Visual graphics for human rights, social justice, democracy and the public good

Vedant Nanackchand
Department of Visual Art
University of Johannesburg
South Africa

Kim Berman email@example.com
Department of Visual Art
University of Johannesburg
South Africa

Visual graphics for human rights, social justice, democracy and the public good
FC9B4BFD7EB595B5123C9CB4DC524D50artdemocracyhuman rightssocial justicethe public goodvisual graphics
The value of human rights in a democratic South Africa is constantly threatened and often waived for nefarious reasons.We contend that the use of visual graphics among incoming university visual art students provides a mode of engagement that helps to inculcate awareness of human rights, social responsibility, and the public good in South African higher education.Visual graphics, the subject of the research project which forms a key component of a Masters dissertation by one of the authors, provides an opportunity to counter a noticeable decline in the students' response and sensitivity to the freedoms entrenched in the South African Bill of Rights.The article presents a study using an action research approach in the classroom between 2005-2010, in order to inculcate awareness of human rights among participating students and deepen their understanding of social responsibility.The method used involved an introduction to specific visual art curricular intervention projects which required incoming first-year students to develop visual responses to address selected human rights violations and, in their second year, to develop their visual voice in order to promote human rights advocacy through civic engagement.The critical outcomes impact positively on the use of graphic images in the curriculum as a visual methodology to re-insert the discourse of human rights as a basic tenet of constitutional democracy in higher education.

Introduction

This article responds to the observation that there is currently a limited critical awareness of human rights-related issues generally among entering visual art and design students at a South African university, where the authors of this article teach.Since 2005 questionnaires have been administered annually with incoming first-year students, recording general attitudes ranging from apathy, indifference and disinterest to a general lack of concern about social and politically related issues.These attitudes have a direct impact on the way in which students might approach critical issues relating to their studies and their potential to engage meaningfully in society.Such limitations among entering visual art students are perceived by the authors as linked to the change in political values experienced in current South African society.This article presents a first-and second-year curriculum intervention conducted from 2005 to 2010 using an action research approach that required students' engagement through reflection, dialogue, and visual responses to the Bill of Rights by means of specific curricular projects.Subsequent classroom projects introduced to the students in their second year of study required them to build on this personal awareness, a level of advocacy through, for example, the design of posters that present a more public engagement with a human rights issue.This article however, focuses primarily on the introductory projects on the Bill of Rights for first-and secondyear visual art students.The methodology of action research in the broader study, underpins 1 the curriculum interventions which draw on Freire's (1996) model of critical pedagogy to effect social change.The article demonstrates ways in which the use of graphic imagery and printmaking in visual art may help to foster awareness of social and visual identities as well as responsiveness to human rights.The authors contend that a critical level of social awareness integrated into the curriculum, over three to four years of study at a higher education institution, can contribute to an ability to make choices as responsible citizens in terms of democratic values and social justice.

Theoretical perspectives

This article draws from the historical legacies of art and human rights advocacy groups as mediators of social change through arts-based practice.This approach is theoretically underpinned by Appadurai's (2004:59-84) model of "the capacity to aspire" as well as Sen's (2004: 37-58 cited in Rao & Walton, 2004:4) belief about "… how does culture matter?" and his positioning of the role of "cultural processes … for positive social and economic transformation, through their influence on aspirations, the coordination of collective action, and the ways in which power and agency work within society", to mediate the power of visual methods towards civic engagement that encompasses human rights.For example, economist Vijayendra Rao and human development advisor Michael Walton ( 2004) also advocate the use of culture as a primary enabling intervention to bring about social change (Nanackchand, 2010).In this regard, visual art practice at higher education level provides key educational strategies for empowering students and practitioners to address the lack of human rights awareness and advocate for citizenship and the public good.Van Heertum (2010:218) draws attention to the political theorist and philosopher Herbert Marcuse  who "… calls for an aesthetic education that can break through instrumental and technological rationality …[via]… ideology critique and consciousness raising, serving as catalysts to reignite the belief that change is possible."This article presents visual arts as potentially effective in introducing human rights values to first-and second-year students in order to catalyse social awareness and the notion of active citizenship that can be integrated into longer term higher education experience.

The theoretical positioning of this article also draws from a framework that advocates for teaching a sense of public good and social responsiveness.Badat (2001), Singh (2001), Soudien (2008) and Waghid (2007;2008) are South African educators who advocate instilling a notion of the 'public good' in higher education.These authors advocate for teaching a sense of public good and social responsibility in order to empower students to think critically, to act in terms of the diverse needs of society and to ensure that the higher education community personifies 'good' in terms of the South African Bill of Rights, in order to act as a conscience of society.

The conceptual framework includes the following: firstly, a definition of the relevance and limitations of human rights in the context of this article; secondly, the suggested interconnectedness between and the relevance of human rights with the sub-disciplines of visual art and critical pedagogy and; thirdly, the relevance of action research as a methodology that facilitates an inculcation of a consciousness of human rights and social justice in the classroom through visual arts.

Human rights, including the emergence of individual and collective rights, help to contextualize the curriculum intervention described here.Heywood (2007) explains that human rights are based on a liberal ideology of constitutional rights that safeguard the liberty and freedom of the individual and society as well as the responsibilities of the state in ensuring these rights.He asserts that individual rights in pursuit of freedom and democracy are fundamental.In addition, Clapham's (2007) explanation of the historical origins of human rights differentiates human rights from the framework of rights and law in general, by referring to Edmundson's (2004:191) assertion that "human rights recognize extraordinary special, basic interests, and this sets them apart from rights, even moral rights, generally."Evans (cited in Abdi & Schultz, 2008:25-37) provides a qualitative and historical analysis of the "human rights project" by focusing on the principle of human dignity within the framework of the United Nations' (2012) Universal Declaration of Human Rights (UDHR) of 1948, which according to Clapham (2007), included such inalienable rights as the right to life, human dignity, freedom of expression, freedom of association and children's rights amongst others.Whereas in 1948 the UDHR laid the foundations for human rights internationally, the National Party government shunned the adoption of the UDHR, resulting in South Africa being governed by apartheid legislation until 1994.The authors maintain that, presently, human rights advocacy is underscored by notions of citizenship, which in effect are based on values of democratic practice.It presupposes taking ownership of one's rights and responsibilities as citizens and to understand the extent of the state's reciprocal right to govern and respond within the spirit of its mandate from the society that elected it.Ramphele (2008) offers critical comment regarding perceptions of societal neglect in South Africa and proposes that what is required is that society adopt ethical standards of behaviour in order to avoid the deep levels of mistrust and anger associated with the past.

