*Article* **Segmentation of Food Consumers Based on Their Sustainable Attitude**

**Michał Gazdecki 1,\*, Elzbieta Gory ´ ˙ nska-Goldmann 1, Marietta Kiss <sup>2</sup> and Zoltán Szakály <sup>2</sup>**


**Abstract:** The proposed study aims to segment consumers based on a sustainable approach to the consumption of food. The shift in consumers' attitudes towards more balanced food consumption can be one of the sustainability drivers for entire food chains and may result in more sustained energy usage in the whole food chain and implementation of farm to fork strategy to the practice. We considered consumers' attitudes as a multidimensional construct. Under this assumption, we asked respondents a series of questions related to the cognitive, behavioral, and affective components of an attitude. Data were collected from a market survey run among 433 consumers. We identified three consumer segments. The "Doers" segment exhibits sustainable behavior to a greater extent than the others. At the same time, they have less knowledge about the concept of food sustainability while the affective dimension was developed on an average level. The "Conscious" segment had well-developed cognitive and affective dimensions (which might indicate their openness to the information about sustainability positive feelings), however, it was not reflected in their behavior. Finally, the "Reluctant" segment, did not show a sustainable attitude towards food consumption in any of the analyzed dimensions. Answering the question of how common sustainable attitudes are may help in determining the market potential and in developing product and promotion strategies.

**Keywords:** sustainable consumption; responsible consumption; consumer segmentation; sustainable attitude; food products; consumers behavior

#### **1. Introduction**

It is hard to disagree that to combat climate changes the current consumption patterns must be altered globally [1–9]. The process of transformation of consumption plays an important role in facing environmental challenges, both globally and locally, and the sense of responsibility for the society, future generations, and the planet [10–20]. The Sustainable Development Goal (SDG) 12 Ensure sustainable consumption and production patterns defined by the European Commission have become a focal point of action until 2030 (Agenda 2030) [21]. This is reflected in the published strategy Farm to Fork [22,23] and the newest edition of the report "The State of Food Security and Nutrition in the World 2020" [24], where it was underscored that consumption patterns conforming to sustainable food consumption (SFC) will be playing a key role in achieving sustainable development, climate goals and satisfying the needs of the ever-growing population. Such processes are crucial nowadays as food markets are assessed as unbalanced. Production of raw materials and food processing requires a high energy input, creating negative side effects, such as greenhouse gas emission, food losses, and environmental burden.

The socio-economic transformation brought about qualitative and quantitative changes in food consumption. Other than by reducing the quantity of used goods and services, SFC can be also achieved by creating more appropriate consumption patterns, all with respect to the basic life needs and aspirations to improve the lives of current and future

**Citation:** Gazdecki, M.; Gory ´nska-Goldmann, E.; Kiss, M.;

Szakály, Z. Segmentation of Food Consumers Based on Their Sustainable Attitude. *Energies* **2021**, *14*, 3179. https://doi.org/10.3390/ en14113179

Academic Editor: Dimitrios A. Georgakellos

Received: 24 April 2021 Accepted: 24 May 2021 Published: 29 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

generations. Any changes to the structure of food consumption require the support of public policy, due to the presence of the lock-in effect [25,26]. Consumption patterns that make food consumption more sustainable should be popularized, and consumers should be perceived as actors playing the main role in creating a transition to a more sustainable food system [20,27–33]. Partha et al. [6] have correctly noted that the success of transition will depend on how individuals and households are convinced to change their consumption patterns and how the state cares, during the transformation of the model, about any vulnerable groups (i.e., those unaware or not motivated to alter their consumption) and indifferent groups [34].

Given the reasonable use of goods and services [26,35], the possibility of elimination of numerous, negative social and environmental effects and the preservation of natural resources for future generations, the sustainable food consumption model is desired [36], and it is necessary to implement strategies aimed at achieving it. This justifies the need to introduce systematic studies on consumers aiming to identify their ability to alter behaviors and a context that fosters such a change [37–41]. The understanding of phenomena present in the area of consumption forms the basis to design processes present in food supply chains, thanks to which they will be able to evolve in the direction of more sustainable processes [14,37,42–45].

Changes in consumption stem from the changing preferences and behaviors of consumers. Vermeir et al. [18] emphasize that although food preferences may be difficult to change, they may still develop in short periods, due to the dynamic transformations happening in the social, cultural, and economic environment. The analysis of behaviors of consumers, while considering the economy of sustainable development, calls for the analysis of signs of behaviors and their causes [3,4,46]. SFC studies, whose scope has been constantly expanding, show that the consumers' interest in SFC is lower than expected [43,47,48], while the scale of signs is not yet large [49,50]. The latest analyses show that despite the popularity of the idea of sustainable consumption and production, Europeans have not yet learned the concept of sustainable development (SD) well and cannot always identify it, with a marginally small number of the surveyed pointing that it is connected with nutrition [51,52]. The share of organic food in EU's market is estimated at 4%, and only 0.3% in Poland (with over 7% in Denmark and 4.4% in Germany) [53,54]. Still, the importance of food's health and environmental aspects are reflected in the growing trends, such as conscious, smart, critical, ethical, green, responsible, ecological, fair, shared, individual consumption, prosumption, deconsumption, food sharing, and freeganism [8,25,48,55–60]. "The Reflection Paper towards a Sustainable Europe by 2030" [61] shows that around 43 million people in the EU still cannot afford to eat a good quality meal every second day.

Further transformations of attitudes and behaviors of food consumers are required for SFC to grow, with such attitudes and behaviors possible to evaluate and identify based on segmentation [47,62]. There are plenty of literary resources discussing the issues of segmentation, with most of them often leading to distinguishing sub-groups, in turn allowing the identification of behaviors and motivations, along with an in-depth analysis of the character of attitudes [37,51]. Although many studies focus on the selection of sustainable food and consumption behaviors, the research efforts on consumer segmentation from the point of view of SFC are still limited [36,41].

Consumer segmentation is performed based on various criteria. Some of the criteria characterize the consumer as an individual and some as a group of consumers. For example, the first group may include consumption patterns, purchasing behaviors, motivation, the manner of perceiving a product, content perception, the level of satisfaction of needs, etc. [47,63,64]. The use of segmentation criteria describing groups of consumers has developed along with the world's globalization and expansion of planning horizons of target markets by international corporations with macro-criteria (such as economic prosperity, political and cultural system, infrastructure), which started to be used for

international market segmentation purposes [65,66] segmentation of groups of potential customers, from abroad.

The identification of consumer segments based on balancing their attitudes towards food consumption is of fundamental importance for the further development of SFC, especially in the practical aspect. Any efforts aiming to alter unsustainable attitudes and behaviors of consumers should be focused on learning and understanding ways to influence consumers to change their behaviors towards more sustainable food, positioning sustainable food, creating effective strategies and information campaigns [7,26,46,56,62,67,68]. The knowledge of consumer profiles allows the creation of a more efficient information and education policy, maintained both by the government and NGOs.

The purpose of this paper is to segment consumers based on a sustainable approach to the consumption of food. The prospects for a more sustainable consumption depend on the ability to improve the innovativeness level of consumers, enterprises, and science and of government and public institutions. Undoubtedly, the creation of a favorable environment that promotes development of consumption sustainability requires a closer cooperation between the academic environment, industry, government, and social organizations. In the context of the topic addressed in this paper, it is important to know consumer attitudes towards sustainable food consumption. The consumer segmentation presented in this paper narrows that gap.

#### **2. Review of Studies on Consumer Segmentation with Regard to SFC**

The review of studies on consumer segmentation in relation to the concept of SFC has been prepared as the result of searching databases of scientific publications. The search covered the articles made accessible from 1990 until March 2020 in Web of Science and Scopus. Publications were selected if the searched phrases appeared in one of the following elements: thematic sections, keywords, titles, and abstracts. The following phrases were selected for searching: "food", "consumption", "segmentation", "segment", followed by "sustainable consumption" and "cluster analysis".

The search of the Scopus base yielded 24 records, while the Web of Science database returned 13 records. When assessing the usefulness of publications using Moher's et al. [69] method, twenty-seven papers published after 2010 were identified (Figure 1).

**Figure 1.** Selection of literary sources.

Dominant subject areas (categories) of the publications were agricultural and biological sciences, environmental sciences, social science, economics, econometrics and finance, business, green sustainable science technology, and environmental engineering. Analyzing the findings by country, it was found that a significant part of them were from Europe, especially Italy, Germany, and the Netherlands. The studies within the analyzed area have been performed, among others, by: Wageningen University and Research Centre (4 publications); Parthenope University of Naples (2); Gent University (2); Aarthus University (2); University of Gottingen (2). The articles have been analyzed and evaluated in terms of usefulness by two, independent scientific institutions (UPP and UD). To be included in the project, an article had to be prepared based on original data. The details of every publication have been identified by the first researcher, then verified by the second researcher and presented in the collective list in Table 1. Any publications that failed to satisfy these criteria have been excluded from analysis.


**Table 1.** Overview of the 27 selected papers.


**Table 1.** *Cont.*


**Table 1.** *Cont.*

The following categories of variables were specified after the analysis of the papers (Table 1): environmental sustainability, socio-demographic, psychographic, economic, behavioral, affective factors, lifestyle, values. Food consumption is a complex issue and calls for a broader perspective [41,92]. In order to explain the consumers' behaviors, the researchers used in their studies wide ranges of variables connected to the lifestyle [71,75], personal traits [7,86], values [80,90,93], preferences, purchasing behaviors [7,26,47,62,70,78, 81,84–86], behaviors related to food consumption, the consumers' attitude to sustainable food consumption [62,75,78,91], environmental issues [68,70,72–74,76,77,79,81,82,88–91] and behaviors that would limit sustainable choices [62,70,72,73,75,76,78,79,81,88,89].

