LB Culture Research Articles

Maize Endophytic Plant Growth-Promoting Bacteria Peribacillus simplex Can Alleviate Plant Saline and Alkaline Stress.

Soil salinization is currently one of the main abiotic stresses that restrict plant growth. Plant endophytic bacteria can alleviate abiotic stress. The aim of the current study was to isolate, characterize, and assess the plant growth-promoting and saline and alkaline stress-alleviating traits of Peribacillus simplex M1 (P. simplex M1) isolates from maize. One endophytic bacterial isolate, named P. simplex M1, was selected from the roots of maize grown in saline-alkali soil. The P. simplex M1 genome sequence analysis of the bacteria with a length of 5.8 Mbp includes about 700 genes that promote growth and 16 antioxidant activity genes that alleviate saline and alkaline stress. P. simplex M1 can grow below 400 mM NaHCO3 on the LB culture medium; The isolate displayed multiple plant growth-stimulating features, such as nitrogen fixation, produced indole-3-acetic acid (IAA), and siderophore production. This isolate had a positive effect on the resistance to salt of maize in addition to the growth. P. simplex M1 significantly promoted seed germination by enhancing seed vigor in maize whether under normal growth or NaHCO3 stress conditions. The seeds with NaHCO3 treatment exhibited higher reactive oxygen species (ROS) levels than the maize in P. simplex M1 inoculant on maize. P. simplex M1 can colonize the roots of maize. The P. simplex M1 inoculant plant increased chlorophyll in leaves, stimulated root and leaf growth, increased the number of lateral roots and root dry weight, increased the length and width of the blades, and dry weight of the blades. The application of inoculants can significantly reduce the content of malondialdehyde (MDA) and increase the activity of plant antioxidant enzymes (Catalase (CAT), Superoxide Dismutase (SOD), and Peroxidase (POD)), which may thereby improve maize resistance to saline and alkaline stress. Conclusion: P. simplex M1 isolate belongs to plant growth-promoting bacteria by having high nitrogen concentration, indoleacetic acid (IAA), and siderophore, and reducing the content of ROS through the antioxidant system to alleviate salt alkali stress. This study presents the potential application of P. simplex M1 as a biological inoculant to promote plant growth and mitigate the saline and alkaline effects of maize and other crops.

Inoculation of chromium-tolerant bacterium LBA108 to enhance resistance in radish (Raphanus sativus L.) and combined remediation of chromium-contaminated soil.

Cr(VI) has been a carcinogen for organisms and a hazard to human health throughout the food chain. To explore a cost-effective and efficient method for removing Cr(VI), a Cr-resistant strain named LBA108 was isolated from the soil of a molybdenum-lead mining area. It was identified as Microbacterium through biochemical tests and 16S rDNA sequence analysis. Following 48 hours of incubation in LB culture medium containing 60 mg L-1 Cr(VI), the LBA108 strain exhibited reduction and adsorption rates for Cr(VI) at 96.64% and 15.86%, respectively. The removal mechanism was subsequently confirmed through Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction analysis. In an experimental setup, radish seedlings were cultivated as test crops under varying levels of Cr stress (ranging from 0 to 7 mg L-1) in a hydroponic experiment. With the inoculation of the LBA108 strain, the fresh weight of radish seedlings increased by 2.05 times and plant length increased by 34.5% under 7 mg L-1 Cr stress. In addition, the plant produced more antioxidant enzymes/enhanced antioxidant enzyme activities such as superoxide dismutase and catalase to prevent oxidative stress. Under Cr stress (6 mg L-1), the accumulation of Cr in rhizomes of radish seedlings increased compared to the control group by 91.44%, while the absorption of Cr by leaves decreased by 52.10%. These findings suggest that the LBA108 strain possesses bioremediation capabilities as a microbial-phytoremediation option for Cr-contaminated soil.

