*2.7. Yogurt Bioactivity*

For the analysis of antioxidant and antihypertensive activities, the supernatant of yogurt was used, which was prepared according to Zhang et al. [30]. For that, 10 g of yogurt samples were centrifuged at 4330× *g* for 5 min at 4 ◦C, and then the supernatants were recentrifuged under the same conditions.

#### 2.7.1. Antioxidant Activity

ABTS Radical Scavenging Activity

The 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was determined using the methods detailed by Zheng et al. [31], with modifications.

The stock solution of the ABTS radical was generated by incubating 7 mM ABTS with 140 mM potassium persulfate in the dark for 16 h at room temperature. Before use, the stock solution was diluted with phosphate-buffered saline (PBS, pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. A 50 μL aliquot of the yogurt supernatant (diluted 1:5 in PBS) was homogenized with 150 μL of ABTS, and after reaction at 30 ◦C for 6 min in the dark, the absorbance was measured at 734 nm. The ABTS radical scavenging activity was calculated as follows: [(*Ac* − *As*)/*Ac*] × 100, where *Ac* is the absorbance of the control, reagents without sample, and *As* is absorbance with sample. The determination was carried out in triplicate.


*2.3. Statistical Analysis*

For statistical analysis, a comparison was made between the values originally collected postoperatively and those collected at the follow-up examination. If the Shapiro–Wilk test did not obtain a normal distribution, the Wilcoxon test for paired samples was used instead. We also used descriptive statistics for data evaluation. The data were evaluated and compared using Origin Pro® 9.0 (OriginLab Corp, Northampton, MA, USA) and SPSS® 26.0 software (IBM Corp, Armonk, NY, USA). OriginLab® was used to create a Kaplan– Meier plot for the endpoints defined as revision for conversion to RSA and revision for any reason. Furthermore, common statistical methods, such as the mean values, medians, effect sizes, ranges, and standard deviations (SDs), were used in this study.


**Author details**

Barbara Pietrucha Department of Immunology, The Children's Memorial Health Institute, Warsaw, Poland

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Profiling Inflammatory Genes and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy**

Yasuko Shibata and Yoshimitsu Abiko

*Department of Biochemistry and Molecular Biology Nihon University School of Dentistry at Matsudo Japan*

### **1. Introduction**

152 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

Sun, Y., Berger, E. J., Zhao, C., Jay, G. D., An, K. N. & Amadio, P. C. (2006). Expression and

Swann, D. A., Silver, F. H., Slayter, H. S., Stafford, W. & Shore, E. (1985). The molecular

Teeple, E., Elsaid, K. A., Jay, G. D., Zhang, L., Badger, G. J., Akelman, M., Bliss, T. F. &

Tilleman, K., Van Beneden, K., Dhondt, A., Hoffman, I., De Keyser, F., Veys, E., Elewaut, D.

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Yokosaki, Y., Matsuura, N., Sasaki, T., Murakami, I., Schneider, H., Higashiyama, S., Saitoh,

Young, A. A., McLennan, S., Smith, M. M., Smith, S. M., Cake, M. A., Read, R. A., Melrose, J.,

Zhang, C. C., Rogalski, J. C., Evans, D. M., Klockenbusch, C., Beavis, R. C. & Kast, J. (2011).

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mapping of lubricin in canine flexor tendon. *J Orthop Res*, Vol.24, No.9, (Sep 2006),

structure and lubricating activity of lubricin isolated from bovine and human synovial fluids. *Biochem J*, Vol.225, No.1, (Jan 1985), pp. 195-201, ISSN 0264-6021 Swann, D. A., Slayter, H. S. & Silver, F. H. (1981). The molecular structure of lubricating

glycoprotein-I, the boundary lubricant for articular cartilage. *J Biol Chem*, Vol.256,

Fleming, B. C. (2011). Effects of supplemental intra-articular lubricin and hyaluronic acid on the progression of posttraumatic arthritis in the anterior cruciate ligament-deficient rat knee. *Am J Sports Med*, Vol.39, No.1, (Jan 2011), pp.

& Deforce, D. (2005). Chronically inflamed synovium from spondyloarthropathy and rheumatoid arthritis investigated by protein expression profiling followed by tandem mass spectrometry. *Proteomics*, Vol.5, No.8, (May 2005), pp. 2247-57, ISSN

M., Drungowski, M., Seyfert, C., Zacher, J., Pruss, A., Neidel, J., Lehrach, H., Thiesen, H. J., Ruiz, P. & Blass, S. (2010). Molecular signatures and new candidates to target the pathogenesis of rheumatoid arthritis. *Physiol Genomics*, Vol.42A, No.4,

Uede, T. (2007). Successful treatment of collagen-induced arthritis in non-human primates by chimeric anti-osteopontin antibody. *Int Immunopharmacol*, Vol.7, No.11,

Y., Yamakido, M., Taooka, Y. & Sheppard, D. (1999). The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved aminoterminal fragment of osteopontin. *J Biol Chem*, Vol.274, No.51, (Dec 1999), pp.

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In silico protein interaction analysis using the global proteome machine database. *J*

Rheumatoid arthritis (RA) is an autoimmune joint disease characterized by inflammation and destruction of the articular surfaces and bone. During joint movement, synovial tissues contribute to mechanical load bearing by changing their shape. These elastic synovial membranes are an early target of rheumatic inflammation, and together with chondrocytes, become a primary source of inflammatory factors (e.g., cytokines) that enter the synovial fluid (Dayer, 2004, Firestein, 2007). The pathophysiological steps leading to RA include inflammation, proliferation of synovial cells, and attachment to and invasion of adjacent cartilage and bone by fibroblast-like cells derived from rheumatoid synoviocytes (RA-FLSs) (Firestein, 1996, Pap et al., 2000, Tolboom et al., 2005, Bartok & Firestein, 2010).