The authors of the analyzed works employed various statistical methods to identify consumer segments. Factor analysis and cluster analysis are among the most used ones [26, 62,68,71–76,79,80,87–90,93]. The other methods included modelling (e.g., logit model [70]; latent class modelling (LCM) [85]; conceptual model [81]; mixed logit model [47], conjoint analysis model [62,84,91]), the use of selected techniques with regard to data mining [78] and multivariate analysis [77]. The following part of the paper presents individual categories of variables identified in the analyzed papers.

#### *2.1. Socio-Demographics*

Socio-demographics have been used in most of the cited works. This type of variable is one of the commonly used variables in consumer behavior studies [7,73,81]; they are

easy to measure and strongly determine consumer's behaviors, which in turn makes them work well as a basis of segmentation.

Gender of consumers was considered in 21 papers (Table 2). For instance, Gen Z segments of the U.S. sustainable food market differ with respect to gender. In addition, Verain et al. [7] stated that "unsustainers" and "product-oriented" consumers were more often male than female compared with "curtailers". Analyzing two sub-samples characterized by different age: "millennials" (18–35 years) and "non-millennials" (36–88 years) revealed that gender is significant and positive only in the category of "non-millennials", while the income level is significant and positive for "millennials". Sogari et al. [81] who ran a study on the vine market noted that "if in the past wine was seen as mainly a male beverage, in recent years more and more females are becoming wine aficionados". Significant gender differences across segments were shown by Su et al. [89], who analyzed consumer groups based on environmental consciousness.

**Table 2.** Socio-demographic factors used in the analyzed papers.


Age of consumers was taken into consideration in 22 papers; in 7 out of the 22 papers noted, significant age differences depended on the consumer segment [7,68,70,73–75,81]. For example, Vanhonacker et al. [68] concluded that the "Unwilling" segment were the youngest on average and significantly younger than the "Active" and the "Uncertain" (the oldest segment). Further, the "Conscious" were significantly younger than the "Uncertain". The "Unwilling" segment was the opposite of the "Active" segment and combined a high personal footprint with a low personal relevance.

Education of consumers was considered in 21 of the analyzed papers. Seven articles showed that education significantly diversifies the consumers' behaviors and attitudes [68,70,72,74,75,79,88]. Vanhonacker et al. [68] found that the large majority of the "Conscious", "Active" and "Unwilling" consumers were higher educated, while a more balanced distribution in education level was found among the "Ignorant" and the "Uncertain". Ghvanidze et al. [88] stated that the "Apathetic" segment had high levels of education and income.

Other variables used in the discussed papers, but on a smaller scale, were, among others: household size [62,70,72,75,76,85], living environment/residence (or type of settlement) [68,74,76–78], family composition/household composition [7,73], and country class [71]. Some of the least used variables in this category included social status, origin/ethnicity, living environment (rural or urban), occupation, family composition, household size, family member, housing tenure, partnership, kids, residential region, country class and leisure time activity.

#### *2.2. Environmental Sustainability*

11 out of the 27 articles used environmental sustainability variables (Table 3) [68,70, 72,74,76–79,81,82,87,90]. Various, detailed variables were identified among these, such as footprint-related, certificates, pro-environmental habitual activity, environmental awareness and related to the product and/or production process.


**Table 3.** Environmental sustainability factors used in the analyzed papers.

Two of the discussed publications focused on the selection and consumption of food and the related impact on the environment measured by the "ecological footprint" [68,76]. Vanhonacker et al. [68] show that numerous consumers fail to see the impact of animal production on the natural environment. They also point to the presence of alternative behaviors in relation to conventional meat consumption, for which eating habits and cultural patterns must be adapted. However, the readiness to pay higher prices is significantly lower than the readiness to consume. Mózner [76], however, has noted that consumers who ate more fruit, vegetables, and milk products did not have a smaller ecological footprint in terms of the entire food consumption.

Three publications examined the issue of food certification [70,77,82]. The used certification systems differ in terms of information value, methods of assigning and conducting monitoring activities. The impact of the origin certificate on consumers has been confirmed

by Bronnmann and Asche [70], who showed that consumers were more willing to pay a higher price and were more eager to make purchases, all in relation to wild fish. Similar results confirming the readiness to pay a higher price have been also obtained by Aprile and Mariani [77], Janßen and Langen [82], and La Lama et al. [79].

The consumers' focus on environmental protection issues have been also considered in the studies by Jakubowska and Radzymi ´nska [87] and by La Lama et al. [79]. Consumers can be divided into ones expressing strong pro-environment attitudes (and health-related values) and ones that do not take environmental aspects into account when selecting food [87]. Similar results have been obtained by Su et al. [89] who stated that the sustainable food market can be segmented according to environmental awareness. Sogari et al. [81] have identified a connection between the selection of a product and the consumer's engagement in the purchasing process and their environmental protection awareness.

#### *2.3. Behavioral Factors*

Behavioral changes have been used in 19 out of 27 articles [7,26,47,62,68,70,72,74,76, 78,80–82,84–86,88,91]. An overview of examples of the factors that have been included in the segmentation studies of the cited authors can be found in Table 4. The behavioral factors consisted mostly of buying behavior and general food choice motives/attribute importance by food category sustainability, general/life attitude, consumption habits, occasional behavior, ethical issues, consumer preferences for product information related to environmental issues.


**Table 4.** Behavioral factors used in the analyzed papers.


**Table 4.** *Cont.*

A few of the issues used for the purposes of establishing consumer segmentation are their behaviors related to purchasing ecological food products and their willingness to pay the price. For example, considering the willingness to pay measures for the respective attributes, it has been noted that the buyers of ecological food were more inclined to pay a premium for ecological products [47,62]. Vanhonacker et al. [68] and Palmieri and Forleo [26] have confirmed that consumers were more inclined to buy products perceived as more sustainable. Consumers exhibiting a more sustainable approach are also more willing to buy innovative products.

An important aspect is also the identification of factors behind the selection of food, with health and quality attributes playing an important role [62]. Risius et al. [85], who segmented fish consumers, emphasized the major importance of the country of origin. Lots of attention was also paid to connecting consumer values with purchasing behaviors. An example could be the attention paid by humans to the natural environment or the wellbeing of animals [93]. Verain et al. [7] found that food involvement and personal norms with regard to healthy and sustainable food positively affect sustainable food choices. Annunziata and Mariani [62] emphasized that the importance of ethical values (i.e., that food was produced with complete observance of human rights or with no abuse to women and children) and environmental values (e.g., that food was produced in an uncontaminated environment, in an eco-friendly manner and with support for local farmers).

The behaviors are related to the level of the consumers' awareness about sustainability, as confirmed by Prokeinová and Paluchová [78] with regard to the younger consumers who choose environmentally friendly and socially acceptable products more willingly than their parents.

#### *2.4. Psychographic Factors*

Psychographic variables have been used as a basis for segmentation in three of the analyzed papers [7,75,86]. Verain et al. [7] have confirmed that social and personal norms, ability, subjective knowledge, and food involvement significantly differentiate meat consumers' behaviors. For example, "product-oriented consumers differ from each other in that the product-oriented attach more importance to social norms and have a higher ability to judge sustainably produced food, and subjective knowledge on sustainable products". Van Huy et al. [75] have focused, among other aspects, on the attention to healthy food, love of cooking, convenience, and love of local and organic food. Wang and Somogyi [86]

have examined the level of acceptability of crustaceans from sustainable production among Chinese consumers. They have found that personal standards significantly influence the purchase intentions of consumers.

#### *2.5. Economic Factors*

A wide range of economic variables, such as net household income per month, financial status of household, and employment status, were used as segmentation criteria or profiling variables. For example, Verain et al. [7] have identified four consumer segments that differ significantly regarding economic factors, e.g., segments named "Curtailers" have lower incomes compared to the "Sustainers" segment. The study by Van Huy et al. [75] has highlighted a nexus between the identified segments and the level of income of the consumers. Yildirim and Candan [93] have concluded that most green product buyers were at a high level of income. These results show that a more prominent presence of sustainable attitudes can be expected from the wealthier consumers.

The employment status has been included in three articles, and only in one study, significant differences across segments have been found by Lavelle et al. [72], who stated that respondents differ in their uptake of occasional and habitual pro-environmental behaviors. Considerable differences exist between the two behavior groups with regard to key socio-demographic variables, such as employment status and income, as well as residential location and housing tenure.

#### *2.6. Affective Factors*

Two of the presented articles considered the affective factors. In one of them, Krystallis [91] has taken into account consumers' assessments related to environmental factors, the perception of processing and well-being of animals in the case of processed meat. On the other hand, Hasanzade et al. [84] have analyzed the connection between ethical product attributes (e.g., ethical criteria of animal welfare, environmental protection, and labor and human rights) with the selected elements of behaviors.