Molecular cloning and isolation of a recombinant alpha-Momorcharin in E. coli against Pyricularia oryzae

Introduction: Alpha-Momorcharin (α-MMC) is a member of the ribosome-inactivating protein (RIP) family that has been widely used as an antitumor, antiviral and antifungal agent. Methods: In this study, the codons of DNA encoding α-MMC were optimized for expression in E. coli and cloned into the pET-28a(+) vector. The protein was then expressed in E. coli strain BL21 (DE3) and purified by nickel affinity chromatography. Results: Under IPTG induction, α-MMC was expressed at approximately 50% of the total protein, showing high-level recombinant protein expression in E. coli. A high amount of purified α-MMC (70 mg) was isolated from 1 L LB culture medium of E. coli BL21 (DE3) with approximately 95% purity. Interestingly, α-MMC inhibited the mycelial growth of Pyricularia oryzae in a concentration-dependent manner. Conclusion: Using a microbial system for α-MMC expression provides a promising method for the design of a new agent against pathogens.

Heat-Stable Antifungal Factor and Expression Patterns of Protease by Chitinase-Producing Bacterium, Pedobacter sp. PR-M6

This study was conducted to investigate the protease expression patterns of the novel chitinase-producing bacterium Pedobacter sp. PR-M6. Protease were detected on SDS-PAGE gel as condition of 4℃, 25℃ and 37℃ to activate of enzymes obtained from incubation of LB, NB and 1% skim milk medium at 4℃, 20℃ and 30℃ after 21 days of incubation, respectively. Pedobacter sp. PR-M6 displayed proteolytic activity on SDS-PAGE gels containing 0.1% (w/v) gelatin, and protease isozymes of PR-M6 were expressed as two major bands (P7 and P8) and seven minor bands (P1, P2, P3, P4, P5, P6, and P9). Protease isozymes of PR-M6 were inhibited at 4℃ and 25℃. The two protease isozymes P5 and P6 were newly expressed in the LB culture medium at 20℃. To investigate the properties of a heat-stable antifungal factor (HSAF), PR-M6 stain was cultured to obtain the products of biosynthesis for incubation in LB medium. HSAF exhibited active biosynthesis at 20℃ for incubation in LB medium. Crude HASF obtained from LB20 medium showed hyphal growth inhibition of Rhizoctonia solani. Inhibition rate of R. solani growth was as high as 67% at 24 h and 47.8% at 48 h after incubation with 1,000 ppm of crude HSAF. Trypan blue stained the inside of the edge for destructed R. solani cell walls after crude HSAF treatment. These results indicated that HSAF and protease from Pedobacter sp. PR-M6 could be used as biocontrol agents against phytopathogens in agriculture.

Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone.

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.

Effect of starter culture sourdough prepared with Lactobacilli and Saccharomyces cerevisiae on the quality of hull-less barley-wheat bread

The aim of the present study was to investigate the microbial and technological characterization of barley-wheat flour (with 25%, 50%, 75% wheat flour added) breads made from hull-less barley sourdough that was prepared with a starter culture of Lb. plantarum SAB15, Lb. brevis SAB31 and S. cerevisiae SAM1-4. The yeasts isolated from spontaneous hull-less sourdough were sequenced with 26S rRNA gene via identification of the dominant species of S. cerevisiae with FTIR. Hull-less barley, with 13.6% protein, 1.47% ash, 66.15% water absorption and 21 ml sedimentation value, was added to wheat flour and protein, water absorption, and ash values decreased while sedimentation values increased. Hull-less barley-wheat bread samples containing starter culture was obtained with higher volume, specific volume and lower hardness than breads containing spontaneous culture. Especially breads with 25% and 50% barley mix were found to be the best ratios for hull-less barley bread. Although bread containing 75% and 100% barley flour scored low, those containing starter culture scored positively in terms of hardness and chewiness, and were accepted by consumers. With more barley added, there was higher a* and lower b* in the bread, while the breads with starter culture were found to be less yellow and red.

