You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Background {#Sec1}
==========

Power plants and large scale industries are located near water bodies because of their need for large amounts of water (for use as coolant). The discharge from soap industries results in the accumulation of sodium palmitate (main constituent of soaps) in these water bodies. When the magnesium ions (present in water bodies) react with sodium palmitate, magnesium palmitate is formed. Bacterial cells use magnesium palmitate as a carbon source for their growth and development (Ashwin et al. [@CR1]). This results in the formation of biofilms since microorganisms exist predominantly as biofilms in water bodies (Costerton et al. [@CR2]). The discharge (of water used as coolant) raises the temperature of the water body in the immediate vicinity of the mixing zone, thereby forming a temperature gradient in the water body (Langford [@CR8]). The formation of a temperature gradient imparts a thermal stress on the bacterial cells, which ultimately influences the formation of biofilm.

Hostacka et al. ([@CR6]) reported the effect of temperature on formation of *Pseudomonas aeruginosa* biofilm by estimating the biomass of biofilm formed. Recently, Zhou et al. ([@CR17]) reported the effect of temperature on formation of *P. aeruginosa* biofilm by estimating the concentration of polysaccharide and protein concentration in biofilm. However a study on the effect of temperature on adhesion force and surface topography of *P. aeruginosa* biofilm is lacking. In this study, the effect of temperature on *P. aeruginosa* biofilm formation was evaluated with respect to three parameters---the mass of biofilm formed, the production of extracellular polysaccharide and the adhesion force. In addition, surface topography of biofilm was also evaluated.

Methods {#Sec2}
=======

The organism *P. aeruginosa* (MTCC 2297) was chosen since it is a model organism used in biofilm research. *P. aeruginosa* grows over a wide range of temperatures. The media for formation of biofilm consisted of M9 minimal media with 1% (w/w) magnesium palmitate as the carbon source (Ashwin et al. [@CR1]). Magnesium palmitate was prepared according to the procedure given by Quraishi et al. ([@CR14]). Equimolar concentration of palmitic acid and potassium hydroxide were mixed and the suspension was heated continuously to yield potassium palmitate. Magnesium chloride, in slight excess of what was required for the stoichiometric reaction, was added to the solution. The product was then cooled and the precipitate was filtered and washed with water and acetone. Magnesium palmitate thus obtained was dried to constant weight.

The samples for inductively coupled plasma (ICP) analysis (to determine the concentration of metal ions) were prepared according to the protocol given by Cruz et al. ([@CR3]). The biofilm sample was centrifuged at 12,500*g* for 15 min and then acid digested in concentrated nitric acid for 1 h. The supernatant (of the digested sample) was diluted using double distilled water and the metal concentration was determined using ICP optical emission spectrometry (Perkin Elmer Optima 5300 DV).

Biofilm biomass was estimated using the protocol of Merritt et al. ([@CR10]). Biofilm samples were prepared according to the procedure given earlier (Ashwin et al. [@CR1]). Biofilm samples were incubated in 0.1% crystal violet solution for 10 min. The samples were then rinsed with water and the bound crystal violet was extracted using 95% crystal violet. A590 of the solubilizing buffer was then measured.

The extracellular polysaccharide produced was quantified using the procedure given by Forde and Fitzgerald ([@CR5]) and Parkar et al. ([@CR13]). The cells were centrifuged at 12,500*g* for 15 min and were then resuspended in 5 ml of sodium hydroxide. The solution was heated for 15 min. The cells were then removed by centrifuging at 12,500*g* for 30 min. 99.9% (v/v) ethanol was added to the supernatant and incubated at 40°C to precipitate the polysaccharides. The precipitated polysaccharides were then dissolved again in 10 ml of sterile water. To 1 ml of this sample, 8 ml of 98% (v/v) sulphuric acid was added and the mixture was cooled for 10 min. 1 ml of 1% (w/v) of cold tryptophan was added to this mixture and heated for 15 min to effect hydrolysis. The amount of polysaccharide was then estimated by measuring A500. AFM images of biofilm were resolved in air using JPK Nanowizard II. The images were acquired in contact mode using Mikromasch CSC38 probes (Additional file [1](#MOESM1){ref-type="media"}: Figure S1). The spring constant of the cantilevers used were 0.03 N/m (Ashwin et al. [@CR1]). A total of eight force-distance curves (on each biofilm sample) were generated to evaluate the biofilm adhesion force. A typical force curve is given (Additional file [2](#MOESM2){ref-type="media"}: Figure S2). All experiments were performed in triplicate.

