Effect of different D-amino acids on biofilm formation of mixed microorganisms

Biofilm, which needs to attach to the surface of some carriers, is an organized membrane polymer secreted by bacteria to form self-protection. In water and wastewater treatment system, biofilm formed by bacteria can increase the cost of operation and maintenance and threaten human health (Li et al. 2020). Therefore, it is necessary to find a non-toxic and low-cost method to inhibit bacterial biofilms.

There are many studies on the bacterial biofilm inhibition by D-amino acid. D-amino acid can inhibit cell wall synthesis of bacterial by altering the structure of peptidoglycan in bacterial walls (Cava et al. 2011, 2014). Kolodkin-Gal et al. (2010) proved that D-amino acids (D-leucine, D-methionine, D-tyrosine, D-tryptophan) could change the physicochemical properties of bacterial cell walls by replacing D-alanine in cell walls. D-amino acid can affect the bacterial biofilm by regulating quorum sensing signal molecule Autoinducer-2 (AI-2). Xu & Yu (2011) indicated that the content of AI-2 in mixed microorganisms (activated sludge) was reduced by the exogenous addition of D-tyrosine. Fluorescence staining experiments on V. Harveyi BB170 confirmed that the synthesis of AI-2 can be inhibited by D-tyrosine (Xu & Liu 2011). Pereira et al. (2013) found that D-amino acid affected the information transmission between Campylobacter jejuni cells by inhibiting the expression of AI-2, resulting in difficulties in the adhesion and formation of bacterial biofilms. The mutation of G92D (a key amino acid determining AI-2 activity) was the reason for the loss of AI-2 activity in Campylobacter jejuni (Plummer et al. 2011). D-amino acids could make the structure of the bacterial extracellular matrix thin and loose by inhibiting the synthesis of extracellular proteins in biofilms. D-amino acid could inhibit the expression of EPS-related genes (such as EPSA and TAPA) in Bacillus subtilis (Leiman et al. 2013).

D-amino acid could not only inhibit the formation of biofilm, but also disintegrate the mature biofilm. Koldkin-Gal et al. (2010) found that a large number of D-amino acids (D-tyrosine, D-tryptophan, D-methionine and D-leucine) existed during the collapse of Bacillus subtilis mature biofilm. Bucher et al. (2016) showed that D-amino acids could be used as a biofilm decomposition agent and the degree of decomposition depended on the culture conditions provided by the medium. For stable and mature biofilms that have been formed, the coupling of norspermidine and D-tyrosine could reduce the content of EPS in microbial aggregates, change the structure of EPS, and promote the dispersion and collapse of biofilms effectively (Si et al. 2014). D-amino acid could also inhibit the initial adhesion of bacteria by reducing hydrogen bonding, changing surface potential and hydrophilicity (Wang et al. 2018).

The influence of D-amino acid on biofilm formation of mixed microorganisms has been studied. Xu & Yu (2011) proposed using D-tyrosine to inhibit the biofilm formation of mixed microorganisms on the surface of hydrophilic nylon membrane. With the effect of D-tyrosine, the yield of AI-2 and EPS decreased significantly. The formation of biofilm on the surface of hydrophilic nylon membrane was inhibited effectively, and the biofilm formed on the surface of the nylon membrane could be dispersed and collapsed. Xu & Liu (2011) found that D-tyrosine had no significant effects on microbial growth, adenosine triphosphate (ATP) and substrate utilization in mixed microorganisms. It had significant effects on carbohydrates and protein contents in EPS, AI-2 and eDNA. The exposure times of D-amino acids in these studies were 1–4 hours.

