Bacterial strain for bast fiber crops degumming and its bio-degumming technique

The research and development of bio-degumming technology is under a slow progress due to the shortage of proper efficient bacterial strains and processes. A degumming bacterial strain—Pectobacterium wasabiae (PW)—with broad-spectrum degumming abilities was screened out in this study. After the fermentation for 12 h, the residual gum contents of kenaf bast, ramie bast, hemp bast, flax bast, and Apocynum venetum bast were all lower than 15%. This bacterial strain could realize the simultaneous extracellular secretion of pectinase, mannase, and xylanase with the maximum enzyme activity levels of 130.25, 157.58, and 115.24 U/mL, respectively. The optimal degumming conditions of this bacterial strain were as follows: degumming time of 12 h, bath ratio of 1:10, temperature of 33 °C, and inoculum size of 2%. After the bio-degumming through this bacterial strain, the COD in wastewater was below 4000 mg/L, which was over 60% lower than that in boiling-off wastewater generated by chemical degumming. This technology achieves higher efficiency, higher quality, and lower pollution.


Introduction
The improvement in human living quality and the shortage of petroleum and forest resources have encouraged various countries in the world to seek for new-type natural plant fiber resources. Cellulose is a renewable natural polymer resource that is rich in nature, and it is a cheap and inexhaustible resource. Among all kinds of plant fiber resources, bast fiber plants such as ramie, kenaf, and industrial hemp have fast growth, high yield, strong adaptability, and environmental protection. Bast fibers are of irreplaceable important development values by virtue of antibiosis, insect prevention, breathable moisture absorption, and natural degradation [3,23,27].
The chemical degumming method centering on soda cooking, which is commonly used at home (China) and abroad, is a process that aims to acquire cellulosic fiber satisfying the follow-up processing requirements by catalyzing the degradation of bast non-cellulosic substances under acidic and high-temperature conditions with some after treatment measures. This process not only seriously pollutes the environment and damages the fibrous quality but also has high processing cost; these limitations restrict its industrial development [13,17]. Bio-degumming is a process that centers on catalyzing the degradation of noncellulosic substances by biocatalysts (enzymes) and aims to obtain the cellulosic fiber satisfying the follow-up processing requirements. Bio-degumming is a cyclic action process of "enzyme producing strains-enzyme degumming-gum culturing strain", and it is a multienzyme collaborative catalytic system in a living organism in essence. The bast fiber crop bio-degumming technology, which has low energy consumption, low pollution, low cost, and high quality, can overcome the drawbacks of the conventional degumming method; it is also the development direction of the bast fiber crop processing industry [5,13,16].
Bast non-cellulosic substances have extremely complicated composition and structure, and the non-cellulosic 1 3 degradation can be realized only through the collaborative action of key enzymes such as pectinase [36,37], mannanase [26], xylanase [2], and lignin-degrading enzyme [7,28]. The bio-degumming technology has been studied for over 50 years at home and abroad, but large-scale productivity has not been formed yet. The primary reason is that no suitable bio-degumming strains have been bred, and this situation is the bottleneck of bast fiber crop bio-degumming technology. At present, very few highefficiency bast fiber crop degumming strains have been reported [1,9,11,12].
A plant putrefying bacterial strain-Pectobacterium wasabiae (PW) (preservation number: CGMCC 14601), which could efficiently degrade bast fiber crops colloids to extract fibers, was reported in this study. This bacterial strain is characterized by a complete enzyme system, high enzymatic activity, fast reproduction, low residual gum content after degumming, and no production of cellulase. It shows favorable degumming effects on ramie, kenaf, and industrial hemp. Moreover, a set of optimized bio-degumming processing technology has been formed and put under pilot-scale test in the enterprise for 1 month and has a good industrial prospect.

Raw materials
Ramie bast: purchased from Yuanjiang City, Hunan Province, China. The ramie bast was dried and shell-less without mildew.

Bacterial strain, classification, and determination
The bacterial strain PW was screened out by the research group.
The morphological and physiological-biochemical characteristics of this bacterial strain were analyzed in accordance with Bergey's Manual of Determinative Bacteriology (9th edition). The 16S rRNA gene of strain PW was amplified with the universal primer pairs 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492 R (5′-TAC GGC TAC CTT GTT ACG ACT-3′) and sequenced by Shanghai Sangon Biotech Company. 16S rRNA genes were used to construct the phylogenetic tree used MEGA by the minimum-evolution method and the neighbor-joining method at the parameter of 10 Program bootstrap values based on 1,000 replications.

Optimization of bio-degumming conditions
Mildew-free kenaf bast was collected and exposed to the sun for 1-2 days. The head and tail parts were clipped by 20

Comparative degumming test
The comparative degumming tests of ramie, kenaf, and industrial hemp were conducted using the chemical degumming method, water retting degumming method, and optimized bio-degumming process. The technical routes were as follows: (1) Technical route of bio-degumming: Bast fiber crop → pretreatment (Drying and removing the dust) → inoculation (The bacteria liquid was added into the raw material without sterilizing according to a certain proportion) → soaking and fermentation (Raw materials were soaked in water with bacteria liquit) → inactivation (NaOH mass concentration: 0.5 g/L) → washing → dewatering → drying → bast fiber.
The residual gum content was detected by referencing the quantitative analysis method of ramie chemical components [15].
(2) COD detection: The COD detection in the fermentation broth after degumming was conducted via COD detector ET99718 (Lovibond ® Group, Germany) by following the specifications.

