臺灣博碩士論文加值系統

微生物附著問題在造紙業中造成產品劣化與巨大的金錢損失。有鑑於此，我們測試四級銨鹽(QACs)、isothiazolone以及2,2-dibromo-3-nitrilopropionamide (DBNPA)等三種紙機常用殺菌劑對於由文化用紙紙機系統所分離的菌株的最低抑菌濃度(MIC)以及最低殺菌濃度(MBC)。本研究使用不同的附著表面—聚苯乙烯培養皿(PS)與不�袗�片(SS)，以及不同組成分的培養液—Mueller-Hinton培養液(MHB)、基礎培養液(BM)、與模擬白水(SWW)，來探討微生物附著的諸變因。

殺菌劑與額外的葡萄糖首先一起被加入培養液中，再與菌液混合，最後與附著表面接觸。生物膜形成過程中最初兩小時的現象被以結晶紫染色法觀測著。

葡萄糖在模擬白水中促進了生物膜的形成：較高濃度的葡萄糖(50-200 mg/L) 對應著較高的生長速率。基礎培養液較模擬白水更能支持生物膜快速生長。在生物膜生長控制方面，則需要較游離態細菌的MIC值高出2-25倍劑量的殺菌劑。在這三種殺菌劑中，四級銨鹽效果最佳，因其使用劑量較輕 (25-75 mg/L)、目標生物膜的比生長速率(specific growth rate)較慢。相較之下，isothiazolone的藥效最低、所需劑量甚高 (625-5000 mg/L)。Morganella morganii並未被觀測到生物膜的生成；在相同的條件下，其他兩株細菌以及紙機雜菌都能產生生物膜。Pseudomonas aeruginosa形成生物膜的速率最高，同時對殺菌劑的感受性最低。另外一株假單胞屬細菌—Pseudomonas putida則產生較少量的生物膜，且容易以DBNPA控制 (1-75 mg/L)。不�袗�片較聚苯乙烯培養皿不易被細菌附著。

Biofouling is responsible for product deterioration and huge financial losses in paper industry. Therefore, we investigated the efficacy of three commonly used disinfectants in paper industry, quaternary ammonium compounds (QACs), isothiazolone, and 2,2-dibromo-3-nitrilopropionamide (DBNPA) by testing their minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against the bacterial culture isolated in our previous work from a fine papermachine system. To elucidate the variables in biofouling in laboratory scale, two adherent surfaces--polystyrene Petri dish (PS) and stainless steel coupons (SS) and three different compositions of media--Mueller-Hinton broth (MHB), basal medium (BM), and simulated white water (SWW) were used.
Disinfectants along with extra glucose were first added to the medium and then mixed with the bacterial suspension before it was allowed to contact with the adherent surface. The initial events within the first two hours of biofilm formation were monitored by using crystal-violet staining method.
Glucose promoted the formation of biofilm on surfaces in simulated white water; higher amounts of additional glucose (50-200 mg/L) responded to higher specific growth rates. Basal medium supported biofilm to grow faster than simulated white water. In biofilm control aspect, it needed high dosage of disinfectants that were 2-25 times higher than MIC value for the same bacterium in planktonic state. Among all the three disinfectants, QAC was the most effective by lighter dosage (25-75 mg/L) and lower specific growth rate of target biofilm. In contrast, isothiazolone was the least effective since dosage as high as 625-5000 mg/L was required. No detectable biofilm was formed by Morganella morganii while the other two strains as well as the mixed culture produced biofilm in the same condition. Pseudomonas aeruginosa produced biofilm at the fastest rate and was least susceptible to disinfectants as well. The other Pseudomonas species--P. putida was otherwise producing less biofilm and could be easily controlled by DBNPA (1-75 mg/L). The stainless steel coupons were less attachable by bacteria than the polystyrene Petri dish.