Currently, there are few studies about the effect of different kinds of D-amino acids on initial adhesion and biofilm formation of mixed microorganisms. In wastewater treatment, it needs long-term cultivation of mixed microorganisms to degrade wastewater, and some treatment processes need to inhibit the formation of biofilm. It's interesting to know that biofilm formation of mixed microorganisms occurs with long D-Amino Acids exposure time. Adhesion and desorption, which are the initial process of biofilm formation, are also crucial steps in biofilm formation. Therefore, this study focused on the effects of different types of D-amino acids on the initial attachment of mixed microorganisms with the exposure time of 24 h. The biofilm formation of mixed microorganisms with different D-amino acids was discussed. Moreover, EPS contents and characteristics were also analyzed to understand the effect mechanism better. As a preliminary investigation on the effect of different D-amino acids on mixed microorganisms, this study provides new ideas for biofilm inhabitation in wastewater treatment.

In this study, activated sludge, which contained many kinds of bacteria and protozoa, was used as mixed microorganism. The activated sludge came from a local sewage treatment plant. First of all, the activated sludge was filtered by a 1 mm screen to remove impurities. Then the activated sludge was planted and acclimated for more than 1 month with simulated wastewater: 252.00 mg/L glucose, 252.00 mg/L soluble starch, 90.50 mg/L urea, 361.80 mg/L sodium bicarbonate, 32.70 mg/L potassium dihydrogen phosphate, 60.40 mg/L magnesium sulfate, 14.90 mg/L calcium carbonate. The trace elements (0.13 mg/L zinc chloride, 17.48 mg/L ferrous sulfate, 0.13 mg/L manganese sulfate) were added once a week.

In order to study the influence of D-amino acids on the biofilm formation of mixed microorganisms, the activated sludge was diluted to a MLSS of 1,200–1,300 mg/L according to the sludge concentration, and placed into 500 mL beakers. 50 μΜ D-amino acid of different kinds were added accordingly. The aeration efficiency was controlled at 60 mL/min, and the culture time was 24 h.

The adhesion substance was a Polyvinylidene Fluoride (PVDF) flat membrane with a diameter of 47 mm and a pore size of 0.22 μm. Before use, the PVDF membrane was soaked in 75% ethanol solution for 30 min, and rinsed with distilled water.

Crystalline violet (CV) staining (Silva et al. 2013) was used to determine the effect of D-amino acids on biofilm formation of mixed microorganisms. Diluted mixed microorganisms were cultured in a 96-well plate for 24 h (Corning, USA). The 96-well plate was dyed with 0.1% CV for 30 min, washed with phosphate buffered saline (PBS) 3 times, and decolorized with 150 μL 95% ethanol. The 96-well plate was measured at 590 nm with a microplate reader analyzer (Biotek, Winooski, VT, United States). Each D-amino acid was set 8 parallel samples, and the average value was taken.

EPS was extracted by thermal extraction (Xiao et al. 2021). The mixed microorganisms cultured for 24 h obtained in 2.1 was centrifuged at 8,991 × g for 5 min, retained for precipitation, re-suspended in the same volume of distilled water, and heated at 80 °C for 30 min. Then the mixed solution was centrifuged at 8,991 × g for 5 min again, filtered by 0.45 μm membrane and the filtrate was EPS. The carbohydrate content was determined by the phenol-sulfuric acid method (Zavřel et al. 2018). The protein content was determined by the Folin method (Berker et al. 2013). Three parallel samples were taken for carbohydrate and protein detection. Excitation-emission matrix spectra (EEMs) (F96S, Lengguang, China) were detected with excitation wavelength (Ex) of 220–450 nm and emission wavelength (Em) of 220–650 nm.

Student's t-tests were employed for analyzing the significance of results at the level of P < 0.05 using SPSS software.

PVDF, a kind of membrane material, is widely used in water and wastewater treatment. Adhesion and desorption of mixed microorganisms on PVDF with and without D-amino acid were investigated (Figure 1). In this study, C₀ was measured as 0.496.