In light of individual rights related to notions of group and community rights, Clapham (2007) also draws attention to Amartya Sen's "… legitimacy critique … which cautions against confusing human rights with legislated legal rights."Sen (cited in Clapham, 2007:12) also points to a "cultural critique", a view that human rights are unfamiliar to some cultures, which could interpret the principles of human rights differently in that they may "… prefer to prioritize other principles such as respect for authority." 2  To support the notion of 'reinserting the public good' as well as the need to improve critical thinking skills related to undergraduate Art and Design education, Jeevanantham's (2005) report on critical thinking skills development is helpful.The work presented in this article proposes an adaptation of action research as a facilitative, enabling and democratic methodology, to inculcate human rights awareness when applied to relevant art processes to support active learning (Stringer, 2008). Musil's (2009) models of the Self Analysis Framework and the Civic Learning Spiral provide valuable approaches that help to develop and assess a critical capacity among students in the curriculum intervention, as they are empowered to work closely together towards shared social and civic outcomes.

The research question

We address the following research question: How can a visual graphics curriculum intervention introduce a discourse of human rights in teaching a sense of public good and social responsiveness to first-entry visual art students at a higher education institution?

The methodological approach In the context of the study, an action research approach as a classroom intervention (historically associated with Kurt Lewin: 1890-1947) has been adapted as a pedagogical approach to lead to social action, using the process of planning, action, analysis and evaluation.This is premised on the Freireian philosophy of dialogical practice, which facilitates an empowering, liberating and enabling educational environment.Paolo Freire's work Pedagogy of the Oppressed (1996), advocated a radical pedagogical approach which is participative and inclusive rather than hierarchical.

The approach of using action research as a method of inserting an intervention into the visual art curriculum can be deemed effective because of the multi-modal nature of creative art practice involving a diverse group of students.Action research is helpful to the envisaged outcomes of the students' projects for two important reasons; first, it is congruent with the pedagogical approach advocated by Freire (1996), which formed the basis for the values and philosophy of the 1995 South African White Paper on Education and Training (Department of Education, Notice 196 of 1995).Second, it ensures that the role of an educator is facilitative rather than directive.In contextualising the four stages (planning, action, analysis and evaluation) that also underscored the primary author's action research study, stage one involved planning the visual art curricular intervention in order to inculcate awareness of human rights among students.Stage two involved an implementation of the first and second year projectbased curriculum interventions, on the basis that action research is a participatory process that benefits from the contribution of willing participants who undertake and validate the outcomes of their particular inquiry; and that the students are able to conduct their independent inquiry within the ambit of the visual art curriculum.The third stage of the action research used a reflective framework to analyse the work of selected students (who are presented as anonymous participants) from the standpoint of aesthetic, conceptual and empirical perspectives.Stage four of the action research process involved an evaluation of the outcome of the curricular programmes in order to understand the extent to which the curricular interventions catalyse awareness of human rights amongst participants.

The notion of inculcating critical personal and civic awareness is drawn from Musil's (2009) Civic Learning Spiral in which she poses five critical questions on self-analysis which are integrated in the design of the curriculum intervention in this study.These include (a) Who am I? (Knowledge of the self), (b) Who are we?(Knowledge of the group or affected party), (c) What does it feel like to be them?(Empathy about the prevalent condition to be studied), (d) How do we talk to one another?(Possible interaction between groups or cultures) and (e) How do we improve our shared lives?The students engage with these questions in the studio, requiring the diverse student group to respond critically, personally and among themselves, though drawings, visual research, collage and cross-cultural dialogue both within and outside of the classroom, to their personal selections from the South African Bill of Rights. 3 The Civic Learning Spiral comprises six areas "(a) The Self, (b) Communities and Culture, (c) Learning and Knowledge, (d) Skills, (e) Values, and (f) Public Action."(Musil, 2009:59-66).These aspects of the learning spiral, combined with an action research methodology, facilitate critical visual inquiry among students and encourage their capacity for reflection and social awareness.Students present and debate their chosen concepts within the group and then refine them into a series of linocuts and narrative statements responding to a selected human right.This approach helps to create awareness among students to the extent that they assume the role of "artist as educator", a theme that relates to how students formulate critical awareness of human rights in order to inculcate a sense of responsibility through their work (Nanackchand, 2010:150).

The following section addresses the planning, action, analysis and evaluation phases of the action research process adopted.

Planning

The findings in the initial questionnaires issued to incoming art and design students, indicate that many students are generally unaware of notions governing the principles of their South African citizenship and, by implication, the South African Bill of Rights.First year students entering the university's undergraduate visual art programme are generally under-prepared to engage critically in debate about human rights.Keet & Carrim (2006) refer to the principle of general under preparedness of learners in the Department of Education's provision of a Peoples' Education and Human Rights Education Policy governing primary and secondary education.Uncritical attitudes have the potential of fostering negative values and a casual acceptance of the status quo regarding questionable aspects of contemporary culture, especially prejudices relating to racism, intolerance, gender inequality, ethnic and national chauvinism, and HIV&AIDS.Even though students may not generally display such tendencies, it would be incorrect to assume that all first year students understand the principles and application of human rights.Where such understanding exists among students, it is generally vague (according to the introductory questionnaires 2005-2010).In such circumstances, a student may not necessarily make the connection with human rights violations in South African society.The approach of introducing human rights through a series of printmaking projects in the first two years of the visual art curriculum, aims to challenge such attitudes to ensure that the curriculum addresses attitudes of ignorance and mediocrity.

The printmaking projects through a directed academic programme can begin to develop civic responsibility among students by their engaging in important aspects of shared histories and current social phenomena.Through this process, visual art students are able to address issues of social relevance and responsibility in a critical and meaningful way.

The curriculum intervention for the first project comprises an introduction to the South African Bill of Rights, which encompasses several broad sub-themes that relate to justice, ethics and moral values.Individual students then interpret these values in terms of their particular culture, heritage, identity and experience.During the students' engagement in the artmaking process, the primary author interprets and assesses social and personal change achieved among the students' through their art making, written statements and research processes recorded in their visual diaries.The students' work is evaluated against their brief to submit their narratives, interviews, observations, and discussions as well as through a process of monitoring conceptual development in their individual artworks.As a result, the primary author is able to gauge, from the critical visual responses the students generate, how the learning process influences the students' awareness and interpretation of human rights.

Action

The approach to the learning process is layered.In the first-year project, students develop their own concepts through engaging in information-gathering procedures such as conducting interviews with peers, family members, friends and neighbours; documenting subjects through photography, drawing, sketching, creating collages and photo-montages, collecting newspaper articles and undertaking library research.The students then present and debate their findings in the classroom.Through this process, students begin to understand the participatory nature of practice-led action research.They also begin to understand the value of the personal experiences of the research topics over the more impersonal information accessed indirectly through secondary sources.Principally, the approach to teaching visual art-practice is open and dialogical, in order to accommodate the variety of students' responses that relate to subject matter, form and style as well as the multi-modal nature of visual methodologies.