The Gene Expression Profile of Uropathogenic Escherichia coli in Women with Uncomplicated Urinary Tract Infections Is Recapitulated in the Mouse Model.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of uncomplicated urinary tract infections (UTIs). UPEC fitness and virulence determinants have been evaluated in a variety of laboratory settings, including a well-established mouse model of UTI. However, the extent to which bacterial physiologies differ between experimental models and human infections remains largely understudied. To address this important issue, we compared the transcriptomes of three different UPEC isolates in human infection and under a variety of laboratory conditions, including LB culture, filter-sterilized urine culture, and the UTI mouse model. We observed high correlation in gene expression between the mouse model and human infection in all three strains examined (Pearson correlation coefficients of 0.86 to 0.87). Only 175 of 3,266 (5.4%) genes shared by all three strains had significantly different expression levels, with the majority of them (145 genes) downregulated in patients. Importantly, gene expression levels of both canonical virulence factors and metabolic machinery were highly similar between the mouse model and human infection, while the in vitro conditions displayed more substantial differences. Interestingly, comparison of gene expression between the mouse model and human infection hinted at differences in bladder oxygenation as well as nutrient composition. In summary, our work strongly validates the continued use of this mouse model for the study of the pathogenesis of human UTI.IMPORTANCE Different experimental models have been used to study UPEC pathogenesis, including in vitro cultures in different media, tissue culture, and mouse models of infection. The last is especially important since it allows evaluation of mechanisms of pathogenesis and potential therapeutic strategies against UPEC. Bacterial physiology is greatly shaped by environment, and it is therefore critical to understand how closely bacterial physiology in any experimental model relates to human infection. In this study, we found strong correlation in bacterial gene expression between the mouse model and human UTI using identical strains, suggesting that the mouse model accurately mimics human infection, definitively supporting its continued use in UTI research.

Enhancement of extracellular bispecific anti-MUC1 nanobody expression in E. coli BL21 (DE3) by optimization of temperature and carbon sources through an autoinduction condition.

Escherichia coli is one of the most suitable hosts for production of antibodies and antibody fragments. Antibody fragment secretion to the culture medium improves product purity in cell culture and diminishes downstream costs. In this study, E. coli strain BL21 (DE3) harboring gene encoding bispecific anti‐MUC1 nanobody was selected, and the autoinduction methodology for expression of bispecific anti‐MUC1 nanobody was investigated. Due to the replacement of IPTG by lactose as inducer, less impurity and toxicity in the final product were observed. To increase both intracellular and extracellular nanobody production, initially, the experiments were performed for the key factors including temperature and duration of protein expression. The highest amount of nanobody was produced after 21 h at 33°C. The effect of different carbon sources, glycerol, glucose, lactose, and glycine as a medium additive at optimum temperature and time were also assessed by using response surface methodology. The optimized concentrations of carbon sources were obtained as 0.75% (w/v), 0.03% (w/v), 0.1% (w/v), and 0.75% (w/v) for glycerol, glucose, lactose, and glycine, respectively. Finally, the production of nanobody in 2 L fermenter under the optimized autoinduction conditions was evaluated. The results show that the total titer of 87.66 µg/mL anti‐MUC1 nanobody, which is approximately seven times more than the total titer of nanobody produced in LB culture medium, is 12.23 µg/L .

Electrochemical Monitoring of Metallic Materials' Surfaces with Biofilm Formation

Biofilms formed on materials by bacterial activity cause many engineering problems. It ranges from those at hospitals to toilets and kitchen at homes in various ways. To solve those problems, it is very important to analyze the phenomenon precisely and also to monitor the formation and growth process. To achieve the purpose, we have had many proposals for biofilm sensors so far. However, there are not so many proposals for rapid, easy and industrial biofilm sensors. In this research, we established the electrochemical system to monitor biofilm formation and growth to measure the equilibrium (static) potential and also dynamic polarization curves of metallic materials. And we discussed on how we should apply the results to evaluate biofilms on metallic materials. We used pure copper, titanium, iron and their alloys as specimen. The lead wire was attached to the tiny coupons (10mm x 10mm) and only the opposite side was exposed as electrode. Specimens (working electrode), helical platinum wire (counter electrode) and silver/silver chloride glass electrode in 3.3M KCl solution (reference electrode) were immersed into LB culture with E.coli (K-12, G6). And those three electrodes were connected to a potentiostat. The static potential change and polarization curves in the solution were measured, while the bacterial growth and biofilm formation/growth were going on. The equilibrium potentials for copper, iron and their alloys were shifted in less noble direction, while those of titanium and their alloys did not change and were almost constant. And the polarization curves were also reflected correspondingly. Those changes were discussed and they could be explained by the biofilm formation and growth.

Complete Genome Sequence of Kosakonia radicincitans GXGL-4A, a Nitrogen-Fixing Bacterium with Capability to Degrade TEX.