#### *2.7. Lifestyle and Values Factors*

As shown by Aydin and Ünal [93], the lifestyle of consumers is related to the sustainability of consumption behaviors. In the publications of Thøgersen [71] and Van Huy et al., [75] the food-related lifestyle (FRL) model developed by Grunert et al. [94] has been used. For example, Thøgersen [71] investigates how the country of residence and FRL interact in shaping (un)sustainable food consumption patterns. The analysis has revealed that the outcome variables vary significantly across FRL segments. Further, after controlling for FRL, the direct effect of country class is highly significant for meatless suppers and marginally significant for buying organic food but non-significant for eating beef and food innovativeness. Van Huy et al. [75] have stated that the FRL model enables better understanding of how consumers employ food and its culinary aspects to achieve certain values in their lives. The acceptance of a specific lifestyle by consumers plays an important role in creating a "green economy" [95].

Consumer value-related approach represents a study by Hölker et al. [80] which developed consumer segmentation based on the human–animal relationship. The segmentation has been prepared according to specific values, such as animal rights, utilitarianism, new contractarian approach, abolitionism, original anthropocentrism, and anthropocentrism with indirect duties. At the same time, Yildirim and Candan [83] have found that green product buyers could be segmented into subgroups according to their personal values and consumption values. Significant differences regarding personal values, especially related to environmental matters, have been confirmed by Jakubowska and Radzymi ´nska [87] as well.

The presented content proves that there are studies on consumer attitudes and behaviors in relation to SFC. Most of the researchers have focused on capturing individual factors or groups of factors allowing the identification of symptoms of sustainable behav-

iors of consumers. The researchers focused on environmental aspects (food choices and practices beneficial to the environment). It is also understood that other factors can also impact attitudes, which are basic determinants of human behavior [96]. Some of the studies have specified the attitude of consumers towards food categories (e.g., organic products), while some other towards products (e.g., fish, seaweed, lettuce, wine). The authors of all studies considered the behavioral component and many studies also accounted for the attitude's affective component [26,71–75,79,86–89]. It is worth noting that accounting for consumer attitudes in three dimensions was an infrequently taken approach, which forms a theoretical basis for the considerations in our article. A similar perspective has also been applied by, for instance, Jakubowska and Radzymi ´nska [87], La Lama et al. [79], Palmieri and Forleo [26], Thøgersen [71], Van Huy et al. [75], Vanhonacker et al. [68].

#### **3. Methodological Approach**

The article is based on results of the authors' own surveys conducted by means of direct interviews. The study aims to identify sustainable consumer behaviors with regard to food consumption.

The general methodological approach is presented in Figure 2. A plan for collecting the data was developed, with the assumption that the goal would be to distinguish consumer segments by the level of sustainability of food consumption. The plan covered: developing a method of selecting consumers for the study and organizing the study itself, namely training interviewers and determining the time frame of the study. Afterward, a questionnaire was developed and put to test, ultimately serving to collect the research material. Once the data had been collected, the raw material was subjected to formal and substantive evaluation. The prepared material was then subjected to clustering by means of a non-hierarchical clustering algorithm. The distinguished segments were then described.

**Figure 2.** Methodological approach of the research.

#### *3.1. Selection of Respondents*

The respondents selected for the study are adults, over 18 years of age, living in the region of Wielkopolska, Poland. The region was selected because: (1) the inhabitants of economically developed regions usually have higher education levels and higher revenues, etc., which means this concept is more widespread in such regions; (2) new directions in food consumption and nutrition usually spread among larger urban agglomerations, inhabitants of regions developed socially and economically, for whom they are certain, sought for a model of food consumption; (3) Wielkopolska is one of the best economically developed regions in Poland (in terms of gross GDP and growth, foreign capital involvement concentration and investment expenses, unemployment rate, revenues, education, and human capital potential) [97].

The quota sampling method was used for the selection of respondents for the study. Public statistics data served as a basis to determine the sample's structure that reflected the structure of Wielkopolska inhabitants in terms of age and gender. The sample was 433 individuals. The social and demographic characteristics of the survey's participants is presented in Table 5.


**Table 5.** Respondents sample structure.

#### *3.2. Questionnaire Development and Data Collection*

Coming from the concept of the approach proposed by Breckler [98], which assumes three-dimensionality of attitudes towards SC, the questionnaire used in the survey has been divided into three parts. Each part contained questions related to the cognitive, behavioral, and affective components, respectively. Questions characterizing food consumption sustainability were asked within each area.

The first version of the questionnaire was subjected to pilot tests. The test consisted of 30 personal interviews with individuals who represented the target group. The purpose of the test was to check the questionnaire:


After collecting remarks from interviewers who did pilot tests and following the analysis of respondents' answers, the necessary corrections were introduced, mainly related to the formulation of questions. The final questionnaire, used in the study, is the result of the introduction of these changes.

The face-to-face interviews for the study were performed between November and December 2019.

#### *3.3. Data Cleaning*

The collected material was subjected to post hoc data cleaning approach. The procedure of data cleaning assumes they are verified by the researchers (Exploratory Data Analysis) [99] and consisted of checking outliers in order to identify any errors that would occur during uploading or digitizing the data; checking the presence of any missing values and replacing them with a median, if found; and checking for any duplicated records in the database and removing such duplicates, if found. The cleaned database was subjected to statistical analysis.

#### *3.4. Data Analysis-Clustering*

We used a k-means cluster analysis (non-hierarchical clustering algorithm), which groups objects on a set of user-selected characteristics. The resulting clusters should exhibit high internal (within-cluster) homogeneity and high external (between-cluster) heterogeneity. Thus, if the classification is successful, objects within clusters will be close together when plotted geometrically and different clusters will be far apart. Cluster analysis was chosen because of the data reduction procedure, which is done objectively by reducing the information from an entire population or sample to information about specific groups, as a large number of observations are meaningless unless classified into manageable groups [100].

Based on selected questions within each of the SC dimensions (cognitive, behavioral, and affective), 3 indices were created, by transforming all variables into dichotomic ones and aggregating sums of answers. There are two types of variables:


Because of the different number of variables used for each dimension, it was necessary to make sure we worked with roughly equal amplitudes within each index. Cluster analysis is sensitive to different scales, as it uses distance measures, therefore by using a mean and standard deviation values of each section, we standardized them with z-score transformation (z = (x − μ)/σ) [101], to eliminate the impact of larger standard deviation.

#### Description of Dimensions

As the three-dimensional model of attitudes was employed, each dimension was reflected by the number of questions in the questionnaire (Table 6). For the cognitive dimension, 6 questions (22 variables in the data) were used to measure and to differentiate respondents by their awareness of the sustainable consumption concept. The behavioral dimension covers 4 questions, representing 28 variables in the data. In the affective dimension, we used 2 questions, based on which 17 variables in the data were created.


#### **Table 6.** Segmentation variables used for the study.


**Table 6.** *Cont.*


#### **Table 6.** *Cont.*

#### *3.5. Clustering*

When running the analysis, the number of clusters was specified as 2, 3 and 4, to compare them and find the best solution. As the 2-cluster solution gave the result of data division into negative values of cognitive, behavioral, and evaluative indices for one cluster and positive for another one, that does not provide an insightful interpretation. As we needed to choose a better solution from two options, i.e., 3-clusters and 4-clusters, we run a validation procedure and assessed differences of cognitive, behavioral, affective indexes between clusters. Considering that in the 4-cluster solution there are more pairs with an insignificant difference, we decided to choose a 3-cluster solution for further interpretation.

The relative centers of each cluster on a standardized scale, are presented in Table 7, and reflect differences between the clusters. The first cluster has the highest values of behavioral aspect, which means that this group represents the most sustainable pattern of behavior. The second one is the only one with positive values for cognitive aspects, so respondents with the highest awareness level. The third group includes respondents with the lowest values for each aspect.

**Table 7.** Final cluster centers.


The ANOVA analysis allows determination of the importance of each index with the relative weight. For the 3 indices, F values are large and all of them are significant (*p* < 0.001), indicating that they have a significant impact on the results for the 3-cluster solution (Table 8).


**Table 8.** ANOVA analysis for 3 clusters.

To validate the analysis, we created cluster membership and compare, whether mean values of each cluster are significantly different within each index. All 3 clusters are significantly different, so we can reject the null hypothesis, that the group means are all equal in all indices:


Nevertheless, we have results showing that not all group means are equal, so we should check whether pair comparison shows a significant difference.

To sum up, there are 3 clusters that describe and differentiate all observations:


#### **4. Results**

As a description of the results, we will first present a synthetic description of segments, followed by the characteristics of their representatives within three attitude dimensions. We identified three segments (clusters) and based on the dominant characteristics named them as: cluster 1—"Doers", cluster 2—"Conscious" and cluster 3—"Reluctant" their characteristics can be presented on the basis of the data in Table 9.


**Table 9.** Consumer segments.


**Table 9.** *Cont.*

Cluster 1—segment "Doers"

This segment included 31% of food consumers. These are action-oriented people, whose consumption-oriented behaviors show a higher number of sustainable behaviors than in the case of other people, despite the fact that their knowledge about sustainable consumption is low and their opinions about this concept are moderately positive. This means that the reasons behind their (sustainable) behaviors may come from other areas, e.g., the socialization or upbringing process, material situation, living environment, etc. This segment shows a significantly higher percentage of people living in rural areas. The members of this segment have diversified incomes, whose distribution is similar to the one in the examined sample. These people are slightly older than the average for the examined group.