Table of Contents
謝誌……………………………………………………..……i
摘要…………………………………………………..….... ..ii
Abstract…………………………..……………………….…iii
Table of Contents………………….………………………...iv
Tables.…………………………….………………………...viii
Figures.…………………………….……………………..….ix

Chapter 1 Introduction
1.1 Microbial Communities of Papermachines……………………….1-1
1.2 Biofilm and Papermaking…………………………………………1-2
1.2.1 Definition of biofilm……………………..………………........………….1-2
1.2.2 Biofilm formation in a papermachine system………………………….....1-3
1.2.2.1 Conditioning layer……………………………………………………1-3
1.2.2.2 Bacterial attachment………………….………………………………1-4
1.2.2.3 Biofilm formation and EPS production………………………………1-5
1.2.2.4 Biofilm maturation……………………………………………………1-5
1.2.2.5 Detachment……………………………………………………………1-6
1.2.3 Implications of biofilms in paper industry……………….………………...1-7
1.3 Biofilm and Biocide………………………………….…………….1-8
1.3.1 Biocide prevalent in paper industry……………………….…………….....1-8
1.3.1.1 Quaternary ammonium compounds…………………………...………1-9
1.3.1.1.1 Structure of QACs……………………………….………………..1-9
1.3.1.1.2 Cationic surface-active agents……………………………………1-11
1.3.1.1.3 Structure-activity relationships…………………………………..1-12
1.3.1.1.4 Mode of action………………………...........................................1-13
1.3.1.2 Isothiazolone………………………………………………...……......1-16
1.3.1.3 2,2-dibromo-3-nitrilopropionamide………………….….…………..1-18
1.3.1.3.1 Properties……………………………………………………….1-18
1.3.1.3.2 Applications…………………………………………………….1-19
1.3.1.3.3 Mode of action………………………………………………….1-20
1.3.2 Antimicrobial control of bacterial biofilm……………………………….1-20
1.3.2.1 Approaches to biofilm control……………………………………….1-20
1.3.2.1.1 Mechanical removal…………………………………………….1-20
1.3.2.1.2 Kill………………………………………………………………1-20
1.3.2.1.3 Inhibit growth………………………….......................................1-23
1.3.2.1.4 Reduce attachment………………………....................................1-23
1.3.2.1.5 Promote detachment…………………………………………….1-24
1.3.2.2 Transport limitation of biocide into biofilm…………………………1-25
1.3.2.2.1 Depletion of antimicrobial agents in the bulk fluid……………..1-25
1.3.2.2.2 Physical barrier………………………………………………….1-25
1.3.2.2.3 Phenotypic adaptation…………………………….……….…….1-27
1.4 Biofilm and Nutrient………………………………………...........1-28
1.4.1 Nutrient analysis in a papermachine system…….......................................1-28
1.4.2 Nutrient limitation and biofilm formation………………….……………..1-29
1.4.3 Reduced growth rate and biocide resistance………………………….......1-30

1.5 Objective.………………………………………………….…........1-31

Chapter 2 Materials and Methods

2.1 Flow Chart of Experimental Design……….………………............2-1 2.2 Materials…………………………………….……………..............2-2
2.2.1 Bacterial strains……………………………………………………………2-2
2.2.2 Disinfectants……………………………………………………………….2-5
2.2.3 Media………………………………………………………………………2-6
2.2.4 Surfaces……………………………………………………………….……2-8
2.3 Methods……………………………………………….………........2-9
2.3.1 Disinfectant tests………………………………………...............................2-9
2.3.1.1 Susceptibility test………………………………..................................2-10
2.3.1.2 Bactericidal test…………………………………..…………...............2-10
2.3.2 Biofilm formation assay…………………………………………………...2-11
2.3.2.1 Biofilm formation in basal medium……………………………..........2-12
2.3.2.2 Biofilm formation in enhanced simulated white water……………….2-13

Chapter 3 Results and Discussion

3.1 Disinfectant Tests……………………………………….………......3-1
3.1.1 Morganella morganii.....................................................................................3-1
3.1.2 Pseudomonas putida………………………………………….…………….3-7
3.1.3 The mixed culture……………………………………...………………….3-14
3.1.4 Pseudomonas aeruginosa…………………..………….………………….3-22
3.1.5 Comparisons between the bacteria…………………………….…………..3-28
3.2 Biofilm Formation in Basal Medium………………………….......3-31
3.2.1 Pseudomonas putida………………………………………………………3-31
3.2.2 The mixed culture…………………………………………………………3-34
3.3.3 Pseudomonas aeruginosa………………………………..………………..3-36
3.3 Biofilm Formation in Simulated White Water………………..…...3-38
3.3.1 Pseudomonas putida……………………………………………………...3-38
3.3.2 The mixed culture…………………………………………………………3-43
3.3.3 Pseudomonas aeruginosa…………………………………………………3-47




Chapter 4 Conclusions

4.1 Efficacies of the Disinfectants………………….…………………..4-1
4.2 Influences of Nutrient……………………………………...……….4-1
4.3 Behaviors of the Bacteria……………………………….…………..4-2
4.4 Bacterial Biofilm and Its Susceptibility to Biocides…….………….4-3