Kosakonia radicincitans GXGL-4A, a free-living nitrogen-fixing (NF) bacterial strain isolated from maize (Zea mays L.) roots was found to have ability to degrade aromatic hydrocarbons. In this study, we describe the main morphological characteristics of bacterium, aromatic hydrocarbon-degrading capability, and the complete genome of K. radicincitans GXGL-4A. The genome is consisted of only one 5,687,681bp linear chromosome with a G + C content of 53.96%. The strain has two genetically distinct nitrogenase systems, one based on molybdenum (Mo) similar to nitrogenase isolated from a wide range of nitrogen-fixing organisms, and the other contains iron (Fe). The differences in transcriptional level of several important nitrogen fixation (nif) genes between LB (nitrogen-rich, NR) and A15 nitrogen-free (nitrogen-limited, NL) culture conditions were detected using Real-time Quantitative Reverse Transcription PCR (qRT-PCR). The bacterial cells of GXGL-4A can grow well in LB liquid medium containing 1% toluene, ethylbenzene or xylene, suggesting a good resistance to the tested aromatic hydrocarbons. The results of GC-MS analysis showed that K. radicincitans GXGL-4A has a good capability to degrade toluene, ethylbenzene, and xylene (TEX). Completion of the genome sequencing will no doubt contribute to the deep exploration and comprehensive utilization of this NF bacterium in sustainable agriculture and bioremediation of aromatic pollutants.

Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm

Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm

Characterization of antibacterial properties of common spices

Antibiotics have long been trusted by physicians to treat bacterial infections. However, due to their overuse, antibiotic- resistant strains of bacteria now threaten the efficacy of many antibiotic types. We tested if 11 commonly-used spices could inhibit growth of the gram-negative bacteria, E. coli . We chose to test cinnamon, clove, thyme, oregano, cumin, garlic, black pepper, rosemary, basil, and ginger, all which have previously been suggested to have antibiotic properties. When these spices were diluted in LB agar, five spices were effective in inhibiting growth completely: clove, cinnamon, garlic, sage, and thyme. After a second round of experimentation using these spices in liquid LB cultures, the most effective spice at inhibiting growth at a low concentration was clove, with four other spices being nearly as effective. The main takeaway from these experiments is that certain spices and herbs have antibacterial effects that inhibit growth of E.coli , and these spices could show similarly promising activity towards other bacteria.

Fortification of milk with plant extracts modifies the acidification and reducing capacities of yoghurt bacteria

The acidification and reducing capacities of yoghurt bacteria were evaluated in different plant extract‐enriched milk samples. The milk samples enriched with thyme and grape seed extracts exhibited the highest values of acidification capacity for Lactobacillus delbrueckii subsp. bulgaricus (LB) (0.0065 pH unit/min) and Streptococcus thermophilus (ST) (0.0068 pH unit/min). The highest values of reducing capacity were observed in thyme (−0.98 mV/min), grape (−1.92 mV/min) and green tea (−0.75 mV/min)‐enriched samples for LB, ST and mixed culture of LB + ST, respectively. The fortification of yoghurt with plant extracts modified the acidification and reducing activities of starters, thus changing the fermentation time and quality attributes of the product.

Electrochemical Behaviors of Some Metallic Materials during Biofilm Formations in LB Liquid Culture with Escherichia coli

Biofilms formed on materials by bacterial activities affect many industrial problems as well as those in daily lives. Therefore, it is very important to monitor biofilm formation/growth processes properly. Biofilms are generally composed of water (more than 80%), exopolymeric substances and bacteria themselves. They exist on material surfaces inhomogeneously. From the electrochemical viewpoint, the interfacial structure would be equivalent to that of metallic electrode/organic solution, when the substrate materials would be metallic ones. In recent years, we have realized that the redox reaction in biofilms would play an important role for many matters relating to biofilms and the effects on industrial/medical/daily problems. And the electrochemical approach to the inside of biofilm and its analysis by micro electrodes have been carried out. However, the analysis from the metallic sides have not been investigated so much. In this study, we focused on the side of metallic electrodes and carried out the electrochemical investigation for some metallic materials, when biofilms by Escherichia coli formed and grew in liquid LB culture. And we analyzed the results and discussed about the application possibility to the evaluation of biofilms and materials deterioration.