Cluster 2—segment "Conscious"

This segment included 30% of food consumers. The individuals in this segment have a high value of indexes within the cognitive and affective dimensions of attitudes, but a noticeably lower intensity of sustainable behaviors. These consumers have a better understanding of and a more positive attitude towards the sustainability concept but have not yet introduced a higher number of sustainable behaviors. However, their higher awareness may be considered a good foundation to grow into a fully sustainable attitude. This segment has a higher representation of women. It is also a segment with a major share of young people and the highest share of individuals with higher education in the identified segments (which may determine the higher level of awareness). The group's higher level of income and satisfaction from material status should be also underscored.

Cluster 3—segment "Reluctant"

This segment included 39% of food consumers. This group consists of individuals with negative attitudes towards the idea of sustainable consumption, manifested both in the low awareness of concepts, negative opinions of them, and low importance of sustainable behaviors. The group is nearly equally represented by men and women, with age distribution similar to the distribution in the examined group. The representatives of this segment may be encountered in towns of various sizes. The profiling data show that this segment is more often populated by individuals with lower education, whose material situation is poor (they declare lower satisfaction with their incomes and that their incomes are lower).

Segment characteristics in the cognitive sphere

Significant differences can be spotted among segments in the cognitive sphere. The individuals in the "Conscious" segment had a high awareness of the terms used for describing sustainable consumption behaviors. In total, 87% of its representatives have met with the term "sustainable consumption". In the case of "Doers" and "Reluctant" segments, this was 36% and 19%, respectively. We can also observe a high activity of the "Conscious" segment individuals in the search for information about food and a high amount of the retained information. The "Reluctant" segment individuals declare looking for information about food in the least degree (17% of the segment's representatives). Additionally, they pay little attention to information about consumption sustainability.

Segment characteristics in the behavioral sphere

The readiness to introduce changes in nutrition is mainly declared by individuals in the "Doers" and "Conscious" segments (67% and 70%, respectively)—however, these changes are of a different nature. The representatives of the "Doers" segment refer to the sustainability concept to a higher degree (e.g., increasing the share of fruit and vegetables in their diets, preparing meals by cooking them themselves). They also more frequently declared limiting undesirable behaviors such as eating fried meals and meals with a highfat content, consuming sugar, salt, and stimulants. The "Doers" representatives limited meat consumption to the highest degree. The members of the "Conscious" segment would introduce similar changes, but they were declared with lower frequency. This segment dominates only in terms of introducing healthy and organic products to the diet. The nutritional changes were the least common in the "Reluctant" segment, declared by around 1/3 of the segment's participants. Moreover, they were related to the sustainable consumption principles to a lower degree than in the remaining segments.

The individuals in the "Does" and "Conscious" segments more often declared changes related to sourcing food for their households. The use of large retail facilities (such as supermarkets and hypermarkets) grew among the "Doers". This could be related to the fact that half of the members of this segment live outside towns, which is where structural changes to retail have been happening in the recent years, leading to changes in purchasing models. Different behaviors are represented by the representatives of the "Conscious" and "Reluctant" segments, both very similar to each other in terms of the place of living. The "Conscious" segment was more eager to use smaller, specialized shops and marketplaces. On the other hand, the "Reluctant" segment preferred large-size retailer and increased the importance of online shopping for food.

The "Does" and "Conscious" segments are changing their behaviors related to sourcing food in the direction convergent with sustainability principles, but they are doing it differently. For example, the "Doers" more frequently declare limiting wastage. For the "Conscious" segment, however, making food products autonomously and purchasing low-processed products are more typical.

Segment characteristics in the affective sphere

Certain similarities emerge between the "Doers" and the "Conscious" segments, and a major difference in relation to the "Reluctant" segment. The two first segments have a positive attitude towards sustainable consumption principles and appreciate the impact of such behaviors on the local communities, environment, and local economy. Still, the individuals in the "Conscious" segment are firmer in this regard than the "Doers". For example, they have a higher propensity to pay more for organic products or spend extra time on sustainable behaviors, e.g., searching for information on nutrition principles. The individuals in the "Reluctant" segment, on the other hand, assess various signs of sustainable consumption as disadvantageous for them.

Contrary to our expectations, not all consumers are interested in food and sustainable consumption. The "Doers" are interested in food for various reasons, which fail to cover the issue of sustainable consumption. There may be an impression that this topic is disregarded by such individuals. The "Conscious" segment consisted of consumers actively looking for information about food and nutrition, sensitive to the issues of sustainable consumption.

#### **5. Discussion**

The results we obtained expand the current consumer segmentation models with regard to attitudes towards sustainability. Similar to several studies conducted in other countries [7,47,62,73–75,79,82,84,89], we proposed three segments of consumers.

Consumers have varying awareness on consumption sustainability (behavioral aspect), buying and eating behaviors reinforcing responsibility towards the planet and future generations (behavioral aspect), the perception of promotional messages related to the balancing of food product choices, and the subjective evaluation of benefits for consumers resulting from sustainable behaviors (affective aspect). The study is one of numerous

papers about profiling food consumers based on a three-element attitude dimension in relation to SFC.

#### *5.1. Cognitive Dimension*

Our studies have revealed that the information about environmental, economic, and social consequences of excessive consumption are factors that foster the popularization of sustainable attitudes. The force of various media should be used to reduce adverse behaviors [16,75,102] by building SC awareness and affecting consumption behaviors, as supported by e.g., Hasanzade et al. [84], Prokeinová and Paluchová [78], Sogri et al. [81], Wang and Somogyi [86], Verain et al. [73]. A better understanding of the characteristics of consumers via segmentation facilitates the preparation of a more effective communication strategy. Public institutions, food producers, and commerce should take steps leading to a better understanding of food and nutrition, potential benefits for the environment, responsibility for the planet and future generations. The existence of a connection between the type of consumers and susceptibility to messages communicated, for example, via social media has been indicated by Sogari et al. [81] claiming that "the greater the importance the consumer places on the product/process dimension of environmental sustainability, the higher the self-selection in market segments". The researchers also pointed to the need to increase the possibilities of communicating the activities of enterprises in environmental protection. Hasanzade et al. [84] have added to the literary references the differentiation of consumer segments due to behaviors resulting from the way they react to messages, additionally showing the need to carefully select information about products (e.g., the product's ethical character). However, not all studies confirm the need to differentiate communication efforts. According to Verain et al. [73] there exist messages that can have a universal character and reach all consumer groups.

#### *5.2. Behavioral Dimension*

Labelling products as environmentally friendly and promoting "new food" may be helpful in reinforcing sustainable consumer choices [74,85,103]. The results of our studies show that marking food products as healthy and organic may be important for the "Conscious" segment. This segment is similar to the "Trendsetters" segment that appeared in the study by Van Huy et al. [75].

Nevertheless, our study fails to provide a detailed insight into the issue of a sustainable diet, which is discussed, for example, by Verain et al. [7,73] who have confirmed that consumer segments are differentiated by the approach to health and eating healthy, organic food products. The interest in healthy eating has been also noted by Van Huy et al. [75], who noted that the interest in healthy nutrition is positively related to organic, locally sourced food.

Verain et al. [73] have proved that individuals oriented toward sustainable growth have been limiting the consumption of meat after learning about the benefits this approach provides both to their health and the environment. This is also confirmed by our study since the inclination to eat meat has been used to differentiate the segments. Meat consumption has been limited to the highest degree by the "Doers", closely trailed by the "Conscious" segment.

The results of our study show a relation between the place of residence of consumers and their sustainable attitudes. It seems that people living in rural areas have "natural" sustainable attitudes (mainly in the behavioral dimension), which may be related to the specific characteristic of the rural environment as a place for living and working. La Lama et al. [79] also point to the role played by the place of residence of consumers, additionally pointing to other factors such as income, lifestyle, access to frequently updated information, and telecommunication technologies.

Vermeir et al. [18] have stated that "many consumers express environmental concern but do not consistently act on it. That is, consumer attitudes toward environmental sustainability are mainly positive, but there is a notable gap between favorable disposition and actual purchase of sustainable food products, i.e., the attitude-behavior gap". Similar conclusions can be formulated on the basis of our segmentation—the "Conscious" segment shows a well-developed cognitive and affective dimension of the attitude, with a less developed behavioral dimension.

#### *5.3. Affective Dimension*

The affective dimension proves the consumers' positive approach towards the concept and principles of sustainable consumption and their commitment to the idea. The results we have obtained show that this is fostered by the consumers' higher material status, although this is not the only condition. The "Conscious" segment is quicker to see health benefits coming from SC than the "Doers". This segment shows similarities to consumers of cluster 3 in the research conducted by Verain et al. [73], who pay close attention to SC attributes. In the segmentation done by La Lama et al. [79] one of the segments is "Skeptical". It consists of consumers with an egocentric approach and little contact with modern food production practices, who are reluctant to pay higher prices for welfarefriendly products. The segment is similar to our "Reluctant" segment, showing a negative attitude towards the sustainability concept. Groups of consumers with a negative attitude towards SC have a significant impact on the development of the market of products covered by this concept. The research conducted by Gerini et al. [47] has shown that consumers may exhibit positive attitudes towards sustainable products, while being reluctant to pay higher prices for them. If most consumers are not willing to pay more for, e.g., organic products, whose production is more costly, such products will not be able to capture a higher share of the market. The growth of the market for sustainable products and services should be supported by good access to information [27,47,104,105]. The factor supporting the growth of this market may include references to consumer ethnocentrism and localism, as discussed, for example, by Van Huy et al. [75] on the basis of studies conducted among consumers in Vietnam, and to personal responsibility [47].