The first year visual graphics programme involves linocut, monotype and drypoint printmaking projects that require students to engage in specific aspects of human rights and social justice challenges in their work.In the first year curriculum, project one focuses on inculcating awareness about the Bill of Rights.By way of illustration, the examples presented in this 4 article are drawn from the introductory linocut printmaking projects which aim to create an awareness of human rights.Such awareness is deepened in the second year of learning and extended in the third year and later years of study. 5 The first year project considered in this article is a linocut printing project presented as part of the Graphic Interpretation 1 module titled: Introduction to the South African Bill of Rights and human rights advocacy.Three examples are presented to illustrate the range of human rights awareness that students engage with.

Student (A)

Student A is a first-year student who lives in a township in southern Gauteng province.His work relates to the disparities between the privileged and poor communities, both in terms of material wealth and discriminatory treatment by society.His journey to and from the university was educational, in the sense that he became aware of the lack of human rights practice among the people who mattered.In his first graphic print based on Article Twenty Four of the Bill of Rights, that is, 'Environment' (Republic of South Africa, 1996:24), Student A explained that this image was as much about social inequity as it was about the environment.In his print (Figure 1), the symbolism is two-fold; firstly, the upper composition depicts the environmental effects of industrialization and globalization, that is, the way in which technological advances in industry and global communications pay scant regard to their effect on the environment.The image addresses the danger of rampant pollution in a fragile natural world, symbolized by the brittleness of an animated tree.Student A juxtaposes technology with poverty symbolized by the group of urban dwellers, which he based in the township of Soweto.The composition addresses the sharp distinction between the level of historical disenfranchisement that contrasts an image of urban poor people against the economic advantage of a growing technology and communications sector.

Student (B)

Student B is a naturalised, black male student originally from Zimbabwe, who completed his high school education in South Africa and subsequently enrolled to study Graphic Design at the university.He produced a linocut print based on Article Twelve of the Bill of Rights In his first year linocut print, he used the metaphor of a tap to symbolise life.The tap, which is imposing in its allusion to a firearm, occupies central space as it gushes references to crime and xenophobia, symbolised by its reference to the South African flag.

The second-year printmaking module of the Visual Graphics course comprises a series of five projects over the year that aim to engender a critical sense of social responsibility.The third example (Student C) referred to in this article is an etching project based on an Introduction of the Civic Learning Spiral.The theme explores an aspect of social justice which introduces notions of civic engagement in the education process.

Student (C)

This example of Student C's response serves to illustrate how human rights awareness is stimulated.His etching project focused on Article Eighteen of the Bill of Rights that is, The Right to Freedom of Association (RSA, 1996:18).In this print, the student depicts two gay lovers in a passionate embrace.A garland of flowers symbolising a celebration of their love, frames their portraits.On the upper section of the composition, the text "Everyone has the Right to Freedom of Association" is integrated with the main body of the image.In evaluating the student's artwork, the student's use of ideology in this image shows how a change in awareness occurs through learning.

Analysis

The authors acknowledge that certain methodological limitations exist.The introduction of human rights in the first year curriculum cannot result in a critical shift in awareness amongst first-year students, but assumes that the development of critical awareness and citizenship is reinforced over time.The selected examples of the first year linocut project show ability by the students to integrate and translate concept and dialogue in relation to the subject.Examples of students' comments in the first and second year 'Bill of Rights' project, as well as an extract from an interview of reflections from the Graphic Design student, reveal their increased engagement with issues of human rights.The visual stimulus often serves as a catalyst for social change wherein each case students apply their knowledge of human rights to specific visual graphic outcomes.Their graphic prints in turn, have the potential to influence public opinion.For example, Student B's comments and artwork reveal ways in which his independent actions in promoting human rights were based on his encounter of human rights as part of his first year undergraduate training in the visual graphics course.Extracts from student interviews record how change takes place.

Student A

Student A depicts an important critique through his exposure of issues of social redress that government and society have not satisfactorily addressed.In addition, the linocut print addresses the socio-historical conditions, which reflect disenfranchisement and poverty.Student A's graphic statement succinctly describes a denudation of the environment and metaphorically, the divide between the haves and the have-nots in South Africa.At level one of the Visual art curriculum, such a display of critical thinking, problem solving, allusion, practical skill, knowledge of the subject and values of social justice, are indications of the way in which human rights education have been integrated into the student's consciousness.

Student B

As a result of engaging with the requirements of the first year printmaking curriculum, Student B was able to formulate a consciousness of human rights by producing a poster design for his third-year portfolio.He based the idea for the poster on his experience of intolerance, prejudice, ethnic chauvinism and xenophobia in his home country as well as in Johannesburg.He affirmed this in a personal interview.In response to the question about the value of the acquisition of adequate technical knowledge and visible skills development that he learnt in first year, he responded that "Research on the module led to an interest in activist graphics" (Personal interview with Student B, 24 October 2008).When asked how he might have benefited from an insight into human rights as part of this module, he stated, "I became more aware of the responsibility an image-maker has in educating about such issues, visually" (Personal interview with Student B, 24 October 2008).This self-evaluation is significant.It shows how this student engages with his role as artist-as-educator and in so doing, realise responsibility in his life.This example demonstrates how, through independent work, visual graphics may help to inculcate awareness of human rights and social injustice among students.This example also demonstrates that exposure to human rights content in image making influenced his subsequent career direction as a graphic designer.

Student C

Discussions with Student C, the second year student who depicted an etching of gay lovers embracing, revealed that he was satisfied with the image because it projects homosexuality in a positive way.He believes that such an image helps to dispel stereotypical attitudes about the gay and lesbian communities, for example, in relation to the subject of HIV&AIDS.Ordinarily, such a statement is normal and is the expectation especially from a student commenting on issues of HIV&AIDS and social significance.Yet, during initial consultation with the student, he acknowledged his aggressively homophobic stance towards the gay community.He explained his attitude by referring to two influential values into which he was socialized.These included his community's traditionally conservative and chauvinistic attitude towards the gay community and his submission to peer-pressure to denigrate gay people at every opportunity.

