22%. This result shows that Neem tree leaves is a good biomass for bioethanol production and pretreatment using Hot water method produced more bioethanol than Alkaline method of pretreatment.