Four Decades of Laccase Research for Wastewater Treatment: Insights from Bibliometric Analysis

Increasing trends of environmental pollution and emerging contaminants from anthropogenic activities have urged researchers to develop innovative strategies in wastewater management, including those using the biocatalyst laccase (EC 1.10.3.2). Laccase works effectively against a variety of substrates ranging from phenolic to non-phenolic compounds which only require molecular oxygen to be later reduced to H2O as the final product. In this study, we performed a bibliometric analysis on the metadata of literature acquired through the Scopus database (24 October 2022) with keyword combination “Laccase” AND “Pollutant” OR “Wastewater”. The included publications were filtered based on year of publication (1978–2022), types of articles (original research articles and review articles) and language (English). The metadata was then exported in a CSV (.csv) file and visualized on VosViewer software. A total of 1865 publications were identified, 90.9% of which were original research articles and the remaining 9.1% were review articles. Most of the authors were from China (n = 416; 22.3%) and India (n = 276; 14.79%). In the case of subject area, ‘Environmental Science’ emerged with the highest published documents (n = 1053; 56.46%). The identified papers mostly cover laccase activity in degrading pollutants, and chitosan, which can be exploited for the immobilization. We encourage more research on laccase-assisted wastewater treatment, especially in terms of collaborations among organizations.


Introduction
The challenge to achieve a sustainable planet has become more difficult with the increase of population resulting in increased hazardous contaminant in water environments [1,2]. Moreover, municipal waste and pharmaceutical waste, especially those deriving from the coronavirus disease 2019 (COVID-19) pandemic, have alarmed researchers in environmental management-related fields due to their harmfulness and persistence [3,4]. This environmental threat has affected the innovation on wastewater treatments, including those using renewable and nontoxic oxidative degrading enzyme-laccase [5,6].
The mechanism of laccase in degrading organic pollutants is dependent on redox reactions involving molecular oxygen and the reduction of Cu 2+ to Cu + nucleus of this enzyme. Laccase was first introduced as an unspecific enzyme observed in Rhus vernicifera sap in Japan [7], and its name was only given a decade later following the success of its isolation and purification [8]. Researchers have isolated the enzyme from various plant, fungal, and bacterial species [9][10][11]. Strategies involved in laccase-related research for wastewater treatment include the isolation of laccase from new sources. Trametes versicolor is a popular source of enzyme with high catalytic degrading activity against dying agent contaminants [12]. Other than the fungus, Pleurotus ostreatus [13,14] and marine fungi (such as Trichoderma asperellum, Stemphylium lucomagnoense, and Aspergillus nidulans [15]) have been reported to produce laccase with the enzymatic degradation activity against organic pollutants. The exploration of laccase-producing microbes has been carried out on wastewater as well [16][17][18].
Rapid progress in genetic modifications have allowed researchers to employ these as strategies to obtain more efficacious laccase. The use of metagenomic data collected from tannery wastewater to obtain thermo-halotolerant laccase has been reported [19]. UV light-induced mutation has also been used to isolate higher efficient laccase from P. ostreatus mutant [20]. Mutation involving polymerase chain reaction has been involved in a research protocol to produce laccase from Bacillus licheniformis mutant [21]. Furthermore, innovation in laccase-themed research also includes the enzyme immobilization to promote its stability and reusability, which is significant to reduce the wastewater treatment cost [22][23][24].
Our work aimed to provide the research landscape of laccase-assisted wastewater treatment based on bibliometric analysis. By utilizing the metadata of published studies obtained from a scientific database (i.e., Scopus), a bibliometric analysis could be performed to map the research trends, especially those from newly emerging fields. The occurrence and incidence of environmental pollution have been analysed using bibliometric analysis against different contexts (i.e., mining effluent) [25,26]. In our previous project, we have employed the same analysis to reveal the trend of COVID-19-related polymer research in [27]. Herein, we present a bibliometric analysis on laccase-assisted wastewater treatment which has not been reported previously.

Study Design and Searching Strategy
This study employed the Scopus database for literature search reporting laccase utilization in wastewater treatment. The keywords used in the Scopus database search were as follows: ("Laccase" AND ("Pollutant" OR "Wastewater")) without constrains set in the publication year. The search resulted in 2073 papers, and was then filtered to include only finalized publications (including original articles, review articles, and any types of publication). Only papers written in English were included. Thereafter, we obtained 1865 papers included in this study. The workflow diagram of the literature search performed in this study has been presented in Figure 1. All metadata were downloaded from Scopus database as a CSV (.csv) file. The metadata were visualized using VosViewer 1.6.17. Crosschecking was conducted to avoid potential disambiguation in the exported data.