The turnaround in his attitude arose after he attended and took part in a group discussion of the printmaking project with his peers.The student indicated a re-interpretation of his attitude in terms of Article Eighteen of the Bill of Rights.He responded to the introduction to this project that presented examples of Gott's (1994) exhibition of the Australian art com-munity's proactive and positive response to the HIV&AIDS crisis.Through a continuous review process of his research and re-conceptualising including self-critique, feedback from the author/lecturer as well as his peers, Student C confirmed his intention and proceeded towards the technical and aesthetic realization of his project.Mitchell (2008:374) affirms an interpretative process such as this as the capacity of visual work to facilitate "… reflexivity in the research process."Characteristically in the second year of the learning process the emphasis shifts towards a more complex interpretation of social justice-related issues that emphasises their social significance.For example, Ryan (2003:12-13;73-77) highlights Rawls's (1971) theory of justice within a set of theories that defines 'social justice' as "… the justice of the way the benefits of social and economic co-operation are allocated."In this case, students generally engage in a framework of 'self-analysis ' that, according to Musil (2009), determines critically how their responses to art may lead to what she refers to as personal and social responsibility.Student C's change in attitude further stemmed from his concern about a relative who was in denial about her HIV status.He used his artwork as the basis for discussion with his relative and found that her response to the work facilitated discussion and disclosure about her condition.This experience represented a leap in confidence of the student's personal learning.

Another indication of change is this student's active involvement in gender politics advocacy programmes after his graduation.He is contracted as a facilitator at Artist Proof Studio in Johannesburg in a gender advocacy project conducted under the auspices of Sonke Gender Justice Network (SGJN).Berman (2012) provides a detailed analysis of the SGJN campaign to advocate for gender equality and justice through critical learning and activism in visual art practice.

Subsequent conversations with the examples of selected visual art students who participated in the two year curricular interventions post graduation, reflect on the catalysing influence of their learning, which they claim, determined their subsequent choice of content in their own artwork, and further led to career choices involving a commitment to human rights development.

Evaluation

An evaluation of the projects emphasize how perceptions of social injustice influence an understanding of human rights among students whose values changed after their active learning experience of visual graphics.Their increased awareness of human rights helps to catalyse personal and social change through the use of visual graphics as an educational intervention.

Other visual art students' responses provide evidence of the inculcating of notions of socio-political awareness through art making (Nanackchand, 2010).Student-based projects using drawing, printmaking, digital photography and photovoice prove instrumental in developing a social consciousness.Another example of this is a project requiring first year students to engage with social documentation of aspects of Johannesburg's inner-city blight.Through such curricular projects, students become aware of the basic principles of political instruments such as the South African Constitution and the Bill of Rights and are introduced to principles of justice, ethics and morality.This type of critical awareness enables them to recognise how social injustice manifests in society, and to comment critically on this by developing their visual voice.Moreover, within the two-year learning cycle, most students' work reflects significant personal change.This outcome relates to the students' capacity to engage critically with aspects of human rights awareness in their work, and to apply knowledge of such an outcome to their personal circumstances.Understanding the impact of human rights awareness that leads to critical shifts amongst students in order to inculcate enhanced citizenship, takes place over time and lies beyond the scope of this article.Given the range of visual graphics outcomes generated by the participants, the authors speculate that important positive shifts in learning occur as a result of the action research project.First in terms of improving their discipline-based knowledge of the subject, conceptualization, methods and techniques of visual art practice.Second, in terms of an enhanced sense of social and civic responsibility which can be reflected on in hindsight, through following the careers of selected post-graduate students.Third, a noticeable degree of awareness occurs by the end of the first year of study which can meaningfully be followed by a continued academic pathway in the second year of undergraduate studies and where the focus on human rights advocacy and civic action is re-emphasised.While the scope of this article looks at first-and second-year studies, the larger research study (see Nanackchand, 2010), documents important developments of students who engage actively in human rights advocacy as a result of their curricular experiences.

Conclusion

Human rights awareness in visual art practice helps to address the lack of social and political acumen and criticality amongst students.In response to Ramphele's (2008) argument regarding the dilemmas of the transformation in South Africa, the philosophical and pedagogical arguments for active learning about human rights through the use of visual methodologies, helps to address very real challenges such as racism, intolerance, and xenophobia facing the current generation of students.Students are able to use the visual graphics approach to address inadequate knowledge about human rights by critically engaging with their personal belief systems.Such arts-based approaches are argued to be effective in developing critical responsiveness amongst the students about the discourse of human rights, democracy and social responsibility.

Notes

1 The broader study advanced in a Master's dissertation by the primary author (Nanackchand, 2010) explores the notion that the visual arts is a useful approach among others, to inculcate social awareness, based on the need for social justice in a society fraught with class and human rights differences.The issues of ethics and trustworthiness are explained in the study.2 In the context of this article, visual art is referred to as a form of cultural practice, where culture is equated with the arts rather than cultural practices more broadly and it is used as a catalyst for inculcating social awareness that can potentially lead towards social change.

The student body comprises a mix of race, ethnicity, class as well as urban and rural backgrounds from diverse educational experiences.Typically the first and second year samples comprise cohorts of 35 and 24 students, respectively, with an equal demographic of race and socio-economic status, although gender is biased in favour of female students.

Figure 1
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Figure 2
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Figure 3
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Figure 4
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Linocut prints in Figures1 and 2. These are first-year projects presented by Students (A) and (B), respectively. The project focuses on inculcating awareness of human rights among students.
 Student (C's)  etching in Figure3is an example of a 2nd year project, which focuses on social responsibility.
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Degradation of azo dyes by Alcaligenes aquatilis 3c and its potential use in the wastewater treatment
10.1186/s13568-019-0788-3Ajaz et al. AMB Expr (2019) 9:64 ORIGINAL ARTICLEAzo dyesDecolorizationA aquatilis 3cHPLCFTIRGC-MS
In the present study, Alcaligenes aquatilis was found to decolorize 82% Synazol red 6HBN after incubation of 4 days at 37 °C and pH 7. Maximum decolorization was found under static conditions by using saw dust and yeast extract as carbon and nitrogen source. It also showed promising potential to decolorize mixture of multiple dyes at a rate of more than 86% in 5 days. Decolorization of dye had positive influence on the growth of bacterium as growth rate was increased along with decolorization. The cleavage of azo bond was confirmed through TLC, HPLC and GC-MS analysis. The dye metabolites produced during bacterial treatment are linked to various pathways including ATP synthesis process. The absence of peaks of wavelength 1612/cm and 1532/cm in bacterially treated FTIR sample demonstrated the cleavage of azo bond. Microbial growth in decolorized dye wastewater shows that bacterially decolorized wastewater is unharmful for the growth of micro-flora. The high decolorization ability of A. aquatilis 3c to convert toxic azo dyes into useful end products may find potential applications in the environmental biotechnology. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Introduction

Industrialization is the backbone for the welfare and development of a country. Apart from its beneficial effects on the economy of a country it also exerts harmful effect on the environment and organisms. The discharge of wastewater from various industries becomes a continuous source of environmental pollution. Synthetic dyes, which are widely used in such industries, are a major portion of this discharged wastewater. These dyes are even more harmful for the organisms in comparison to other pollutants because of their resistance potential (de Souza et al. 2010).