Results and discussion {#Sec3}
======================

The effect of temperature on *P. aeruginosa* biofilm formation was evaluated with respect to three parameters---the mass of biofilm formed, the production of extracellular polysaccharide and the adhesion force. Quantification of biofilm biomass was done using the dye crystal violet (Merritt et al. [@CR10]). Biofilm biomass formed at temperatures 28, 33, 37 and 42°C were 1.25 (O.D), 2.20 (O.D) 2.80 (O.D) and 2.30 (O.D) respectively (Table [1](#Tab1){ref-type="table"}). The results show that biofilm biomass was highest for biofilm sample formed at 37°C. This is due to increased production of extracellular matrix material by *P. aeruginosa* cells at 37°C.Table 1Biofilm formation evaluated at different growth temperature supporting informationGrowth temperature (°C)Adhesion force (nN)Surface roughness R~a~ (nm)Biofilm biomass (O.D)Extracellular polysaccharide (µg)Concentration of metal ions in biofilm (mg/l)426.6 ± 0.1802.30 ± 0.11240 ± 40Ca, 0.055\
Mg, 1.583710.8 ± 0.21392.80 ± 0.11570 ± 57Ca, 0.10\
Mg, 2.36335.4 ± 0.21182.20 ± 0.11210 ± 35Ca, 0.046\
Mg, 1.42284.7 ± 0.11851.25 ± 0.1Ca, 0.03 \
Mg, 0.78

Adhesion force of biofilm is a measure of adhesiveness of biofilm (probe-biofilm interaction force). Adhesion force of biofilm grown at 28, 33, 37 and 42°C were 4.7, 5.4, 10.8 and 6.6 nN respectively (Table [1](#Tab1){ref-type="table"}).

The results indicate that biofilm adhesion force was highest for biofilm sample formed at 37°C. This is due to increased production (and accumulation) of extracellular matrix material by *P. aeruginosa* cells at 37°C (Ashwin et al. [@CR1]; Oh et al. [@CR11], [@CR12]; Fang et al. [@CR4]). The increased production of extracellular polymeric material is due to higher metabolic rate of *P. aeruginosa* cells at 37°C.

Extracellular polysaccharides play an important role in biofilm formation since it is only when extracellular polysaccharides are produced that cell aggregation and biofilm formation occur. This is because the extracellular polysaccharides act as 'cement' in holding the cells together (Sutherland [@CR15]).The amount of extracellular polysaccharide produced by *P. aeruginosa* cells grown at 28, 33, 37 and 42°C were 890, 1,210, 1,570 and 1,240 µg respectively (Table [1](#Tab1){ref-type="table"}). The results show that extracellular polysaccharide production was highest for biofilm sample formed at 37°C.

The higher amount of calcium and magnesium ions incorporated in the biofilm (formed at 37°C: Table [1](#Tab1){ref-type="table"}) suggests that biofilm (formed at 37°C) would have a higher mechanical stability (than biofilms grown at 28, 33 and 42°C). This is because calcium and magnesium ions have been reported to bind with negatively charged polysaccharides and have also been implicated in cell--cell binding mechanisms (Korstgens et al. [@CR7]; Lattner et al. [@CR9]).

Surface topography of biofilm was characterized by evaluating its surface roughness. Surface roughness of biofilm is an important parameter that is characteristic of growth conditions and has been reported to have an effect on the rate of diffusion of nutrients, thickness of dissolved oxygen boundary layer and mass transfer resistance (Zhang et al. [@CR16]). Surface roughness of biofilm was evaluated using parameter R~a~. This parameter measures the arithmetic average of deviation about the mean profile line (Ashwin et al. [@CR1]). Table [1](#Tab1){ref-type="table"} also shows that biofilm sample grown at 28°C had the highest surface roughness, although no specific trend was observed. This indicates that growth temperature has a significant effect on the surface topography of *P. aeruginosa* biofilm.

The aforementioned results show that *P. aeruginosa* biofilm (strain MTCC 2297) formed at 37°C produce higher amount of extracellular polysaccharide and also have a higher biomass, higher adhesion force and greater mechanical stability than biofilms formed at other temperatures (in the temperature range 28--42°C).

Conclusion {#Sec4}
==========

The effect of temperature on *P. aeruginosa* biofilm formation was evaluated with respect to three parameters---the mass of biofilm formed, the production of extracellular polysaccharide and the adhesion force. The results indicate that biofilm biomass (2.8, A590), extracellular polysaccharide production (1240 ± 40 µg) and adhesion force (10.8 ± 0.2 nN) were highest at 37°C. The results also suggest that biofilms formed at 37°C would have a higher mechanical stability (than biofilms grown at 28, 33 and 42°C). The results indicate that temperature has a significant effect on formation of *P. aeruginosa* biofilm.

Additional files {#Sec5}
================

Additional file 1:**Figure S1.** AFM (Vertical Deflection) images of biofilm formed at different temperatures: A- 28°C. B- 33°C. C- 37°C. D- 42°C.Additional file 2:**Figure S2.** A typical force curve.

AK carried out the experiments. AK and PG drafted the manuscript. Both authors have read and approved the final manuscript.

Acknowledgements {#d30e528}
================

The authors would like to thank the Department of Biotechnology and Department of Science and Technology for their continued financial support. The funding agency had no role in the design of experiments, data collection and analysis, decision to publish or preparation of the manuscript.

Compliance of ethical guidelines {#d30e533}
================================

**Competing interests** The authors declare that they have no competing interests.