Enzymatic activity in the degumming process
The enzymatic activity was defined as the enzyme dosage needed to degrade 1 μmol of substrate per min. The dinitrosalicylic acid (DNS) method was used to detect the enzymatic activity of the bacterial strain [8,25].

Analysis of monosaccharides formed during the bio-degumming process
From 0 h when the bast fiber crop was inoculated and oscillated until 20 h when the degumming was completed, the fermentation broth sample was collected every other 4 h and separated using 0.2 μm film coating (Vivaflow 200). The filtered solution was used to detect the monosaccharide content. The monosaccharide components generated after the bast fiber crop hydrolysis were systematically analyzed through 1-phenyl-3-methyl-5-pyrazolone precolumn derivatization high-performance liquid chromatography (HPLC) method. After the bast fiber was hydrolyzed using trifluoroacetic acid, C 18 chromatographic analysis column and UV detector were used to detect the monosaccharides in the hydrolysate and the components and contents of their derivatives [14,32].

Microscopic analysis of plant fibers after degumming with P. wasabiae
After degumming for 8 h, slices were made and observed under an electron microscope JEOL-1230 (JEOL Ltd., Japan) through thousand-fold amplification. The fibers were observed under KH-2700 3D video microscope by 100-fold amplification.

Morphological and physiological-biochemical characteristics of bacterial strain PW
After activated-state bacterial solution grew in the broth medium for 8 days, the bacterial strain was rod-shaped without spores, and the average size was 0.6 μm × 1.5 μm (Fig. 1a). After growing on the broth plate for 20 h, PW became round, white, and humid with micro-bulges and transparent edges (Fig. 1b). The P. wasabiae is Gramnegative, did not hydrolyze starch and did not produce urease. Tested positive for casein, catalase, indole and nitrate reduction.
The 16S rRNA gene sequence of PW was a nucleotide sequence with the full length of 1375 bp, and it was submitted onto GenBank to acquire accession number GU097456. On the basis of the 16S rRNA gene sequence of PW, the minimum-evolution method was used to construct the phylogeny tree diagram (Fig. 2). Meanwhile, another phylogeny tree diagram was established through the neighbor-joining method, and the results were consistent in essence. The results showed that the bacterial strain PW belonged to Pectobacterium sp. and had a high similarity to 16S rRNA gene sequence of Pectobacterium wasabiae (NR 026047). By combining the physiological and biochemical characteristics, PW was identified as Pectobacterium wasabiae, which was preserved in China General Microbiological Culture Collection Center (CGMCC), with the preservation number of CGMCC No. 14601.

Bio-degumming fermentation parameters
After the treatment of kenaf bast under different fermentation conditions, the test results of raw material weight loss ratio are shown in Table 2, and the analysis of variance (ANOVA) and multiple comparisons results are listed in Tables 3 and  4. According to the ANOVA results, the sample:solution (m:v) and time significantly influenced kenaf weight loss ratio, which was insignificantly affected by the inoculum size or temperature. From the multiple comparisons of the parameters in Table 4, the influencing degrees were sorted in a descending order as time, sample:solution (m:v), temperature and inoculum size. Their minimum influencing degrees on kenaf weight loss ratio were the optimal levels. Thus, the combinational optimal levels were obtained as follows: time of 12 h, sample:solution (m:v) of 1:10, temperature of 33 °C, and inoculum size of 2%.
In contrast to the traditional water retting degumming and rain and dew degumming, one of the important features of bio-degumming is short degumming time. The traditional water retting degumming of ramie and kenaf and rain and dew degumming of hemp and flax generally take 7-30 days, and they are greatly affected by the external environmental and climatic conditions [18,35]. However, the bio-degumming can complete the degumming process within 1 day, and the bacterial strain can finish the degumming of kenaf bast within 12 h.

Enzymatic activity in fermentation broth (unit: U/ mL)
Pectinase, mannanase, and xylanase could be detected in the whole degumming process; the pectinase activity during the process was increased from 12.52 U/mL at 0 h to 130.25 U/ mL at 15 h, mannanase activity was from 14.82 U/mL to 157.58 U/mL, and xylanase activity was from 11.74 U/mL to 115.24 U/mL; the growth rates were high within 0-9 h and slowed down after 12 h, but they still kept growing (Table 5).