Azo dyes are the largest class of synthetic dyes which are aromatic in nature structurally having one or more -N=N-bond. These dyes are rapidly used in various industries like textile, cosmetics, paper, food and pharmaceutical industry while the textile industry is its largest consumer (Alalewi and Jiang 2012). Their extensive commercial use is because of the reason that they are easily synthesized and economical for the users (Saratale et al. 2009a).

The loss of dyes from fabrics ranges from 2% for basic dyes to as high as 50% for reactive dyes as all the dye does not bind to the fabric (O'Neil et al. 1999;Pandey et al. 2007). The discharge of dye contaminated wastewater in aquatic ecosystems arises many serious problems. It causes obstruction in light penetration; oxygen transfer is reduced in water bodies, poses acute toxic effects on aquatic flora and fauna and also causes severe environmental problems (Solis et al. 2012).

There is an extensive use of physicochemical techniques like coagulation and flocculation worldwide to treat industrial effluents (Verma et al. 2012). Certain disadvantages are associated with the use of such techniques like usage of chemicals, sludge production which serve as source of secondary pollution, production of by products and high cost (Jadhav et al. 2007). In comparison to these physiochemical processes, use of variety of microorganisms including yeast, fungi and bacteria is more feasible as they are environmental friendly and their use result in the cleavage of azo bond which leads to formation of colorless aromatic amines (Kaushik and Malik 2010;Das and Mishra 2016;Meerbergen et al. 2018). The resulting products such as aromatic amines are further degraded to simpler non-toxic forms by multiple-step bioconversion occurring aerobically or anaerobically (Shah et al. 2012;Singh et al. 2014).

The current study was aimed at to isolate and characterize bacterium from industrial effluents capable to decolorize azo dyes. Optimum growth conditions of the organism were determined and bacterially degraded dye products were also analysed through TLC, HPLC, FTIR and GC-MS.

Materials and methods

Sample collection and bacterial isolation

Industrial effluent samples were collected in autoclaved screw-capped bottles from Kot Lakhpat industrial estate, Lahore, Pakistan. Physicochemical parameters such as pH, temperature, as well as color of the samples were also noted at the time of sample collection. The samples were serially diluted and plated on the L-agar plates. L-agar medium was prepared by dissolving tryptone (10 g), yeast extract (5 g), NaCl (5 g) and agar (15 g) in 1000 ml of distilled water. pH of the medium was adjusted to 7.

Evaluation of dye degrading potential

Dye degrading potential was determined by inoculating the bacterial isolate in 250 ml Erlenmeyer flask having 100 ml of mineral salt medium (MSM) whose composition is (g/l): (NH 4 (Parshetti et al. 2006) with glucose and yeast extract (1% each) used as carbon and nitrogen source respectively. The medium was supplemented with dye (Synazol red 6HBN) at a concentration of 50 mg/l for 4 days of incubation at 37 °C. Synazol red 6HBN, Congo red, Methyl red, Phenol red, Brilliant black and Navy blue were purchased from Sigma-Aldrich. All other chemicals were of analytical grade purity.

Bacterial characterization

The bacterial morphological parameters and biochemical tests were performed according to protocols given in Cappucino and Sherman (2008). For 16S rRNA ribotyping, DNA was isolated (Masneuf-Pomarade et al. 2007) and 16S rRNA gene was amplified through PCR by using universal bacterial primers (Turner et al. 1999). PCR was performed according to Rehman et al. (2007) and PCR product was purified by Fermentas Gene Jet Gel Extraction kit (#K0691). The purified product was sequenced and submitted to GenBank for obtaining accession numbers. Phylogenetic analysis was done using MEGA7 (Kumar et al. 2016). Phylogenetic tree was constructed using neighbor joining method with 1000 replicates.

Determination of optimal growth conditions

The physical parameters which were favorable for the bacterial growth i.e., temperature and pH were analyzed by growing bacterium at different temperature and pH. For optimum temperature determination, bacterial isolate was grown in LB broth and incubated at different temperature i.e., 20, 30, 37 and 45 °C and for optimum pH, the bacterial isolate was grown in LB broth with pH values i.e., 5, 6, 7, 8, 9 and 10. After 24 h of incubation, absorbance was recorded at 600 nm by using spectrophotometer. In order to determine growth pattern of bacterial isolate, LB medium was inoculated with log phase grown bacterial culture (1 ml). Optical density was determined by spectrophotometer at 600 nm at the time of inoculation and then after regular interval of 4 h up to 28 h of growth at optimum temperature and pH.

Decolorization experiments

In order to optimize the decolorization conditions decolorization experiments were performed at various conditions i.e., temperature (20, 30, 37, 45 and 50 °C), pH (5, 6, 7, 8, 9, 10), incubation condition (static and shaking), carbon sources (saw dust, sugarcane baggase and wheat bran) and nitrogen source (yeast extract, beef extract and peptone) source, inoculum percentage (2%, 4%,6%, 8% and 10%) and dye concentration (3, 5, 7, 10, 20 and 50 mg/l). A volume of 100 ml of MSM was taken in 250 ml of Erlenmeyer flask which was then inoculated with 2% of bacterial suspension and incubated at respective condition. The stock solution of dye was added into the MSM to obtain a final dye concentration of 50 mg/l. Aliquot was taken out at 0 h and after 3 days in order to measure initial and final absorbance, respectively. The aliquot was centrifuged before measuring the optical density at 465 nm. Decolorization percentage of the sample was measured by using following formula. All the treatments and controls were carried out in triplicates.
Decolorization (%) =

Effect of decolorization on growth of bacterial isolate

In order to check the effect of decolorization on growth of bacterial isolate, the bacterium was grown in MSM containing dye concentration of 50 mg/l. The aliquot was obtained after every day up to 5 days and optical density was measured at 600 nm in order to find out growth ratio. Decolorization was also calculated by measuring the optical density of supernatant at 465 nm after centrifugation of the sample.

Decolorization of multiple dyes

The bacterial isolate was checked for its ability to decolorize a mixture of azo dyes i.e., Congo red, Methyl red, Phenol red, Brilliant black and Navy blue. The initial concentration of each dye was maintained at 50 mg/l. The optical density of centrifuged sample (supernatant) was taken and finally percent decolorization was calculated by using above mentioned formula (Kalyani et al. 2008).