Appendix： Relationships between Numbers of Attached Bacteria and the Intensity of Staining
A1 Pseudomonas putida…………………..……………….………...A-1
A2 The Mixed Culture…………………….……………….…….…..A-2
A3 Pseudomonas aeruginosa………………..……………….……...A-2

References……………………………………………….…....R-1
















Tables

Table 1 Selected biofilm resistance factors to antimicrobial killing………….1-22
Table 2 MIC and MBC values for the 3 disinfectants against
Morganella morganii in different media……………………………….3-1
Table 3 MIC and MBC values for the 3 disinfectants against
Pseudomonas putida in different media………………………………..3-7
Table 4 MIC and MBC values for the 3 disinfectants against the mixed
culture in different media……………………………………………..3-14
Table 5 MIC and MBC values for the 3 disinfectants against
Pseudomonas aeruginosa in different media…………………………3-22
Table 6 MIC and MBC values for QAC in different media..............................3-28
Table 7 MIC and MBC values for DBNPA in different media.........................3-29
Table 8 MIC and MBC values for isothiazolone in different media.................3-29
















Figures

Figure 1 General structure of QACs…………………………………………..1-9
Figure 2 Structures of cetrimide, domiphen bromide, bezalkonium chloride
and cetylpyridinium chloride………………………………………..1-10
Figure 3 Rate of leakage of intercellular materials from bacteria exposed to
a membrane-active agent………………………………………........1-14
Figure 4 Chemical structures of isothiazolones……………………………....1-16
Figure 5 Structure of 2,2-dibromo-3-nitrilopropionamide…………………....1-18
Figure 6 Flow chart of experimental design…………………………………...2-1
Figure 7 Effects of QAC on Morganella morganii in different media………...3-2
Figure 8 Effects of DBNPA on Morganella morganii in different media……...3-3
Figure 9 Effects of isothiazolone on Morganella morganii in different media..3-4
Figure 10 Comparisons between the efficacy of the 3 disinfectants
against Morganella morganii in basal medium…………………. …..3-5
Figure 11 Comparisons between the efficacy of the 3 disinfectants
against Morganella morganii in Mueller-Hinton broth………………3-6
Figure 12 Effects of QAC on Pseudomonas putida in different media………....3-8
Figure 13 Effects of DBNPA on Pseudomonas putida in different media………3-9
Figure 14 Effects of isothiazolone on Pseudomonas putida in different media.3-10
Figure 15 Comparisons between the efficacy of the 3 disinfectants
against Pseudomonas putida in simulated white water……………..3-11
Figure 16 Comparisons between the efficacy of the 3 disinfectants
against Pseudomonas putida in basal medium……………………...3-12
Figure 17 Comparisons between the efficacy of the 3 disinfectants
against Pseudomonas putida in Mueller-Hinton broth………….…..3-13
Figure 18 Effects of QAC on the mixed culture in different media…………...3-15
Figure 19 Effects of DBNPA on the mixed culture in different media…….…..3-16
Figure 20 Effects of isothiazolone on the mixed culture in different media.......3-17

Figure 21 Comparisons between the efficacy of the 3 disinfectants against
the mixed culture in simulated white water……………………....3-18
Figure 22 Comparisons between the efficacy of the 3 disinfectants against
the mixed culture in basal medium………………………….....…3-19
Figure 23 Comparisons between the efficacy of the 3 disinfectants against
the mixed culture in Mueller-Hinton broth…………………….…3-20
Figure 24 Effects of QAC on Pseudomonas aeruginosa
in different media…………………………………..…………....3-23
Figure 25 Effects of DBNPA on Pseudomonas aeruginosa
in different media……………..…………………..……………..3-24
Figure 26 Effects of isothiazolone on Pseudomonas aeruginosa
in different media………………………..………………………3-25
Figure 27 Comparisons between the efficacy of the 3 disinfectants against
Pseudomonas aeruginosa in basal medium……………………....3-26
Figure 28 Comparisons between the efficacy of the 3 disinfectants against
Pseudomonas aeruginosa in Mueller-Hinton broth……………....3-27
Figure 29 Effects of QAC on the attachment of Pseudomonas putida on
stainless steel coupons in basal medium….……..………………..3-31
Figure 30 Effects of QAC on the attachment of Pseudomonas putida on
polystyrene plate in basal medium…………………………..…..3-32
Figure 31 Effects of surfaces on the formation rate of Pseudomonas putida
in the initial 2 hours…………………….………………………..3-33
Figure 32 Effects of QAC on the attachment of the mixed culture on
stainless steel coupons in basal medium…………………………3-34
Figure 33 Effects of QAC on the attachment of the mixed culture on
the polystyrene plate in basal medium…………………...............3-34
Figure 34 Effects of surfaces on the formation rate of the mixed culture
biofilm in the initial 2 hours………………………………………3-35
Figure 35 Effects of QAC on the attachment of Pseudomonas aeruginosa
on stainless steel coupons in basal medium…………………........3-36