As shown in Figure 1(a), the adhesion efficiency of the control group was 24.33 ± 0.92%. The adhesion efficiencies of D-tyrosine, D-leucine, D-aspartic acid and D-tryptophan groups were 7.23 ± 0.74%, 22.15 ± 1.19%, 19.14 ± 1.82% and 17.25 ± 1.14%, respectively. The addition of D-amino acid was effective in inhibiting the adhesion of mixed microorganisms on PVDF. D-tyrosine had the greatest effect. Yu et al. (2016) found that D-tyrosine caused the attachment efficiencies of E. coli, B. subtilis and P. aeruginosa to decrease significantly. Xu and Liu (Xu & Liu 2011) found that D-tyrosine inhibited microbial adhesion to glass and polypropylene. The results obtained in this study showed that D-amino acids could inhibit the adhesion of mixed microorganisms to PVDF membranes effectively.

As shown in Figure 1(b), the desorption efficiency of the control group was 41.19 ± 2.88% (Figure 1(b)). The desorption efficiencies of D-tyrosine, D-tryptophan, D-aspartic acid and D-leucine groups were 88.23 ± 1.80%, 74.47 ± 2.49%, 58.88 ± 1.99% and 52.42 ± 2.64%, respectively. It showed that all D-amino acids had promoting effects on the desorption of mixed microorganisms on the PVDF membrane, of which D-tyrosine had the best effect. The higher desorption efficiency indicated more reversible adhesion. The weaker adhesion suggested attached microorganisms fall off more easily with the effect of external force.

D-amino acids inhibited the attachment of mixed microorganisms to the surface of PVDF membrane and reduced the firmness of adhesion. Among all amino acids, D-tyrosine has the greatest effect on adhesion and desorption. Adhesion, which is the first step of biofilm formation, plays an important role in biofilm formation. The possibility of biofilm formation is reduced directly by the reduction of mixed microorganisms' adhesion efficiency.

Biofilm formed by mixed microorganisms was stained by CV method. Then the A₅₉₀ value was measured with a microplate analyzer (Figure 2).

As shown in Figure 2, the value of A₅₉₀ in the control group was 2.35 ± 0.02. The values of A₅₉₀ in the D-tryptophan, D-aspartic acid, D-leucine and D-tyrosine groups were 1.94 ± 0.04, 2.00 ± 0.05, 2.27 ± 0.03 and 1.78 ± 0.06, respectively. Compared with the control group, the addition of D-amino acid generally inhibited the biofilm biomass. The value of A₅₉₀ in the D-tyrosine group was 24.37% lower than that of control group, indicating that D-tyrosine had the best inhibitory effect on biofilm biomass of mixed microorganisms. Yu et al. (Yu et al. 2016) studied the effect of D-Tyrosine on biofilm formation of P. aeruginosa and B. subtilis. Both high (5–200 μM) and low (0.005–0.5 μM) concentrations of D-Tyrosine could significantly inhibit the biofilm formation of P. aeruginosa and B. subtilis. The results of this study showed that 50 μM D-amino acids inhibited the biofilm formation of mixed microorganisms, which was consistent with previous study. More than 90% of biofilms are EPS (Maruzani et al. 2019; Felz et al. 2020). Therefore, it is necessary to detect the EPS of mixed microorganisms to analyze the reasons of biofilm inhibition.

Carbohydrates and protein contents in mixed microorganisms EPS with and without D-amino acid were measured (Figure 3). The three-dimensional fluorescence characteristics of EPS were investigated (Figure 4).

Figure 3(a) showed that the protein content in control group was 38.71 ± 1.24 mg/g. The protein contents in D-tryptophan, D-aspartic acid, D-leucine and D-tyrosine groups decreased by 8.21%, 7.65%, 3.51% and 11.31%, respectively. As shown in Figure 3(b), carbohydrates content in the control group was 30.27 ± 1.03 mg/g. The carbohydrates contents in D-tyrosine, D-tryptophan, D-aspartic acid and D-leucine groups decreased by 29.53%, 21.44%, 14.60% and 10.54%, respectively. All the D-amino acids inhibited the secretion of carbohydrates and protein in mixed microorganisms EPS. D-tyrosine showed the best inhibition effect.