Analysis of dye degraded products HPLC, TLC and FTIR

The analysis of dye degraded products was done by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR). The metabolites were extracted from dye degraded sample (100 ml, 5 days) by mixing it with an equal volume of ethyl acetate. The extracts were then dried on anhydrous Na 2 SO 4 and evaporated in rotary evaporator. The dried powder thus obtained was dissolved in HPLC grade methanol and used for analysis. TLC was performed to analyze the degraded products on silica gel using mobile phase solvent system n-propanol, methanol, ethyl acetate, water and glacial acetic acid in the ratio 3:2:2:1:0.5 (Kalyani et al. 2008) and the results were visualized under UV illuminator at 254 and 366 nm. HPLC was performed at (Waters model no 2690) C18 column having symmetry 250 × 4.6 mm using methanol as mobile phase with a flow rate of 1.0 ml/min for 15 min and UV detector at 254 nm (Telke et al. 2009).

FTIR (Bruker, alpha-P) was performed to observe the change in structure of dye before and after decolorization. The FTIR analysis was done in the mid IRF region of 400-4000/cm. Prior to analysis sample was mixed with pure KBr in the ratio of 5:95 and pellet were then fixed in the holder for analysis (Saratale et al. 2009b).

Gas chromatography mass spectrometry

The collected samples were centrifuged for 10 min at 4 °C and the supernatant collected were extracted thrice with an equal volume of ethyl acetate, dried with Na 2 SO 4 and further concentrated in the rotatory evaporator. GC-MS analysis of metabolites was carried out using gas chromatograph system (GCMS-QP2010 Ultra, Shimadzu) equipped with capillary column (DB-5 ms). The samples were diluted 1:100 and injected 1.0 µl sample into GC-MS in split mode with injector temperature of 290 °C. Helium was used as a carrier gas with flow rate of 1.02 ml/min under 54.9 kPa inlet pressure. The column temperature was set to 50 °C and hold for 1 min with subsequent increase to 280 °C with ramp rate of 30 °C/min without holding and finally to 310 °C with ramp rate of 15 °C/min by holding it for 8 min. The peaks were identified by comparing with NIST27.LIB mass spectra library of GC-MS database.

Effect of decolorized dye wastewater on microbial growth

The extent of bacterially treated wastewater effect was determined on some useful micro-flora including Bacillus megatarium (z-28), Bacillus cereus (T358-2) and Bacillus subtilis (z-66) according to Mali et al. (2000). The plates (L-agar) were swabbed by bacteria; a 2 mm well was made and filled with decolorized dye wastewater. The plate's incubation was done at 37 °C for 24 h and the zone surrounded the well indicating the toxicity index of degraded dye sample.

Results

Physicochemical parameters and isolation of dye degrading bacterium

The temperature of wastewater samples ranged from 28 to 36 °C while the pH ranged between 8 to10. The color of the samples was black, navy blue and maroon. A total of 14 bacteria were isolated from 3 wastewater samples. On the basis of dye degrading potential, one bacterial isolate designated as 3c, showing 82% decolorization after 4 days of incubation at 37 °C, was selected for further research work (Additional file 1: Table S1). The significant visual difference between control and dye containing wastewater is shown in Fig. 1.   Fig. 1 Change in color of Synazol red 6HBN as compared to control in flask containing culture of bacterial isolate 3c from the dye contaminated wastewater

Bacterial identification

The morphological and biochemical characteristics are given in Table 1. The optimum growth conditions of the bacterium were 37 °C and pH 7 (Additional file 1: Fig. S1). The organism showed maximum growth (O.D) after 16 h of incubation in LB medium (Additional file 1: Fig. S2). The 16S rRNA gene sequence of bacterial isolate showed 95% homology with 16S rRNA gene sequence of Alcaligenes aquatilis. Then this 16S rRNA gene sequence was submitted to GenBank under accession number of KY009932. The bacterial strain has also been deposited at First Fungal Culture Bank of Pakistan and has been assigned accession number FCBP-B-728. The phylogenetic tree (Additional file 1: Fig. S3) which was constructed through MEGA7 shows the homology of A. aquatilis 3c with members of Alcaligenes sp. The frequency of appearance of completely identical sequence among the strains was indicated by number in the parenthesis.

Optimization of dye decolorization

The result showed that the A. aquatilis 3c showed maximum decolorization at 37 °C (Fig. 2a), pH 7 (Fig. 2b) and at static condition (Fig. 2c). The bacterium efficiently utilized saw dust and yeast extract as carbon and nitrogen sources, respectively (Fig. 2d, e) to show maximum percentage of decolorization. Moreover, efficient decolorization was determined when inoculum percentage was 6 and dye concentration was 10 mg/l (Fig. 2f, g).

Effect of decolorization on growth of bacterial isolate

It was determined that with the passage of time growth rate of A. aquatilis 3c was also increased along with the increase in decolorization percentage (Fig. 3a).

Multiple dyes decolorization

When A. aquatilis 3c was grown in the medium containing multiple dyes, it showed 27, 38, 62, 80 and 86% decolorization after incubation of 1, 2, 3, 4 and 5 days (Fig. 3b).

Dye degraded products analysis TLC

TLC analysis showed two bands with Rf value of 0.96 and 0.94 as compared to the control (original dye) band with Rf value of 0.83 when visualized under UV range of 254 and 366 nm (Additional file 1: Fig. S4).

HPLC

The chromatogram of the untreated dye sample showed three detectable peaks at retention time of 1.80, 2.88 and 5.00 min (Fig. 4a) while A. aquatilis 3c treated dye sample showed peaks at retention time of 1.99, 2.30, 2.95 and 3.76 min after 5 days of incubation (Fig. 4b).

FTIR

A comparison between the FTIR spectrum of control dye and extracted metabolites was given in Fig. 5. Two specific peaks of wavelength 1612/cm and 1532/cm are shown in FTIR spectrum of control dye which are due to the presence of -N=N-stretching. The peak with wavelength 1395/cm indicates C-O-H bending. The peak with a wavelength 1037/cm is due to C-O stretching. There is a variation in the peaks in the FTIR spectrum of metabolites extracted from bacterial decolorized sample of dye when compared to the control dye spectrum. The absence of peaks with wavelength 1612/cm and 1532/cm indicates the reductive cleavage of azo bond. The peak with wavelength 2850/cm and 2923/cm is due to stretching of alkanes. The peaks with 1755/cm and 1717/cm wavelength indicate the presence of C=O group. A peak of wavelength 1289/cm depicts the presence of carboxyl group. Two peaks with wavelength 1104/cm and 1037/ cm show the presence of amines (C-N). The presence of peak with wavelength 769/cm indicates the presence of conjugated C=C.

GC-MS analysis

In the present study, it is confirmed through GC-MS analysis that azo dye enzymatically converted into various end products. These metabolites are used in different pathways for example pyrrolo[1,2-a]pyrazine-1,4-dione derivative can be used as a substrate in amino acid metabolism. The amino acid catabolism can synthesize 3C compound (pyruvate) which can be converted into acetyl-CoA. The acetyl-CoA further undergoes Krebs cycle to generate reduced molecules (NADH 2 and FADH 2 ). Moreover, another end product, phthalate derivative can be changed into different fatty acids and aldehydes and these molecules can directly/indirectly enter into fatty acid β-oxidation reactions to produce NADH 2 and FADH 2 . These reduced molecules may be used in the process of ATP synthesis (Fig. 6).