Manufacture of low-fat Cheddar cheese by exopolysaccharide-producing Lactobacillus plantarum JLK0142 and its functional properties

This study aimed to evaluate the effect of exopolysaccharide (EPS)-producing Lactobacillus plantarum JLK0142 on the ripening characteristics and in vitro health-promoting benefits of low-fat Cheddar cheese. Three batches of cheese were made by employing a non-EPS-producing cheese starter (control), in combination with Lb. plantarum JLK0142 as an adjunct and the purified EPS as an ingredient. Lactobacillus plantarum JLK0142 survived well in cheese, with counts of 7.99 log cfu/g after 90 d of ripening. All experimental cheeses (with adjunct culture or EPS ingredient) had higher moisture, proteolysis, and sensory scores, and lower hardness and cohesiveness compared with the control cheese. Water-soluble extracts from the experimental cheeses outperformed that of the control in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and hydroxyl radicals, and inhibiting α-amylase, angiotensin-converting enzyme, and HT-29 tumor cell growth. Therefore, incorporation of the EPS-producing culture of Lb. plantarum JLK0142 is promising for improvement of low-fat cheese quality and bioactivities.

One-step production of bioactive human lipopolysaccharide binding protein from LPS-eliminated E. coli

Human lipopolysaccharide (LPS) binding protein (LBP) is a ∼60 kDa glycosylated protein that mediates potent innate immune against invading Gram-negative bacteria by recognition of LPS in their outer membranes. To date, there is no method for efficient production of bioactive LPS-free LBP at sufficient amounts through prokaryotic expression system. Here we present a simple approach for rapid preparation of human LBP from a LPS-eliminated E. coli strain named ClearColi BL21 (DE)3. Combined with the usage of an ultra-high-affinity CL7/Im7 purification system, we achieved one-step purification of recombinant human LBP with over 90% purity at a yield of ∼4 mg/L when using LB culture medium. The produced LBP retains full LPS binding activity which was validated by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Collectively, we develop a valid method that can be applied to cost-effectively produce and purify LPS-free proteins.

Induction of Viable but Nonculturable Escherichia coli O157:H7 by Low Temperature and Its Resuscitation.

Viable but non-culturable (VBNC) cells are alive bacteria cells, but lose their culturability in conventional culture media, usually escape detection by the plate count method and pose a serious threat to food safety and public health. Therefore, it is urgent to study the VBNC status, and to provide theoretical basis and scientific basis for food processing and safety control caused by pathogenic microorganisms. In this study, Escherichia coli O157:H7 was induced to the VBNC state at two different temperatures (-20°C and 4°C) and its resuscitation and morphological changes under different nutritional conditions were studied. The initial inoculum of 2.1 × 107 CFU/mL E. coli O157:H7 cells were induced into the VBNC state in normal saline, distilled water, LB broth at -20 °C after 176, 160, 80 days, respectively. The results showed that E. coli O157:H7 reserved at -20°C, and LB culture medium were easier to enter VBNC state than others conditions, the cells still had metabolic activity and the cell morphology changed from the typical rod shape to short rod and the cell size decreased. The resuscitate ways including the direct warming resuscitation, gradual warming resuscitation, adding chemical substance resuscitation, and adding nutrients resuscitation were studied. The results showed that the optimal conditions of 5% Tween 80 and 3% Tween 80 acculated the resuscitation of E. coli O157:H7 VBNC state cells induced by low temperature LB medium and low temperature saline. E. coli O157:H7 VBNC state failed from resuscitation when incubating in LB broth, respectively using direct warming and adding nutrients substance. This study provides new insights into induction and resuscitation of VBNC E. coli O157:H7 and offers an approach for investigating the formation mechanism of VBNC foodborne pathogens in food safety.

Portable bacteria-capturing chip for direct surface-enhanced Raman scattering identification of urinary tract infection pathogens.