#### **6. Conclusions**

Conclusions in two areas may be formulated on the basis of our work. The first conclusion applies to the review of papers on food consumer segmentation based on the sustainability concept. The second one covers the segmentation and characterization of such segments in the region of Wielkopolska, Poland.

It may be concluded, on the basis of the literature review, that eight categories of variables are used for the purposes of segmentation of consumers with regard to food consumption. These were: environmental sustainability, socio-demographic, psychographic, economic, behavioral, affective factors, lifestyle, and consumers' values. The factors related to the environment and consumption behaviors should be considered the dominating category. The multi-faceted nature of consumer attitudes leads researchers to use a wide range of variables related to lifestyle, values, preferences, and consumers' attitude towards sustainable food consumption and environmental issues. Given the significant diversity of the used factors, it may be also observed that individual papers most usually put emphasis on one of the dimensions of the attitude. The theoretical foundation of the approach to segmentation proposed by us is the concept of a three-element attitude structure, thanks to which factors that may be indicators of sustainability can be approached comprehensively. The results we obtained prove that such an approach is efficient in the segmentation of consumers.

The consumers' segmentation model proposed in that paper contributes to the knowledge about consumers' sustainable behavior and might be used for further research development and as well as by practitioners and consumer policymakers. Identified segments represent a different potential for adoption of sustainable behaviors what implies the necessity of implementing various methods of promoting the idea of sustainability among them.

Two of the identified segments ("Doers" and "Conscious") represent a certain escalation of sustainable attitudes, and the characteristics of these segments show that they may

grow in the future. The representatives of the third segment ("Reluctant") are negatively oriented towards the sustainability concept. Taking into account the distribution of the size of individual segments, it may be assumed that around 60% of consumers make up a group that may positively modify their attitudes towards sustainable consumption. Consequently, this group is the target of activities that may be taken to support the development of sustainable attitudes. A universal set of activities promoting sustainable food consumption attitudes should cover activities in two main areas. The first one would be to build consumer awareness in relation to the concepts of sustainability and sustainable consumption, along with the derived benefits. This area should mainly impact the "Doers"—thanks to the reinforcement of the consumers' knowledge it will be possible to achieve the effect of further intensification of behaviors. The second area would be activities incentivizing to take sustainable activities, on the basis of the already developed consumer awareness.

Understanding and recognizing consumers' attitudes and behaviors is useful for industrial practitioners and decision-makers making efforts to transition into more sustainable food systems. Information and communication strategies should be built upon full knowledge about food consumers from a given region, taking into consideration the three-dimensional nature of their attitudes. By adapting the content of messages to the profiles of specific consumer segments, emphasis should be applied to informing about benefits coming from the consumption of sustainable food, in order to motivate the sensitive segments and to raise the consumers' awareness about the benefits stemming from the pursuit of the sustainable food consumption model.

#### *Limitations and Recommendations for Further Research*

A conclusion can be drawn from our literary research that the food consumption segmentation proposition presented in the article is one of very few propositions that apply to the concept of making food consumption attitudes sustainable and is based on a three-element attitude concept. Maintaining such an approach in the future will make result comparison possible, leading to a better understanding of the consumers.

Our study is limited by a relatively low sample size and its regional reach, which implicates the need to continues similar studies, but in pan-regional and international scope. It is also worth expanding the future studies with qualitative elements, for a better understanding of mechanisms of shaping sustainable attitudes.

The knowledge of signs of sustainable consumption and factors that determine it is still emerging and needs intensive studies. It would be beneficial to direct such future studies at methods of combating any factors that hinder the popularization of sustainable attitudes. Examples of valuable directions would be the impact of retail structure on making food consumption behaviors sustainable, or the characteristics of one's living environment as a factor determining the sustainability of attitudes. Additionally, depending on the type of food considered by consumers, their attitudes can be differentiated.

**Author Contributions:** Conceptualization M.G., E.G.-G., M.K. and Z.S.; methodology M.G., E.G.-G., M.K. and Z.S.; software M.G. and E.G.-G.; validation M.G. and E.G.-G.; formal analysis M.G. and E.G.-G.; investigation M.G. and E.G.-G.; resources M.G., E.G.-G., M.K. and Z.S.; data curation M.G. and E.G.-G.; writing—original draft preparation M.G. and E.G.-G.; writing—review and editing M.G., E.G.-G., M.K. and Z.S.; visualization M.G. and E.G.-G.; supervision M.G. and E.G.-G.; project administration M.G. and E.G.-G.; funding acquisition, M.G. and E.G.-G. All authors have read and agreed to the published version of the manuscript."

**Funding:** This research received no external funding.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Microorganisms as New Sources of Energy**

**Jasminka Talapko \*,†, Domagoj Talapko †, Anita Mati´c and Ivana Škrlec \***

Faculty of Dental Medicine and Health, Josip Juraj Strossmayer University of Osijek, HR-31000 Osijek, Croatia

**\*** Correspondence: jtalapko@fdmz.hr (J.T.); iskrlec@fdmz.hr (I.Š.)

† These authors contributed equally to this work.

**Abstract:** The use of fossil energy sources has a negative impact on the economic and socio-political stability of specific regions and countries, causing environmental changes due to the emission of greenhouse gases. Moreover, the stocks of mineral energy are limited, causing the demand for new types and forms of energy. Biomass is a renewable energy source and represents an alternative to fossil energy sources. Microorganisms produce energy from the substrate and biomass, i.e., from substances in the microenvironment, to maintain their metabolism and life. However, specialized microorganisms also produce specific metabolites under almost abiotic circumstances that often do not have the immediate task of sustaining their own lives. This paper presents the action of biogenic and biogenic–thermogenic microorganisms, which produce methane, alcohols, lipids, triglycerides, and hydrogen, thus often creating renewable energy from waste biomass. Furthermore, some microorganisms acquire new or improved properties through genetic interventions for producing significant amounts of energy. In this way, they clean the environment and can consume greenhouse gases. Particularly suitable are blue-green algae or cyanobacteria but also some otherwise pathogenic microorganisms (*E. coli*, *Klebsiella*, and others), as well as many other specialized microorganisms that show an incredible ability to adapt. Microorganisms can change the current paradigm, energy– environment, and open up countless opportunities for producing new energy sources, especially hydrogen, which is an ideal energy source for all systems (biological, physical, technological). Developing such energy production technologies can significantly change the already achieved critical level of greenhouse gases that significantly affect the climate.

**Keywords:** bioenergy; biomass waste; hydrogen; microorganisms; renewable energy sources

#### **1. Introduction**

The basic feature of life is oxidoreduction, which creates energy from matter [1]. However, some microorganisms can embed solar energy in very complex mechanisms of production of low-energy compounds from so-called nature pollutants caused by natural pollutants created by the technology of processing oil, sugar cane, and natural oils (harmful technologies) [2]. In addition to photosynthesis, some microorganisms, such as cyanobacteria, can decompose water into the desired oxygen and even more desirable hydrogen, and some can directly produce hydrogen via anaerobic processes [3]. Some, in turn, can convert classic environmental pollutants into very highly potent energy compounds (methane, alcohol) [4]. Thus, the genotypic and phenotypic traits of many species of microorganisms can direct the production of energy products to more perfect and efficient technologies and environmental purifiers [5].

Current technologies of energy production (energy) are a big problem (technical, environmental, and financial), because in addition to environmental pollution, they require significant investment (initial research, adaptation of new technologies, remediation as the final stage of production) [6]. However, natural pollutants (in terms of quantities, environmental impact, and permanent need for disposal) significantly reduce the benefits of conventional energy from fossil fuels (oil, gas, coal) and represent a subsequent often unsolvable problem of the remediation of CO2, NO2, SO2, and other oxides [7]. Technologies

**Citation:** Talapko, J.; Talapko, D.; Mati´c, A.; Škrlec, I. Microorganisms as New Sources of Energy. *Energies* **2022**, *15*, 6365. https://doi.org/ 10.3390/en15176365

Academic Editor: Dimitrios A. Georgakellos

Received: 8 August 2022 Accepted: 29 August 2022 Published: 31 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are being developed to use waste products (biorefinery) to produce renewable energy, as they permanently pollute the environment in the repeated energy production cycle [8]. Thus, microorganisms are undoubtedly crucial in developing waste purification and use strategies [9]. Bioenergy research is the center of scientific and technological research in the strategy of finding cost-effective biorefineries [10] as a way out of the current high level of air, water, and soil pollution to find photosynthetic and non-photosynthetic microorganisms that can produce clean energy (directly) or clean hydrogen [11].

An increasingly common research target of potential raw materials for biofuel production is microalgae obtained from adjusted wastewater. However, this may also significantly impact the environment, especially when compared with other renewable energy sources [12]. This can be particularly important when disposing of farm wastewater, representing an increasing environmental issue [13].

The present review aims to demonstrate the activity of biogenic and biogenic– thermogenic microorganisms that produce methane, alcohols, lipids, triglycerides, and hydrogen and contribute to creating renewable energy sources from waste biomass.