Impact of decolorized dye wastewater on microbial growth

The plates were overlaid with microbial growth showing no inhibition zones and indicating that the decolorized dye wastewater is unharmful for the microbial growth (Fig. 7). Likewise, A. aquatilis 3c treated dye wastewater was tested for microbial toxicity assessment and was also found unharmful for microbial growth.

Discussion

Numerous microbes, counting fungi, yeasts, bacteria as well as algae, may decolourize and even cause whole mineralization of several azo colorants underneath sure ecological circumstances. Several assessments are accessible on the physiochemical as well as microbiological approaches for azo dyes decolourization Malik 2010, 2011;Singh et al. 2014;Das and Mishra 2016;Liu et al., 2018;Meerbergen et al. 2018). Aftab et al. (2011) reported a Corynebacterium sp. which decolorizes Reactive black 5 up to 60% and Reactive yellow 15 up to 76% within a period of 4 days when the initial concentration was 100 µg/ml. Shah (2014) reported three bacteria namely Pseudomonas putida, P. aeruginosa and B. subtilis capable of degrading multiple dyes i.e., Blue RR, Black B, Red RR, Yellow RR and Navy blue. P. putida showed maximum decolorization of Blue RR (95%), P. aeruginosa of Black B (93%) and Navy blue (70.58%) and B. subtilis of Yellow RR (65%) and Red RR (91%). Joe et al. (2008) reported a bacterium, Clostridium biofermentans, which was able to decolorize dyes Reactive red 3B-A, Reactive black 5, and Reactive yellow 3B-A, by over 90% within a period of 36 h. Likewise, Kaushik and Malik (2011) reported that the high dye removal efficiency (99.97%) and high uptake capacity (97.54 mg/g) of Aspergillus lentulus FJ172995 in 24 h using optimum process variables.

In the current study, A. aquatilis was found to decolorize 82% Synazol red 6HBN after incubation of 4 days at 37 °C and pH 7. Saha et al. (2017) reported two A. faecalis species namely A. faecalis E5.Cd and A. faecalis Fal.3 which decolorize up to 93% of Blue H/C and Red 3B dye at pH 7 and 94% of Yellow 3R at pH 8 within a period of 96 h. Both the strains decolorize up to 91% of dye at 35 °C, 92% with 50 ppm initial dye concentration and 93% with 20% inoculum size and supplementation of 1% co-substrate respectively. Shah et al. (2013) reported that Bacillus species i.e., B. cereus and B. megatarium having 95% and 98% dye decolorizing potential respectively under optimum conditions. Optimal condition for B. cereus was found to be 37 °C, pH 7, 1% sucrose, 0.25% peptone and 8% inoculum and that for B. megaterium was found to be 37 °C, pH 6, glucose 1%, 0.25% yeast extract and 10% inoculum. Different bands with Rf value were visualized in Direct orange 16 treated with Micrococcus luteus strain SSN2 and untreated samples when visualized under UV range of 254 nm (Singh et al. 2015). Likewise, a band with Rf value of 0.71 was visualized in the TLC chromatogram of decolorized sample of Direct red by Enterococcus faecalis YZ66 as compared to the untreated original dye which had a band with Rf value of 0.97 (Sahasrabudhe et al. 2014). Similar results were obtained against Amarnath mono azo dye decolorized by Acinetobacter calcoaceticus NCIM 2890 (Ghodake et al. 2011). In the present investigation, TLC analysis showed two bands with Rf value of 0.96 and 0.94 as compared to the control dye band with Rf value of 0.83 when visualized under UV range of 254 and 366 nm. Lade et al. (2015) observed three major peaks with retention time 2.521, 3.241 and 3.564 and two minor peaks of retention time 3.123 and 3.910 when extracted metabolites from Provedencia rettgeri decolorized C.I reactive blue 172 were analyzed through HPLC. The HPLC analysis of the control dye results in the presence  (Prasad and Rao 2014). In this study, A. aquatilis 3c treated dye sample showed peaks at retention time of 1.99, 2.30, 2.95 and 3.76 min after 16 days of incubation ( Fig. 4b) while dye sample showed three detectable peaks at retention time of 1.80, 2.88 and 5.00 min.

In the present study, two specific peaks of wavelength 1612 and 1532/cm are shown in FTIR spectrum of control dye while such peaks are absent in bacterium treated dye sample indicates the reductive cleavage of azo bond. The absence of peak of wavelength 1631/cm in FTIR spectrum of Marinobacter sp. strain HBRA treated Direct blue-1 sample as compared to the spectrum of control dye clearly demonstrated the degradation of azo bond (Prasad et al. 2013). Shyamala et al. (2014) reported the breakdown of Methyl orange by halotolerant Bacillus sp. as peaks of wavelength 1567/cm and 1424/cm which were present in FTIR spectrum of control dye were absent in spectrum of bacterially treated sample. Hungerer et al. (1996) reported that the azo-dye degraded compound 1,2-benzene dicarboxylic acid, butyl 8-methylnonyl ester was cleaved to form phthalic acid and palmitic acid. Our findings are similar with other studies that reported that the predominant compounds found in the final dye treated sample were 3-Aminobutanoic acid, pyrrolo pyrazine-1, 4-dione and palmitic acid (Harwood and Parales 1996;Walker and Van der Donk 2016;Shanmugam et al. 2017). In the current research, it is confirmed through GC-MS analysis that azo dye  Synazol red 6HBN dye enters into the cell (A. aquatilis 3c) by unknown mechanism. Upon entrance into the cell the dye is enzymatically processed into various end products. Most probably, azo group (-N=N-) is reduced, followed by cleavage reaction and different end products are produced. Secondly, desulfonation and oxidative deamination results in synthesis of pyrrolo[1,2-a]pyrazine-1,4-dione derivative which can be used as substrates in amino acid metabolism. The amino acid catabolism can synthesize pyruvate (3C compound) which can be converted into acetyl-CoA. The acetyl-CoA undergoes Krebs cycle to produce NADH 2 and FADH 2 (substrates of electron transport chain). Moreover, dye desulfonation, oxidative deamination and carboxylation lead to produce phthalate derivatives, which can be transformed into different fatty acids and aldehydes. The phthalate, fatty acids and aldehydes can directly/indirectly enter into fatty acid oxidation reactions (β-oxidation) to produce acetyl-CoA, NADH 2 and FADH 2 (See figure on next page.) Ajaz et al. AMB Expr (2019) 9:64 enzymatically converted into various end products which may be involved in the synthesis of energy yielding processes.