Analysis of monosaccharides formed during the degumming process of kenaf by fermentation with P. wasabiae
In the supernatant of kenaf bio-degumming fermentation broth, the detected hydrolysates included mannose, rhamnose, galacturonic acid, glucose, galactose, and xylose, while glucuronic acid was not detected. Figure 3 illustrates the monosaccharides detected by liquid chromatography in the fermentation broth of kenaf bast with Pectobacterium wasabiae and Table 6 shows the monosaccharide contents during 15 h of fermentation. As the fermentation time progressed, the content of galacturonic acid showed a sustainable and slow growth, while the contents of other monosaccharides were first increased and then reduced. Among them, the contents of mannose, xylose, rhamnose, glucose, and galactose all reached the maximum values at 9 h. The initial concentration of glucose was high, which might be correlated with the glucose components contained in the culture medium. The content of galacturonic acid, which was the pectin degradation product, was low and continuously increased. The reason might be that pectinase was a key enzyme with timely and radical microbial degradation and great demand. Its residual content was also not high in the supernatant of the fermentation broth. This finding proved that Pectobacterium wasabiae PW continuously released pectinase, mannanase, and xylanase in the kenaf bast degumming process.

Microscopic analysis of kenaf bast fibers after bio-degumming
The kenaf fibers generated by bio-degumming (Fig. 4a) and chemical degumming (Fig. 4b) were observed under 3D video microscope by 100-fold amplification. As observed, the microfibers of kenaf generated by bio-degumming were intersected and warped, while those generated by chemical degumming were nearly straight. The enzymes secreted by microorganisms selectively degraded colloids and reserved the inherent fiber morphologies and structures. In the chemical degumming process, strong alkali destructed the chemical and hydrogen bonds with Description The results proved that Pectobacterium wasabiae PW continuously released pectinase, mannase, and xylanase in the kenaf bast degumming process weak structural force while hydrolyzing the colloids. Thus, the fibrous structure tended to be stable, and the excessive degradation decomposed bundle fibers into short single fibers. As a result, the mass of kenaf bundle fibers was reduced. The observation results under the electron microscope showed that, after 4 h degumming, the microorganisms infected the colloids by a large area and local degradation occurred (Fig. 5a). After 10 h, the single fibers were under an obvious discrete state, the fiber surface was smooth, and most colloids already peeled cellulose off (Fig. 5b).

Comparison of kenaf degumming effects
The residual gum content of kenaf bast, fiber strength, and COD in the fermentation broth after oscillating fermentation of PW under optimized conditions for 12 h are shown in Table 7. The kenaf sample experiencing 15-day water retting degumming was collected for the control. Under sample:solution (1:10, m:v), temperature of 33 °C, and inoculum size of 2%, the bast fiber crops were washed after 12 h PW degumming. The residual gum content was 11.38%, which was 12.13% higher than that in the traditional water retting degumming. The raw material weight loss ratio was 4.51% lower than that in the traditional water retting degumming. However, the fiber strength in bio-degumming was 28.62% higher than that in traditional water retting degumming, and the COD in bio-degumming was 15.0% lower than that in traditional water retting degumming. The pectin removal rate of bio-degumming was 31.11% higher than that of water retting degumming, but the hemicellulose and lignin removal rates were 21.43% and 3.24% lower than those of traditional water retting degumming, respectively (Table 8).

Wide spectrality of PW degumming function
PW is of good wide spectrality in the aspect of bast fiber crop degumming. After the fermentation for 12 h, the residual gum contents of kenaf bast, ramie bast, hemp bast, flax bast, and Apocynum venetum bast were all lower than 15%, and the raw material weight loss rate was 28.54-34.70%. The residual gum content of Apocynum venetum was the minimum (12.57%) and that of flax was the maximum (15.07%). PW could complete the degumming of ramie bast, kenaf bast, hemp bast, flax bast, and Apocynum venetum bast. Therefore, it had excellent degumming wide spectrality. The fiber counts (The length of a fiber or yarn of a certain quality, m/g) were greatly different. Specifically, those of kenaf and Apocynum venetum were 272 m/g and 1002, respectively. The COD ranged from 2945 mg/L to 3582 mg/L. In particular, the COD of ramie was the highest and that of kenaf was the lowest. In the traditional chemical soda cooking degumming process, the COD in boiling wastewater reached as high as 10,000 mg/L (research progress of wastewater treatment technology of ramie chemical degumming), which was much higher than that of bio-degumming wastewater. Among the current reported degumming strains, few can simultaneously realize the degumming of ramie, kenaf, hemp, and flax, but PW has favorable wide spectrality (Table 9).

Conclusions
A degumming bacterial strain Pectobacterium wasabiae PW with broad-spectrum degumming abilities was screened out in this study. After the fermentation for 12 h, the residual gum contents were lower than 15%. This bacterial strain could realize the synchronous extracellular secretion of pectinase, mannanase, and xylanase with the maximum activity levels of 130.25, 157.58, and 115.24 U/mL, respectively. The optimal degumming conditions of this bacterial strain were as follows: time of 12 h, sample:solution (1:10, m:v), temperature of 33 °C, and inoculum size of 2%. The COD in bio-degumming wastewater was below 4000 mg/L, which was over 60% lower than that in boiling wastewater of chemical degumming.