#### **2. Microbial Technologies for Biofuel Production**

The main reason for the increased interest in biomass as an energy source is the application contribution to the sustainable development paradigm. In addition, biomass sources are often present at the local level, and conversion into biofuel is possible with low initial costs [14]. Per the Renewable Energy Directive (EU Directive 2018/2001) [15], a common framework for the promotion of energy from renewable sources in the EU was established, setting a binding target for final gross consumption in the EU, with the total share of energy from renewable sources having to be 32% by 2030. This directive also promotes using non-food crops to produce biofuels and limits the number of biofuels and bioliquids produced from food or animal feed [15,16]. Methane is the so-called greenhouse gas produced indirectly by organic waste landfills (mainly in anaerobic processes) [17] and directly produced by all living beings (especially ruminants) [18]. There are two known sources of methane production: biological and non-biological. Non-biological methane is formed as a result of some geological processes. However, most methane (over 90%) is produced by the action of microorganisms and is a biological way (source) of methane production. This process of biological methane production is called methanogenesis, and microorganisms that carry out the same process are called methanogens [19,20]. Methanogens belong to the *Archaea* domain, which differs from bacteria because they do not possess peptidoglycan in the cell wall. Still, in *Methanosarcina*, it is a protein; in *Methanosarcina*, it is a heteropolysaccharide; and in *Methanobacterium* and *Methanobrevibacter*, it is replaced by pseudomurein [21]. The most crucial methanogen in the rumen is *Methanobacteriales ruminantium*, which contains pseudomurein in its cell wall. It needs formate, coenzyme M, hydrogen, and carbon dioxide for methane production [22]. For the process of methanogenesis, coenzymes F420, M, and HSHTP and lipids that methanogens have as cofactors are essential [23]. Cofactor F420 is necessary for the activity of hydrogenase as well as the formation of dehydrogenase enzymes, while coenzyme M acts as a terminal methyl carrier in the process of methanogenesis [24].

It is estimated that microorganisms annually produce and consume about one billion tons of methane [25]. However, the methane removal process can also occur in biological and non-biological ways. The most significant is the non-biological pathway in the Earth's atmosphere (specifically, the troposphere and stratosphere), where various chemical reactions under ultraviolet radiation decompose methane. In chemical reactions, the issue is associated with the breaking of the covalent bond in methane–carbon-hydrogen, which is one of the strongest bonds among all hydrocarbons [26]. Regardless, methane is used in a process called the catalytic steam reforming of methane, where methane is first converted into synthetic gas, i.e., into a mixture of hydrogen and carbon monoxide. Then, it serves as a raw material for producing hydrogen, methanol, and other chemicals, where the catalyst is nickel, and the reaction takes place at temperatures from 700 ◦C to 1100 ◦C [27].

Pyrolysis is the process of burning methane, in which formaldehyde (HCHO or H2CO) is formed in the first step, to which the HCO radical is added, after which carbon monoxide (CO) is formed [28].

The photocatalytic oxidation of methane is similar to the natural atmospheric process that oxidizes CH4 to CO2 [29]. Ultraviolet light is used to split the oxygen molecule into two free radicals that react with methane to produce products such as CH3OOH, CH3OH, HCOOH, CO2HOCH2OOH, and water. In photocatalytic reactors, catalysts increase the formation of free radicals and thus the rate of the methane reaction [30].

$$\text{CH}\_4 + \text{O}\_2 \rightarrow \text{CO} + \text{H}\_2 + \text{H}\_2\\\text{OCH}\_4 + \text{H}\_2\text{O} \rightarrow 700 \text{-} 1100 \text{ }^\circ \text{C} \,\text{NiCO} + 3 \text{H}\_2\text{O}$$

Unlike non-biological methods, biological methods of methane decomposition are carried out by the action of microorganisms called methanotrophs, and the process is named methanotrophy [19] (Figure 1). Methanotrophs can use methane to produce methanol, and *Geobacter sulfurreducens* and *Shewanella oneidensis* can use the mechanism of specific electron transfer from the membrane's outer surface to visible surfaces. This phenomenon can be used in bioelectrochemical devices to produce biohydrogen [31]. In addition, methanotrophs have a significant role in reducing the production of large amounts of greenhouse gases via their formation below the surface of the Earth (below the ground) and the utilization of methane produced in the soil conversion of methane emissions into the atmosphere [32,33]. Methane oxidation begins with reducing oxygen to peroxide and then to methanol with the action of the monooxygenase enzyme (MMO) [34].

**Figure 1.** The process of formation and decomposition of methane via biological and nonbiological means.

#### **3. Production of Ethanol and Butanol**

One of the most immediate and vital applications of biomass is the fermentation of biomass and the production of bioethanol, the most common renewable fuel today. Various microorganisms can be involved in the fermentation process for bioethanol production (Table 1) [35]. Bioethanol is the leading liquid biofuel, with a global production of 29 billion gallons in 2019. The top producers are the United States and Brazil, which account for 84% of global production [36].

**Table 1.** Bioethanol yield from different microorganisms.


Yeasts can produce ethanol via the direct decarboxylation of pyruvate formed via biomass oxidation [44]. At the same time, bacteria (*E.coli*) with coenzyme A activate the acyl group during the decarboxylation of pyruvate and convert it to ethanol (reduction) (Figure 2) [45].

**Figure 2.** Ethanol formation from glucose in yeasts.

Ethanol production via direct decarboxylation (*Saccharomyces cerevisiae*) is more efficient than that of *E. coli*. Butanol can be commercially similarly produced from sugar–starch biomass [46] (Figure 3). In addition, it can be made from so-called polysaccharides; from acetone–butanol fermentation (anaerobic process); and from and acetone, CO2, and hydrogen (*Clostridium acetobutylicum*) [47].

Some microorganisms are used in the gasification process. They can partially oxidize biomass by means of air or oxygen at about 800–1000 ◦C, whereby microalgae biomass is converted into a gaseous product—syngas—which means that microalgae are a suitable raw material for gasification [48,49]. Syngas is a mixture of hydrogen, CO, CO2, methane, and nitrogen [50]. It is used as a turbine fuel but primarily as a feedstock for producing methanol, ethanol and synthetic hydrocarbons, butanol, methane, butyric, and acetic acid [51]. Microalgal bio-oils also contain metals (Fe, Mg, Ni, Zn), which can be removed via heat treatment [52]. Microbial oil can be obtained from microalgae, yeasts, and molds, and triglycerides (oleic, linoleic, and palmitic acid) can also be obtained [53]. These raw materials can be used to produce biodiesel. Some rare yeast and fungus species can

yield various substrates (lignocellulosic biomass, industrial waste glycerol, whey fat, and molasses) [54]. Some microorganisms can grow in sewage sludge and wastewater [55].

**Figure 3.** Ethanol formation from glucose in bacteria. AdhE—bifunctional CoA-dependent ethanol/aldehyde dehydrogenase.

#### *Main Metabolic Pathways for Ethanol in the Most Prominent Microorganisms*

The most common microorganisms used in ethanol production are yeast *Saccharomyces cerevisiae* and bacterium *Escherichia coli*. They possess specific metabolic pathways and different types of catalytic enzymes for producing biofuels [56]. For example, *Saccharomyces cerevisiae* produces ethanol via the direct decarboxylation of pyruvate, while *E. coli* activates the acyl group during the decarboxylation of CoA, which is then reduced to ethanol [57,58].

A more efficient route in the production of ethanol is considered to be a route without the use of CoA. This pathway can also be achieved in other microorganisms by means of genetic engineering techniques. However, this method represents a significant challenge because synthesizing an artificial metabolic pathway requires highly sophisticated tools to control mRNA and protein levels for the synthetic pathway to be functional [59].

*Zymomonas mobilis* is another well-studied strain with a known genome that produces ethanol. It is important to note that it has a significantly higher ethanol yield than *Saccharomyces cerevisiae*. Furthermore, ethanol yield considerably increases after genetic manipulation, i.e., after introducing genes encoding catabolic enzymes mannose and xylose, so the theoretical yield of ethanol within 72 h can reach 89.8% [60].

The costs of bioethanol production from lignocellulosic raw materials are high, so for commercial reasons, this production method is still not used [61,62]. However, the production of second-generation bioethanol is in the development phase. For this production, microbial strains that can produce ethanol from glucose and xylose, the main fermentable sugars, are necessary [63].

#### **4. Biodiesel Production**

Biodiesel is the first alternative fuel and, at the same time, the most widespread biofuel in Europe. It is obtained from oil and fat through the transesterification process and is very similar in composition to mineral diesel fuel [64]. Biodiesel production is an inherently complex system that requires optimization, keeping profitability and environmental sustainability in mind [65]. Recent studies suggest an unquestionable benefit for the environment. Economic profitability depends on feedstock sources and choice, technological process and production capacities, and transport to the consumer [66,67]. Sources of third-generation feedstock, microalgae, have an unquestionable advantage over other sources [68].

Regarding the need for increasing the amounts of energy (due to direct use in internalcombustion engines or the production of heat and electricity), guided by the imperative to reduce CO2, modern science and technology are giving the first positive results [69]. These are the so-called biofuels produced as a product of microorganisms from biological materials and even from organic waste biomass [70]. In addition to the already considered bioenergy agents (methane, methanol, ethanol), we especially highlight the importance of biodiesel production. The European Union is at the forefront of applying such technologies in biodiesel consumption, which is about 105 billion liters—about 53% of the total world biodiesel consumption [71].

One of the most promising biodiesel production methods is the production of lipids, triglycerides, and other oil molecules from rapeseed, soybeans, and some other specialized plants, which incorporate this otherwise undesirable greenhouse gas into lipid molecules via photosynthesis [72]. At the same time, these plants have different types of fixatives (nitrofixatives, *Azotobacter* sp. and *Azospirillum* sp.; phosphofixatives, *Acinetobacter junii* and *Pseudomonas fluorescens*) in the soil, from which they benefit via the rhizome system [73]. Rhizome nodules fix nitrogen and phosphorus, conducive to plant growth and the formation of products (lipids, triglycerides) [74].