Acute urinary tract infections (UTIs) are one of the most common nosocomial bacterial infections, which affect almost 50% of the population at least once in their lifetime. UTIs may lead to lethal consequences if they are left undiagnosed and not properly treated. Early, rapid and accurate uropathogens detection methods play a pivotal role in clinical process. In this work, a portable bacteria-grasping surface-enhanced Raman scattering (SERS) chip for identification of three species of uropathogens (Escherichia coli CFT 073, Pseudomonas aeruginosa PAO1 and Proteus mirabilis PRM1) directly from culture matrix was reported. The chip was firstly modified with a positively charged NH3+ group, which enables itself grasp the negatively charged bacterial cells through the electrostatic adsorption principle. After the bacterial cells were captured by the chip, concentrated Ag nanoparticles (NPs) were used to obtain their Raman fingerprint spectra with recognizable characteristic peaks and good reproducibility. With the help of chemometric method such as discriminant analysis (DA), the SERS-based chip allows a rapid, successful identification of three species of UTI bacteria with a minimal bacterial concentration (105 cells ml−1) required for clinical diagnostics. In addition, this chip could spot the bacterial SERS fingerprints information directly from LB culture medium and artificial urine without sample pre-treatment. The portable bacteria-grasping SERS-based chip provides a possibility for fast and easy detection of uropathogens, and viability of future development in healthcare applications.

Biovalorization of glucose in four culture media and effect of the nitrogen source on fermentative alcohols production by Escherichia coli

ABSTRACTGlucose is one of the most abundant monosaccharides and the easiest carbon source to be consumed by bacteria. In this study, four culture media (LB, M9, M63 and MOPS) were supplemented with glucose at three different concentrations (4, 12.5 and 25 g/L) in the presence of a genetically modified strain of Escherichia coli with the purpose of selecting the most suitable culture medium to obtain ABD (acetoin (A) and 2,3-butanediol (2,3-BD)). The selected medium was M9, the cheapest culture medium, since the ABD yields obtained fermenting 12.5 and 25 g/L of glucose in M9 culture medium at 37°C, atmospheric pressure, initial pH 6.5, 100 rpm and 10% (v/v) of inoculum were similar compared to the ABD yields obtained using M63 and LB culture media. The influence of nitrogen on ABD yield was tested adding sodium nitrate (NaNO3) or urea ((NH2)2CO) to M9 culture medium at three different nitrogen concentrations (2.5, 5.0 and 7.0 g N/L). Adding urea (7.0 g N/L) to M9 supplemented with 25 g/L of glucose improved by 23% the ABD yield at 96 h compared to M9 without urea, reaching a value of 27.2% (g ABD/g glucose). In contrast, the use of NaNO3 had no significant effect on the ABD yield.

Electrochemical Responses of Graphene with Biofilm Formation on Various Metallic Substrates By Using Laboratory Biofilm Reactors

We have confirmed that graphene is very sensitive to biofilm formation by environmental biota and by some special bacteria (E.coli and S.epidermidis). Therefore, graphene is not recommended for biomaterials used in the human body. Graphene’s high sensitivity could lead to infections. On the other hand, this high sensitivity might allow grapheme to serve as a sensor for detecting biofilm formation at a very early stage. To obtain more data, we investigated the change of electrochemical responses for graphene on various substrates (while biofilms were forming on these samples). Biofilms were formed on metallic substrates. The procedure is briefly described. A mechanical exfoliation method was repeated by using scotch tape. For each final stage, the graphene on the scotch tape was transferred to the surface of a metallic sheet-type specimen. (Various specimens were used.) The graphene formed on the metallic specimens was confirmed by using Raman spectroscopy and by staining. For the staining, 0.1% crystal violet was used. We expected that the extent of graphene formation would increase with the repeated number of attachments between tapes. Therefore, some transferred specimens, with different repeated numbers than others, were prepared as specimens. Then they were immersed in culture liquids (LB culture liquids and HI ones) including E.coli and Staphylococcus epidermidis. The specimens were also immersed into rotational-type reservoirs filled with culture liquids including bacteria. Each specimen was fixed with a special jig and the culture liquids were stirred continuously. Two laboratory biofilm reactors (a static type and a flow type reactor) were used for this work. Measurements of electrochemical responses (Cyclic voltammetry, immersion potential etc.) were made for all samples, while the changes of Raman shifts with time on those specimens were observed too. Also the final states of the specimens were checked by using crystal violet. Finally, behaviors were analyzed and the applicability to sensing biofilm formation was discussed.