The addition of D-amino acids inhibited the production of carbohydrates and protein in EPS. This may be the reason why D-amino acids reduced the adhesion of mixed microorganisms on PVDF membrane, promoted its desorption, and reduced the biofilm biomass. D-tyrosine had the strongest inhibitory effect on the production of carbohydrates and protein in EPS, and showed the best inhibitory effect on biofilm formation. The properties of EPS, EEMs were further monitored.

EPS of mixed microorganisms was characterized by three-dimensional fluorescence spectra (Figure 4 and Table 1).

There were three fluorescence peaks in EPS (Figure 4 and Table 1), which were tyrosine-like protein (Ex/Em): 280/310–320 nm, tryptophan-like protein (Ex/Em: 285/340–360 nm) and humic-like acids (Ex/Em: 340–360/440–460 nm), respectively. Tyrosine-like protein fluorescence intensity of control group was 340.9, while tyrosine-like protein fluorescence intensity in other three groups was lower than that in the control group. Only tyrosine-like protein fluorescence intensity in D-tyrosine group was higher than that in the control group, with a fluorescence value of 417.8. One reason might be that part of the D-tyrosine was not utilized. Another reason might be that the addition of D-tyrosine promoted the synthesis of tyrosine-like protein. Considering the results obtained in Figure 3(a) and the EEMs of pure D-tyrosine solution (Fig. S1), it's more likely the first reason. The tryptophan-like protein fluorescence intensity in the control group was 496.2, while that in the other groups was lower than that in the control group. Only tryptophan-like protein fluorescence intensity in the D-tryptophan group was higher than that in the control group, with a fluorescence value of 559.5. The fluorescence intensity of humic-like acid in the control group was 274.6, and fluorescence intensity in the other groups was lower than that in control group.

Compared with the control group, all tyrosine-like protein in D-amino acid groups showed blue shift. All tryptophan-like protein and humic-like acid in D-amino acid groups showed red shift. The blue shift was caused by the decrease of aromatic rings of organic compounds, molecular weight, and the polar groups such as carboxyl group, amino group and hydroxyl group (Makarska-Bialokoz 2018). The red shift was caused by the increase of polar groups (Hudson et al. 2010). These results suggested that the addition of amino acids decreased the aromatic rings of organic compounds, molecular weight, and the polar groups in tyrosine-like proteins. It increased polar groups in tryptophan-like protein and humic-like acid. That is to say, the structural properties and fluorescence intensities of the tyrosine-like proteins, tryptophan-like protein and humic-like acid might be changed by D-amino acids, which might be one reason for the decrease of biofilm biomass and adhesion efficiency, and the increase of desorption efficiency.

The addition of D-amino acids reduced the adhesion efficiency of mixed microorganisms on the PVDF membrane, and increased the desorption efficiency, indicating the possibility that biofilm formation is reduced directly. The biofilm biomass of mixed microorganisms decreased with the addition of D-amino acids. Moreover, D-amino acids inhibited the secretion of carbohydrates and protein in mixed microorganisms EPS. The aromatic rings of organic compounds, molecular weight, and the polar groups in tyrosine-like proteins were decreased, and the polar groups in tryptophan-like protein and humic-like acid increased with the effect of D-amino acids.

D-amino acids can provide a potential low-cost, non-toxic and non-biological sterilization method to inhibit biofilm formation. In this paper, the inhibition of D-amino acid on mixed microorganisms biofilm was preliminarily confirmed by batch experiment. Further studies should be carried out to investigate the biofilm inhibition of D-amino acids on continuous-running reactors (such as MBRs) and the effect on the operation of the reactors, so as to promote the application of D-amino acids in wastewater treatment.

This study was supported by Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. QA202136) and the Youth Innovation Talent Support Scheme of Harbin University of Commerce (2020CX04).

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