Data Analysis and Visualization
Microsoft Excel 2016 was used to analyze the selected papers and to export the graphs and tables needed to present the paper type, source journals, top 10 organizations or countries and the most cited papers. Data were double-checked using Biblioshiny (https://www.bibliometrix.org/, accessed on 17 December 2022). Furthermore, VosViewer

Data Analysis and Visualization
Microsoft Excel 2016 was used to analyze the selected papers and to export the graphs and tables needed to present the paper type, source journals, top 10 organizations or countries and the most cited papers. Data were double-checked using Biblioshiny (https: //www.bibliometrix.org/, accessed on 17 December 2022). Furthermore, VosViewer 1.6.17. was used to conduct the bibliometric analysis such as the co-authorship/co-occurrence analysis and to generate data visualization.

Characteristic of Identified Papers on Laccase and Pollutant or Wastewater
A total of 1865 papers of original articles (n = 1697, 90.9%) and review articles (n = 168, 9.1%) regarding laccase and pollutant/wastewater were identified through a Scopus database search with the publication year ranging from 1978 to 2022. A majority of the identified papers (n = 1413, 75.76%) were published in the past decade (2012-2022), showing the increase of the study of lacasse with regard to its use in dealing with pollutants or wastewater ( Figure 2).

Data Analysis and Visualization
Microsoft Excel 2016 was used to analyze the selected papers and to export the graphs and tables needed to present the paper type, source journals, top 10 organizations or countries and the most cited papers. Data were double-checked using Biblioshiny (https://www.bibliometrix.org/, accessed on 17 December 2022). Furthermore, VosViewer 1.6.17. was used to conduct the bibliometric analysis such as the co-authorship/cooccurrence analysis and to generate data visualization.  Environmental Science (n = 1052, 56.4%), Biochemistry, Genetics and Molecular Biology (n = 581, 31.15%), and Chemical Engineering (n = 561, 30.08%) were the most studied subject areas among papers related to laccase and pollutants or wastewater (Table   0   20  Environmental Science (n = 1052, 56.4%), Biochemistry, Genetics and Molecular Biology (n = 581, 31.15%), and Chemical Engineering (n = 561, 30.08%) were the most studied subject areas among papers related to laccase and pollutants or wastewater (Table 1). A total of 165 journals published 1865 papers related to laccase and pollutants or wastewater, with only 16 journals publishing more than 20 papers (Table 2). These 16 journals (9.69% of total journals) published 701 papers (37.58% of total papers). Bioresource Technology (n = 108, 5.79%) was the leading journal publishing the most papers, followed by the Journal of Hazardous Materials (n = 95, 5.09%) and Chemosphere (n = 89, 4.77%). These three journals were also the only ones to publish more than 50 papers, contributing to 15.65% of total papers published ( Table 2).

Mostly Cited Article
There was a total of 74,833 citations for 1865 papers, averaging 40.12 citations per paper and 1700. 75 citations per year (1978-2022). The most cited paper was Biodegradation aspects of Polycyclic Aromatic Hydrocarbons (PAHs): A review, authored by Haritash, A.K. and Kaushik, C.P. in 2009, which was cited in 2072 papers, almost twice as many as Technologies for the removal of phenol from fluid streams: A short review of recent developments, written by Busca, G., et al. in 2008, cited in 1046 papers. There were only three papers cited more than 1000 times. The trend in citations continued to rise across the years, and peaked in 2022 with 11249 citations. The data of the top 10 most cited papers have been presented (Table 6).

Results from Co-Authorship Networking Analysis
A co-authorship analysis was conducted to evaluate the relationship among authors, organizations and countries according to the number of papers they published together. This analysis indicates the trends of collaborations and identifies the leading authors, organizations, and countries in terms of the related study, which in this case was laccase and pollutant/wastewater. Using VosViewer software, a visualization map will be generated which will also show nodes of various sizes representing the author, organization, or country. These nodes will also be connected by a line called Link Strength (LS), where thickness determines the link strength corresponding to the number of co-authored papers and how strong the collaboration is between the two authors, organizations, or countries. All links of a node will be accumulated as Total Link Strength (TLS), representing how strong the author, organization, or country is connected to others [38].
To analyze the authors' co-authorship, the minimum number of papers per author was set to three and the minimum times of citations of an author was set to 0. Of 5573 identified authors, 689 met the thresholds. The network visualization and overlay visualization maps are shown in Figure 3 Meanwhile, an organizational' co-authorship analysis was conducted on 4165 identified organizations, with the minimum number of papers per organization set to three and the minimum times of citation of an organization was set to 0. Of 4165 organizations, 97 met the thresholds, however only 11 organizations were found to be linked to each other. The network visualization and overlay visualization maps are shown in  Finally, countries' co-authorship analysis was conducted on 89 identified countries, with the minimum number of papers per country set to three and the minimum times of citations of a country set to 0. The results of this analysis have been presented in Figure 5.  Finally, countries' co-authorship analysis was conducted on 89 identified countries, with the minimum number of papers per country set to three and the minimum times of citations of a country set to 0. The results of this analysis have been presented in Figure 5