Biodiesel can then be produced directly from the vegetable oils of the above-mentioned plants [75] (Figure 4). Likewise, a biodiesel composition similar to vegetable oils can be obtained via the transesterification of *Rhodotorula glutinis* oil, and *Yarrowia lipolytica* can be used to produce microbial oil [76]. Genetic engineering can increase the tolerance of lipids or fatty acids in microorganisms, and some types of bacteria and fungi can tolerate higher amounts of accumulated triacylglycerol [77]. In terms of efficiency, bacteria show significantly more favorable properties than fungi (higher growth rate, easier maintenance, and the possibility of genetic adaptation) [78], because it is known that bacteria are subject to genetic improvement and possess the property of rapid growth, which can be used for the highly efficient production of microbial oil. Because of this, even *Escherichia coli*, under certain circumstances, can directly produce biodiesel in the form of fatty acid esters and can ferment biomass from renewable carbon sources (specialized or waste biomass) [79,80]. Thus, microbial oils are becoming a very likely source (raw material) in biodiesel production mainly due to the faster growth of microorganisms, easier maintenance, and the possibility of genetic adaptations [79,81].

The possibilities of direct electricity production by means of biochemical treatment systems are also being studied very intensively, and microbial fuel cell (MFC) devices convert chemical energy into electricity (without the Carnot cycle) from biodegradable raw materials and even from wastewater [82,83]. Furthermore, potentially electrogenic bacteria can be identified in the MFC device; in it, microorganisms feed on organic compounds, releasing electrons to the electrode, thus generating electricity [84]. In summary, it is clear that modern science and technology have efficient responses to increasing environmental pollution (especially greenhouse gases—CO2 and methane) [85,86].

#### *Main Metabolic Pathways for Biodiesel in the Most Prominent Microorganisms*

Biodiesel belongs to the group of renewable energy sources and represents an ideal replacement for petroleum-based diesel fuels. It is produced using transesterifying fatty acid sources with short-chain alcohols, giving monoalkyl esters of long-chain fatty acids [87]. For this process to be realized, it is essential that microorganisms can produce fatty acids and short-chain alcohols that are available for transesterification and that they possess acyltransferases with more significant activity for short-chain alcohols. Genetic engineering significantly transforms microorganisms into forms that produce biodiesel with high efficiency [88].

**Figure 4.** Biodiesel production pathway.

Microalgae attract particular attention as a raw material for biodiesel production (Figure 5). Namely, it is an economically profitable raw material for oil. They are characterized by easy cultivation, diverse metabolic activities, and a high content of fatty acids [89,90]. The results of the research study by Huang et al. suggest that the problems with fossil energy could be alleviated by the additional processing of microalgae residues after the lipid extraction process, using the pyrolysis process [91]. A moderately fast pyrolysis temperature (~700 ◦C) is essential for higher bio-oil production and a lower limit of pollutants [92].

**Figure 5.** Pathways of processing microalgal lipids into biodiesel. Modified according to [93]. Reprinted with permission from ref. [93]. 2021 © Portal hrvatskih znanstvenih i struˇcnih casopisa—Hrˇ ˇ cak.

Among the numerous autotrophic algae are *Botryococcus braunii*, *Chlorella vulgaris*, *Crypthecodinum cohnii*, *Dunaliella primolecta*, *Navicula pelliculosa*, *Scenedsmus acutus*, *Crypthecodinium cohnii*, *Monallanthus primolecta*, *Monallanthusocornussel olia*, and *Teallanthus chloridea sul*. The oil content in microalgae varies from 1 to 70% (Table 2).



The cultivation conditions include the composition of the nutrient medium, pH, temperature, the efficiency of light delivery to the cells of microalgae, the intensity and wavelength of light, the speed and method of mixing the nutrient medium in the bioreactor, and the ratio of the concentration of dissolved oxygen and CO2 in the nutrient medium [96,97]. Accordingly, the biotechnological production of microalgal lipids is determined by the physiological potential of the microalgae (Table 3), that is, the conditions and procedure of conducting the bioprocess in the bioreactor system [98]. Therefore, it is considered that microalgae are an inevitable trend in the development of future biodiesel, provided that for the industrial production of biomass, i.e., lipids of microalgae, the optimal conditions, and procedures for running the bioprocess in the bioreactor system are chosen so that an ecologically and economically sustainable bioprocess of the production of biomass, i.e., lipids of microalgae, is obtained for the production of biofuels (biodiesel) [99].

Numerous studies have established that residual biomass contains carbohydrates from which ethanol can be produced through fermentation. It is the specific share and yield during biodiesel production [100].


**Table 3.** Yields of lipids from different microorganisms.

#### **5. Hydrogen**

Biohydrogen represents an essential factor in solving global energy problems [102]. It is a substitute for primary fossil fuels and their derivatives. Its main advantage is that the product of its combustion with oxygen is water, and not CO and CO2, which are greenhouse gases [103]. Therefore, it is expected to play a crucial role in the future energy infrastructure. Biohydrogen has a gross energy or heat value of 142 MJ/kg, which is significantly higher

than those of natural gas or crude oil, whose values are 52 and 45 MJ/kg [102], while petrol has a value of 44 MJ/kg [103]. The global demand for hydrogen is predicted to increase from 70 million tons in 2019 to 120 million tons by 2024. Hydrogen development should also fulfill the seventh United Nations goal on affordable and clean energy [104].

Hydrogen is the first atom from which everything in the universe was created. The energy produced by fusion reactions (stars) was sufficient for forming all other elements, which created the conditions for evolution and the creation of life [105–108]. Traces of the life of the most primitive microorganisms (recognized today through the simplest microorganisms such as prions and viruses) used oxidation–reduction processes in which, under anaerobic conditions, hydride was oxidized to sulfides, nitrides, and phosphides and generated enough energy to start the process (which is still insufficiently explained) that could constitute life [109].

Modern life requires unimaginable amounts of energy [110,111], and fire is the simplest form of clean energy. In the same way, hydrogen is slowly and rapidly introduced into our daily lives [112]. Moreover, all mineral fuels provide energy by burning hydrogen (wood, coal, oil) [113], while nuclear processes, such as fusion, use hydrogen as fuel [114,115]. However, other products of hydrogen combustion (organic hydrocarbons, oxides) are today putting in question the continuation of life as we know it [116]. In addition, so-called greenhouse gases threaten civilization to such an extent, arousing the necessity for the creation of new mechanisms for increasing amounts of needed energy [117].

Microbial universality through life-saving adaptations has created natural reactors for producing biofuels and future fuels, i.e., hydrogen [118,119]. Moreover, they can produce it (extract it) from hydrocarbons, thus launching a more certain perspective for civilization [120]. The development and selection of microbial biorefineries are the result of the creation of syntrophic communities (a symbiotic form of joint metabolism) [121,122]. One example of syntrophy is methanogenic communities in which reducing equivalents, e.g., hydrogen and formate, are transferred among syntrophic partners [20,123]. In the coal seam, the anaerobic fermentation of organic matter includes hydrolysis, acidogenesis, acetogenesis, and methanogenesis [22,124]. In the first phase, bacteria hydrolyze macromolecules into simple sugars, amino acids, and fatty acids [125,126]. Then, acidogenic bacteria decompose them into propionic acid, butyric acid, and alcohol [127,128]. Microorganisms capable of acetogenesis then convert them to acetic acid, hydrogen, and CO2, and ultimately methanogenic microorganisms can produce methane [129]. Thus, the production of carbon-based biogas significantly improves protection against the formation of unwanted gases (primarily sulfur). As noted, coal conversion into methane requires the synergistic action of three groups of microorganisms (syntrophic community) [130,131]. They are mainly from genera *Clostridium*, *Enterobacter*, *Klebsiella*, and *Citrobacter* [118,132]. Methanogenic bacteria, based on mcrA and the phylogeny of ribosomal genes, are classified into seven orders, among which *Methanopyrales*, *Methanococcales*, *Methanobacteriales*, *Methanomicrobiales*, and *Methanocellales* comprise hydrogenotrophic methanogens. At the same time, the *Methanomasiliiicoccales* guild is obligated to perform methylogenotropic respiration [20].

A more complex form of microorganisms can be considered as the factory of electrochemical devices for producing electricity and biohydrogen [133,134]. The mechanism of biohydrogen generation can start from wastewater and some other types of organic matter [135–137]. By creating an electro-biofilm, the mechanism of electron transfer to conductive surfaces is triggered [138,139]. These electrons can then be used to produce electricity and hydrogen [140], similar to the so-called electrochemical cells (BECs), molecular machines that transfer electrons from a microbial membrane [141,142]. Microalgae can further produce hydrogen via the reaction of the photolysis of water, i.e., by converting water into hydrogen ions and oxygen, after which they convert these hydrogen ions into hydrogen, all under anaerobic conditions [143,144]. Likewise, photosynthesis can produce hydrogen through two stages [145]. The first stage is created via photosynthesis, in which acid production is separated from hydrogen production. In the second stage, microalgae

are denied access to sulfur and are forced to change their cellular metabolism for survival by forming starch, from which they produce hydrogen [146,147]. As a result, the amount of hydrogen produced gradually reduces, but this process does not create undesirable and harmful by-products [148]. Biological hydrogen can also be made via the fermentation of lignocellulosic raw materials and cotton-sludge hydrolates. It can be produced by bacteria isolated from higher organisms (such as fish and termites) [149]. These are predominantly *Enterobacter*, *Klebsiella*, *Clostridium*, and *Citrobacter* [150]. Several metabolic pathways exist for biohydrogen production, and anaerobic fermentation is the most efficient and rapid way to produce it [151,152] (Table 4).