In the current work, A. aquatilis 3c treated dye wastewater was tested for microbial toxicity assessment and was also found unharmful for microbial growth. Aspergillus niger and Nigrospora sp. degraded dye end products containing wastewater was found safe for the growth of soil micro-flora (Ilyas and Rehman 2013). The degradation products of Sudan 1 by B. circulans BWL1061 and S. putrefaciens CN32 showed a decreased toxicity to E. coli BL21 and B. subtilis 168 (Liu et al. 2018). Similarly, Shah et al. (2012) reported the degradation of Reactive Orange 13 through enzyme assay and GC-MS analysis. The final products, naphthalene and 6-[(4-chloro-1,3,5triazin-2-yl) amino]-2-iminonaphthalen-1(2H)-one were non-toxic revealed by phytotoxicity study.

In conclusion, A. aquatilis 3c has a promising potential to decolorize Synazol red 6HBN (82%) after incubation of 4 days at its optimum growth conditions. The maximum dye decolorization was found under static conditions by using saw dust and yeast extract as carbon and nitrogen source. The bacterium also showed promising potential to decolorize multiple dyes at a rate of 86% in 5 days and dye degradation had positive effect on the growth of organism. This degradation was confirmed through TLC, HPLC, FTIR and GC-MS analysis and the end products are used in various metabolic pathways including ATP synthesis process. It was tested that bacterially decolorized dye wastewater is safe and sound for useful microbial flora. By using this promising decolorization ability of A. aquatilis 3c wastewater can be ameliorated and used at least for crops irrigation.

Fig. 2
2Optimization of decolorization conditions for A. aquatilis 3c a temperature, b pH, c incubation condition, d carbon source, e nitrogen source, f inoculum percentage, and g dye concentration

Fig. 3 a
3Effect of growth (O.D) on decolorization (%) of A. aquatilis 3c incubated for 5 days. b A. aquatilis 3c potential to decolorize multiple dyes incubated at 37 °C for a period of 5 days

Fig. 4
4HPLC analysis of reactive Synazol red 6HBN with a mobile phase of profile of methanol at 1.0 mL/min. The column was C-18 (250 × 4.6 mm) a represents dye chromatogram while b represents A. aquatilis 3c degraded dye products extracted after 5 days of incubation at 37 °C of one major peak with retention time of 2.702 min three minor peaks at retention time of 2.125, 2.801 and 3.394 min. HPLC analysis demonstrated the presence of two major peaks at retention time 2.45 and 2.68 and two minor peaks at retention time 3.50 and 6.50 min when B. halodurans decolorized Acid black-24. The chromatogram of original dye had only one peak at retention time of 2.81 min

Fig. 5
5FTIR spectrum of control dye (Synazol red 6HBN) and A. aquatilis 3c treated sample

Fig. 6
6Fig. 6 Synazol red 6HBN dye enters into the cell (A. aquatilis 3c) by unknown mechanism. Upon entrance into the cell the dye is enzymatically processed into various end products. Most probably, azo group (-N=N-) is reduced, followed by cleavage reaction and different end products are produced. Secondly, desulfonation and oxidative deamination results in synthesis of pyrrolo[1,2-a]pyrazine-1,4-dione derivative which can be used as substrates in amino acid metabolism. The amino acid catabolism can synthesize pyruvate (3C compound) which can be converted into acetyl-CoA. The acetyl-CoA undergoes Krebs cycle to produce NADH 2 and FADH 2 (substrates of electron transport chain). Moreover, dye desulfonation, oxidative deamination and carboxylation lead to produce phthalate derivatives, which can be transformed into different fatty acids and aldehydes. The phthalate, fatty acids and aldehydes can directly/indirectly enter into fatty acid oxidation reactions (β-oxidation) to produce acetyl-CoA, NADH 2 and FADH 2

Fig. 7
7Bacterial decolorized broth used for incubation of a B. subtilis (z-66), b B. megatarium (z-28) and c B. cereus (T358-2) at 37 °C for 48 h. No zone of inhibition was observed in any case

)SO 4 , 0.28; MgSO 4 ·7H 2 O, 0.04;NH 4 Cl, 0.23; KH 2 PO 4 , 0.067; FeCl 3 ·6H 2 O, 0.005; CaCl 2 ·2H 2 O, 0.022; yeast extract, 0.2; NaCl, 0.15; NaHCO 3 , 1.0 and 1 ml/l of a trace element solution containing (g/l): MnCl 2 ·4H 2 O, 0.1;ZnSO 4 ·7H 2 O, 0.01; CuSO 4 ·5H 2 O, 0.392; NaB 4 O 7 ·10 H 2 O, 0.177; CoCl 2 ·6H 2 O, 0.248 and NiCl 2 ·6H 2 O, 0.02

Table 1
1Morphological and biochemical characteristics of A. aquatilis 3c+ve: positive, -ve: negativeMorphological characteristics
A. aquatilis 3c

Shape
Spherical

Size
1 mm

Color
Pale yellow

Elevation
Non elevated

Edges
Smooth

Texture
Sticky

Gram's staining
Gram negative

Biochemical characteristics

Fermentation

Lactose
-ve

Sucrose
-ve

Dextrose
-ve

H 2 S production
-ve

Nitrate reduction
-ve

Indole production
-ve

Methl red reaction
-ve

Voges Proskauer reaction
-ve

Citrate use
±

Urease activity
-ve

Catalase activity
+ve

AcknowledgementsNot applicable.Additional fileAdditional file 1:Table S1. Decolorization (%) of bacteria isolated from 3 industrial samples.Figure S1. Growth of A. aquatilis 3c at various (a) temperature and (b) pH after incubation of 16 h.Figure S2. Growth of A. aquatilis 3c in LB medium. Optical density was taken at 600 nm after regular time interval.Figure S3. Phylogenetic tree constructed through MEGA7 to show homology of A. aqualitis 3c with Alcaligenes sp.Figure S4. TLC chromatogram of extracted metabolites of A. aquatilis 3c decolorized dye samples visualize UV range of (a) 254 and (b) 366 nm.Figure S5. Chromatograms of extracted metabolites of A. aquatilis 3c decolorized dye sample through GC-MS analysis.Authors' contributionsMA performed experiments and analyzed the results. AR designed and supervised the study and helped in manuscript preparation. SZH helped in conducting experiments. ZK and MAN helped in analyzing the data and manuscript preparation. All authors read and approved the final manuscript.FundingThis is to acknowledge the support of University of the Punjab (Grant no. Env-67), Lahore, Pakistan to accomplish this research work.Availability of data and materialsPlease contact author for data requests.Ethics approval and consent to participateNot applicable.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.
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