Keyword Co-Occurrence Analysis
Keyword co-occurrence analysis is a visualization approach to indicate how frequent and high the connected terms or words used in papers are related to a specific field of research. This analysis is also able to identify the development trend of the research focus, generally grouping the keywords into clusters. Using VosViewer software, each node indicates the keyword, while the link line connecting the nodes indicate the keyword relationship and their use on a paper. Both nodes and link lines are varied in size, showing the popularity of the keyword.
For this study, a total of 3777 keywords were identified, with the minimum number of occurrences of a keyword set to 10. Of 3777 keywords, 100 met the threshold. The most used main keyword in the study on laccase and pollutant/wastewater was laccase (occurrences = 753), while the other included keywords were biodegradation (occurrences = 189), decolorization (occurrences = 165), bioremediation (occurrences = 141) and immobilization (occurrences = 102). These five keywords were also the only five keywords to pass the 100 occurrences-mark. The network visualization shown in Figure 6a shows different colours based on the keywords' closeness to a certain research theme. The keywords coloured in red indicate their closeness to laccase immobilization; in blue-biodegradation; in green-redox enzymes similar to laccase (i.e., peroxidase); in purple-ligninolytic enzyme; in yellow-laccase effectiveness against various pollutants based on decolorization; and in cyan-types of pollutant which could be effectively removed by laccase (including lignin, catechol, phenolic compounds, and olive oil mill wastewater). Nonetheless, it is not clear what research theme is indicated by the keywords cluster in orange colour.   The density visualization was altered to show the keyword change trends across the past 20 years, as the default range (1978-2022) did not show any significant change over the keywords use (Figure 6b). The analysis revealed that in the last 10 years, laccaseassisted wastewater treatment mostly involved the enzyme immobilization indicated by its performance in biodegradation and decolorization. Chitosan, a supporting polymer material, was found as the most connected keyword (Figure 6b).
Density visualization of the keywords that occurred in laccase-assisted wastewater treatment research has been presented in Figure 6c. The analysis indicated that the keywords in red occurred in papers with the highest density, followed by keywords grouped in yellow, green, cyan, and blue. The analysis suggested that biodegradation was the keyword with the highest density. Immobilization, decolorization, and biosorption were the only keywords presented in yellow coloured zone. Types of pollutant are found localized in the green coloured area, namely bisphenol, dye, wastewater, and azo dye.