**Table 4.** Methods for hydrogen production and their efficiency.

The production of hydrogen using distinct methods and using various feedstock implies different capital investment costs and the costs of hydrogen production itself, as shown in Table 5. The level of production-technology innovation, the accessibility of existing infrastructure, and the feedstock cost significantly impact this cost.

**Table 5.** Hydrogen production costs by different methods.


#### *5.1. Main Metabolic Pathways for Hydrogen in the Most Prominent Microorganisms*

For bacteria that participate in the production of biohydrogen, such as *Geobacter sulfurreducens* and *Shewanella oneidensis*, it is significant that they possess specific molecular mechanisms that facilitate the transfer of electrons from the outer membrane of the microorganism to visible surfaces, after which this feature can be used to produce biohydrogen and, accordingly, bioelectric energy [156]. Thus, such bioelectrochemical cells (BECs) represent an exceptional significance in the potential production of bioenergy from wastewater and organic biomass [157].

Microbial electrolysis cells (MECs) and microbial fuel cells (MFCs) are primarily used to produce biohydrogen and bioelectricity. Based on a biological perspective, both species function in a similar manner, and accordingly, common microorganisms can be used for bioenergy production. These microorganisms are called electrogenic or exoelectrogenic [158]. However, it is essential to note that the output energy from MECs and MFCs is insufficient for practical application and commercialization [159].

#### *5.2. Hydrogen Production via Photofermentation with Photofermenting Bacteria*

Biological methods for producing hydrogen are in increasing focus because they can use renewable raw materials such as the remains of plant biomass, organic waste, and sunlight [160]. There are two main ways in which microorganisms produce hydrogen: photosynthesis and fermentation. The process of photosynthesis is dependent on light and includes direct biophotolysis, indirect biophotolysis, and photofermentation. On the other hand, dark photofermentation is essentially anaerobic fermentation, and this process is not dependent on light [161]. Microorganisms produce hydrogen at room temperature and pressure, significantly reducing the need for additional energy. Photosynthetic hydrogen is produced by microorganisms, such as photosynthetic bacteria, algae, and cyanobacteria. Fermentative hydrogen production is carried out by fermentative microorganisms, such as strict anaerobes, e.g., strains of *Clostridium* sp. thermophilic rumen bacteria and methanogenic facultative anaerobes or mixed cultures [162].

Based on the available data, the conclusion is that fermentative hydrogen production has more potential for practical application than photosynthetic hydrogen production. Hydrogen production via fermentation is currently more profitable in energy gain than photosynthesis [163]. This is supported by the facts that fermentation bacteria have fast growth and that oxygen does not affect the anaerobic process to a large extent; they do not need light; and they have a higher level of hydrogen production. It is also important to note that there is a large selection of substrates and that the methods (techniques) of setting up bioreactors are simple [164–166]. The production of hydrogen by means of dark fermentation can be carried out under different thermodynamic conditions so that it can be carried out under mesophilic, thermophilic, and hyperthermophilic conditions. However, the production degree is still more favorable at higher temperatures [167].

It is known that there is a significant difference between theoretical and practical energy yield, which can be seen from the following example:

Theoretical yield—12 moles of H2 can be produced from each mole of glucose.

Practical yield—a maximum of 3.8 moles of H2 can be produced from each mole of glucose.

Yields can be increased by combining two metabolic pathways and using compatible mixed bacterial cultures [79].

#### **6. Cyanobacteria**

Hydrogen from bacteria is also produced via photosynthesis, because bacteria do not consume the created hydrogen but rather retain it [168]. Namely, the process of photosynthesis of cyanobacteria begins with the fixation of solar energy due to water splitting, so this energy is stored by hydrogenase in sugars [169,170]. Electrons undergo a series of reactions produced by the ATP energy carrier and the reduction equivalents of NADPH, which are required for CO2 fixation or sugar production [171]. These electrons and protons (energy) can be diverted to primarily produce hydrogen, that is, by linking the photosystem to hydrogenase. Modified cyanobacteria produce less sugar than unmodified ones at the expense of greater hydrogen production [169,172]. This fusion can function indefinitely. It is also transmitted during cell division, and since the oxygen created is blocking the processes, metabolism is transferred to anoxygenic photosynthesis. All these processes offer a new concept for the production of green hydrogen [173].

In addition to genetic modifications that create unique and desirable traits in some bacteria, some bacteria are also subject to natural phenomena that can still produce everything necessary for growth and reproduction under unfavorable conditions [174]. Extremophiles, which live where there is no liquid water and no solar energy, can use hydrogen from the air as a fuel to create water for their needs, specifically, by capturing hydrogen and oxygen from the air [175]. Thus, hydrogen drives chemosynthesis with enzyme RuBisCo, which otherwise uses sunlight to capture CO2 [176].

Thus, about 400 species of evolutionarily adapted microorganisms live without free water and can use inorganic energy sources (hydrogen, CO) as sources that drive metabolism (chemosynthesis) [177,178]. Some marine bacteria work similarly. Seawater has sufficient amounts of hydrogen and CO, which can be sources of energy for bacteria from families *Rhodobacteraceae*, *Flavobacteriaceae*, and *Sfhingomonodaceae* [179,180]. A representative of this

group of ultramicrobacteria, *Sfinopyxis alascensis*, grows mycotrophically on hydrogen by expressing NiFe hydrogenase [181,182].

Thus, one of the possible solutions for obtaining new renewable energy sources is hydrogen (H2), which could be obtained via photosynthesis, which requires sunlight, water, and cyanobacteria. However, in doing so, it is necessary to consider the development of cost-effective production technologies [183].

#### **7. Future Perspective**

The biggest challenge in employing microorganisms to produce biofuels is producing a considerable amount of fuel more cheaply and efficiently than traditional fossil fuels. To replace petrol with bioethanol should be cheaper, which is very demanding to cover the necessary daily quantities. For example, in the USA, about 19 million barrels of petrol are consumed daily, and producing this amount on an industrial scale is challenging. Therefore, future biofuel productivity should be prioritized to increase microbial biofuel's acceptability [184]. Some of the most common advantages and disadvantages of the biofuel production process are shown in Table 6. Due to the politically increasingly unstable oil market, many countries are turning to renewable energy sources. Biofuels (bioethanol and biodiesel) are a sustainable energy source due to their high chemical similarity, carbon neutrality, and comparable energy content, and microorganisms are crucial to their synthesis. Depending on the feedstock's evolutionary hierarchy and the manufacturing technique, biofuels are divided into four generations. Biofuel production advances with each generation toward achieving sustainability and financial success in energy production. They are created to most effectively address the issues of the energy crisis, pollution, global warming, and waste management. Microorganisms used to be mere biomass decomposers, but because of advancements in biotechnology, gene editing, and synthetic biology, they now produce biofuel [185].


**Table 6.** Advantages and disadvantages of biofuel production.

The production processes for the second and third generations of biofuels are quite complex, which results in high energy costs. Additionally, the feedstock for the third generation has very complex requirements for structure, storage, and content. The expenses mentioned above explain the capital intensity of manufacturing second- and third-generation fuels, respectively, and the decision by most nations to choose first-generation biofuels [188]. The microbial lipids produced by microorganisms are the ideal feedstock for biodiesel synthesis due to their high production rate and independence from environmental conditions such as soil and climate. In the study by Wang et al., several ideas for generating biodiesel using microbes from inexpensive lignocellulosic biomass are addressed [79]. A country that intends to develop alternative fuels must have enough land to prevent a food shortage and enact stringent controls limiting the proportions of raw materials provided to the food and fuel markets. The ratios in which a blend of biodiesel with diesel and bioethanol with petrol can be created must also be governed by state standards. Developing second- and third-generation biofuel production, which uses significantly less land and is mostly not arable, despite having a higher capital during production, needs help and subsidies [188].

#### **8. Conclusions**

Numerous studies are being conducted based on the growing need to find new renewable energy sources that could replace gas and oil and reduce harmful effects to the ecosystem. As a result, scientists are increasingly turning to biofuels based on microorganisms. In doing so, it is necessary to use increasingly advanced genetic engineering technologies. The imperative of preserving and surviving civilization can be met—using enough species of microorganisms that we can find in our immediate environment. In this way, microorganisms are ready to lead us into new human–environmental relationships to be our companions in a more confident and secure future.

**Author Contributions:** Conceptualization, J.T. and D.T.; writing of the manuscript, J.T., D.T., A.M. and I.Š.; updating of the text, J.T. and I.Š.; literature searches, J.T., D.T., A.M. and I.Š.; figure drawings, A.M.; critical reviewing of the manuscript, J.T. and I.Š.; organization and editing of the manuscript, J.T. and I.Š. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research study was funded by a grant from the Croatian Ministry of Science and Education and dedicated to multi-year institutional financing of scientific activity at Josip Juraj Strossmayer University of Osijek, Faculty of Dental Medicine and Health, Osijek, Croatia, grant number IP11-FDMZ-2021.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