Figure 36 Effects of QAC on the attachment of Pseudomonas aeruginosa
on the polystyrene plate in basal medium………….....................3-36
Figure 37 Effects of surfaces on the formation rate of
Pseudomonas aeruginosa biofilm in the initial 2 hours………....3-37
Figure 38 Effects of QAC and glucose on the formation of
Pseudomonas putida biofilm during 2 to 6 hours
on polystyrene plate in simulated white water…………………….3-38
Figure 39 Effects of DBNPA and glucose on the formation of
Pseudomonas putida biofilm during 2 to 6 hours
on polystyrene plate in simulated white water…………………….3-40
Figure 40 Effects of isothiazolone and glucose on the formation of
Pseudomonas putida biofilm during 2 to 6 hours
on polystyrene plate in simulated white water…………………….3-40
Figure 41 Effects of QAC and glucose on the formation of
Pseudomonas putida biofilm in the initial 2 hours
on stainless steel coupons in simulated white water……………...3-41
Figure 42 Effects of DBNPA and glucose on the formation of
Pseudomonas putida biofilm in the initial 2 hours
on stainless steel coupons in simulated white water……………….3-41
Figure 43 Effects of isothiazolone and glucose on the formation of
Pseudomonas putida biofilm in the initial 2 hours
on stainless steel coupons in simulated white water……………….3-42
Figure 44 Effects of QAC and glucose on the formation of
the mixed culture biofilm during 2 to 6 hours
on polystyrene plate in simulated white water……………………..3-43
Figure 45 Effects of DBNPA and glucose on the formation of
the mixed culture biofilm during 2 to 6 hours
on polystyrene plate in simulated white water……………………..3-44
Figure 46 Effects of isothiazolone and glucose on the formation of
the mixed culture biofilm during 2 to 6 hours
on polystyrene plate in simulated white water……………..………3-44
Figure 47 Effects of QAC and glucose on the formation of
the mixed culture biofilm in the initial 2 hours
on stainless steel coupons in simulated white water……………….3-45
Figure 48 Effects of DBNPA and glucose on the formation of
the mixed culture biofilm in the initial 2 hours
on stainless steel coupons in simulated white water…………….…3-45

Figure 49 Effects of isothiazolone and glucose on the formation of
the mixed culture biofilm in the initial 2 hours
on stainless steel coupons in simulated white water…………….…3-46
Figure 50 Effects of QAC and glucose on the formation of
Pseudomonas aeruginosa biofilm during 2 to 6 hours
on polystyrene plate in simulated white water………………….…3-47
Figure 51 Effects of DBNPA and glucose on the formation of
Pseudomonas aeruginosa biofilm during 2 to 6 hours
on polystyrene plate in simulated white water…………………….3-48
Figure 52 Effects of isothiazolone and glucose on the formation of
Pseudomonas aeruginosa biofilm during 2 to 6 hours
on polystyrene plate in simulated white water…………………….3-48
Figure 53 Effects of QAC and glucose on the formation of
Pseudomonas aeruginosa biofilm in the initial 2 hours
on stainless steel coupons in simulated white water………………3-49
Figure 54 Effects of DBNPA and glucose on the formation of
Pseudomonas aeruginosa biofilm in the initial 2 hours
on stainless steel coupons in simulated white water………………3-49
Figure 55 Effects of isothiazolone and glucose on the formation of
Pseudomonas aeruginosa biofilm in the initial 2 hours
on stainless steel coupons in simulated white water……………….3-50
Figure 56 Relationship between numbers of attached cells and intensity of
staining for Pseudomonas putida.......................................................A-1
Figure 57 Relationship between numbers of attached cells and intensity of
staining for the mixed culture............................................................A-2
Figure 58 Relationship between numbers of attached cells and intensity of
staining for Pseudomonas aeruginosa...............................................A-2

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