Discussion
Laccases (EC 1.10.3.2), a member of the multicopper oxidases family, have been reported for their efficacy in removing phenolic or non-phenolic contaminating agents which can be obtained through an isolation from plants, bacterial, or fungal species [39]. Despite its variety in structure (as isolated from different organisms), they have highly conserved region amino acids which are covalently bonded with the copper centres [40,41]. Laccase induces oxidative reaction against a phenolic molecule involving one electron from one of the enzyme's Cu centres (Cu 2+ /Cu + ) to produce a phenolic radical. This initiates a long chain reaction which eventually induces the cleavage of the organic contaminant and the reduction of molecular O 2 into H 2 O [42]. In terms of efficiency, an immobilized laccase was reported to yield a 90% or higher decontamination rate [22,[43][44][45][46]. As many strategies have been employed to improve the utilization of laccase in wastewater treatment, we have performed a bibliometric analysis on a big and reliable scientific database, Scopus [47].
A bibliometric analysis was performed on the 1865 identified papers reporting the utility of laccase in pollutant or wastewater treatment. This study aimed to provide a thorough assessment of the study status according to the source journals, leading organizations, countries and the collaborations between each other. Bioresearch Technology published the highest number of papers about laccase and wastewater or pollutants (5.79% of 1865 papers), followed by the Journal of Hazardous Materials (5.09% of the papers). Govindwar, S.P. published the most number of papers and also recorded the highest number of citations. An author co-authorship analysis showed that Iqbal, H.M.N. and Bilal, M. had the highest number of collaborations between each other.
The organizations publishing papers on laccase-assisted wastewater treatment were centred in China and India, with Shivaji University (India), Jiangnan University (China), and Tianjin University (China) producing the most papers compared to other institutions. China published the largest number of papers (22.3% of papers), almost twice as many as India (14.79% of papers). These two countries were also the only ones who managed to pass the 10% mark. China's dominance may occur due to its large funding sources, one of them being the National Natural Science Foundation of China, which was also the largest funding source with regard to papers about laccase and pollutants or wastewater. The aforementioned funding source contributed to 13.67% of 1865 papers, followed by the Natural Sciences and Engineering Research Council of Canada, which contributed funding to 2.57% of papers. Of the 10 top funding sources, both Asia and Europe had three funding sources, while the rest were two funding sources from both North America and South America. The number of collaborations remained an issue among organizations, with only 0.26% of 4165 organizations having collaborations.
Between 1978 and 2022, publications on laccase and pollutant or wastewater reached a total of 1865, of which 90.9% were research articles and the remaining 9.1% were review articles. The total number of identified papers is still significantly smaller compared to the 14,057 total papers that studied laccase (a search was conducted using the same term excluding the specific keywords used in this bibliometric analysis). This shows the dominance of experimental work on laccase and pollutants or wastewater. However, the top papers found in this present study (indicated by the number of citations) were mostly review papers. Whilst most papers specifically discussed laccase activity in degrading pollutants, a paper authored by Rodríguez Couto and Toca Herrera highlighted chitin and chitosan characterizations [31]. We also found that chitosan as the only biopolymer shown in the keyword networking analysis. Taken together, chitosan is the most studied polymeric support for laccase immobilization. This is due to the fact that chitosan has functional groups that can be manipulated for the immobilization reactions or involved interactions [48][49][50][51]. Moreover, chitosan could play a part in contaminant removal via the adsorption on the polymer surface [52][53][54].
A visual networking analysis of the keywords on papers published from 1978 revealed that biodegradation has the highest density, followed by immobilization, decolorization, and biosorption. This suggests that the research is dominated by the utility of non-isolated laccase for the wastewater treatment [55][56][57]. As mentioned previously, immobilization could enhance the removal efficiency whilst improving its working condition ranges and reusability [55,[58][59][60]. Biosorption is a keyword usually used to explain the adsorption phenomenon on bio-based materials. Coconut and rice husks are among the growing substrates that have been reported to immobilize T. versicolor and could also perform biosorption [61][62][63].

Challenges and Future Prospects
To get an insight on the constrains of laccase-assisted wastewater treatment, we performed a literature search on review articles using the same previously used keyword combination, but the search was refined to only include those published between 2018 and 2023. Challenges and recommended topics for future research have been presented in Table 7. The high-costs of enzyme production along with its stability and reusability limit its application in wastewater treatment [64][65][66][67][68][69][70][71]. Research on gene editing and molecular engineering could overcome the stability and improve the production yield of the enzyme [64][65][66][67][68][69][70][71]. Using a low-cost solid substrate would also reduce the production cost [13,[72][73][74]. Immobilizing laccase onto a supporting material could enhance the stability, reusability and contaminant removal performance [70,71,75,76]. However, laccase immobilization creates other challenges such as enzyme leakage, catalytic site blockage (hence reducing enzymatic activities), and additional production costs [64,71,75]. Therefore, it is important to consider the appropriate immobilization technique and the type of supporting materials. Another challenge in laccase research for contaminant removal is our lack of understanding regarding its catalytic degradation mechanisms, especially because the mechanism is dependent on the type of the contaminants. Screening the enzymatic activity against different pollutants could be assisted by an in silico approach [6,66,70,77].

Strengths and Limitations
Being the first study to research the bibliometric analysis on the study of laccase and pollutants or wastewater trends, this study used VosViewer to analyse the inputted data searched from the Scopus database, offering more accountability and objectivity. It is worth mentioning that we did not include book chapters, conference paper/reviews, editorial notes, erratum, letters, short surveys, or retracted documents. This study was also based on the Scopus database, and did not include other databases such as Web of Science (WoS). We also found difficulties in the visual networking analysis, including the incomplete name of affiliation in the visualization, an issue which cannot be resolved by the user manually.

Conclusions
Laccase utilization in degrading pollutants is mostly studied by scientists in China (22.3%) and India (14.8%). The leading journal publishing research about laccase utilization in wastewater treatment was Bioresource Technology. Environmental Science was the most reported subject area, indicating the urgency of laccase use on wastewater treatment. Laccase immobilization is on the research spotlight, where the high occurrence of the keyword 'chitosan' has been found. This research, however, was still lacking in terms of collaboration between organizations, which needed to be strengthened to further ensure the betterment in terms of treating wastewater or pollutants.
There are also several recommendations for the bibliometric analysis. Despite being user friendly, the visualization using VosViewer software restricts the user to editing the name in the presentation. In our case, we found the affiliation being presented with an incomplete name, and there was no option for manual editing. There should be a tool to analyze the quality of the paper rather than being based solely on the quantity. More importantly, the bibliometric analysis should be validated with a different approach, and in our case we used Biblioshiny.