In the healthy synovium, one to three layers of synoviocytes, the macrophage-like type A and the more abundant fibroblast-like type B (also referred to as synovial fibroblasts), form the synovial lining layer separating the synovial sublining layer of loose connective tissue from the joint cavity (Iwanaga et al., 2000). The joint damage observed in RA is mainly mediated by macrophage/macrophage-like cell-derived cytokines, such as interleukin (IL)- 1, IL-6 and tumor necrosis factor (TNF)-, which induce neutral protease production by FLSs and articular chondrocytes (Houssiau et al., 1988, Feldmann & Maini, 1999, Dayer, 2002). Although IL-1 and TNF- share many biological activities that are relevant in RA, early studies of experimental arthritis models have demonstrated that IL-1 plays a predominant role in cartilage destruction via inflammatory processes that include the activation of matrix metalloproteinases (MMPs) (Borghaei et al., 1998) and the inhibition of the synthesis of extracellular matrix (ECM) molecules (Mauviel et al., 1988). In particular, IL-1 is readily detected, long after the onset of RA, at high levels in the synovial fluid of RA patients. IL-1 can significantly alter the expression of a variety of genes, including inflammatory mediators such as cytokines and MMPs (Sun & Yokota, 2002, Suzuki et al., 2010). In addition, IL-1 stimulates bone resorption by activating osteoclasts (Goldring, 2003). That said, there is little comprehensive information available on the effects of IL-1 on fibroblast-like synoviocytes (FLSs).

RA-FLSs not only mediate tissue destruction, but also are considered to play a major role in initiating and driving RA in concert with inflammatory cells (Huber, 2006, Bartok &

Profiling Inflammatory Genes

was signaling-related (Fig. 1).

significant.

**2.1 IL-1-induced inflammatory signaling pathways**

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 155

Our aims were to identify potential mediators of RA inflammation and joint destruction induced by the inflammatory cytokine IL-1 and to investigate the mechanisms responsible for their stimulation. Of the 8,746 genes on the Focus array HG-8500 GeneChip, 4,909 from untreated cells and 5,073 from IL-1-treated cells were classified as marginally expressed or present. After the cut-offs for induction (≥2.0-fold) and suppression (≤0.5-fold) by IL-1 were applied, 120 genes (74 upregulated and 46 downregulated genes) were identified as being affected by IL-1. When we then classified the IL-1-inducible gene expression in MH7A cells with respect to six functionalities based on BiologicalProcess words, we found that IL-1 conspicuously increased the expression of inflammation and apoptosis/proliferation-related genes, although the largest group of genes affected by IL-1

The genes that were upregulated (n=74) or down-regulated (n=46) by IL-1 were classified into 6 groups based on the GO-BiologicalProcess terms listed in the GeneSpring 6.2 software program. Fig. 1. Classification of genes whose expression in MH7A cells was affected by IL-1.

We used IPA to gain a better understanding of how differentially expressed genes were integrated into specific regulatory and signaling networks. A data set containing gene identifiers adhering to the 2-fold (120 genes) change used in microarray experiments was uploaded into the IPA software application. Each gene identifier was mapped to its corresponding gene object and then overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. The networks of these genes were then algorithmically generated based on their connectivity, after which a functional analysis of the networks identified the biological functions that were the most

We attempted to visualize the IL-1-induced genes within the canonical IPA pathway, which involves IL-1, IL-6, and TNF- signaling (Fig. 2A, canonical pathway name: IL-6 signaling). Four IL-1-inducible genes (>2.0-fold), IL-1, IL-6, IL-8, and IB, were situated within this atlas. As shown in Fig. 2A, activation of NF-B signaling is also important for the secretion of IL-8, IL-1 and IL-6. We previously used an ELISA to show that TNF- was not expressed in MH7A cells, regardless of whether they were exposed to IL-1 (0.1 unit/ml) (Shibata et al., 2005). Likewise, the results we obtained using the GeneChip microarray indicated no expression of TNF- in MH7A cells under our experimental conditions. IL-1 did induce its own expression, which may be important for its accumulation during the inflammation in RA.

Furthermore, high levels of NF-IL6 (CEBPB) expression were consistently observed.

Firestein, 2010). However, the original roles of single RA-FLS, and their relationship with macrophage-like type B cells in the rheumatoid synovium are poorly understood, although many case reports of RA patients, and many experimental reports using the synovium tisuues/cells from RA patients have been published. The utilization of cells such as the MH7A human RA-FLSs (Cell Bank, Riken Bioresource Center, Ibaraki, Japan), which are established cells isolated from the knee joint of an RA patient that retain the morphological and functional characteristics of primary synovial cells should provide guidance to clarify the mechanisms of onset of RA and to the development of useful treatments for RA. The MH7A cells are an immortalized RA-FLS line that stains positively for IL-1R, intercellular adhesion molecule-1 (ICAM-1), CD16, CD40, CD80, and CD95 and has been used extensively to investigate the molecular mechanisms underlying RA (Miyazawa et al., 1998). It has been shown, for example, that IL-1 enhances the production of IL-6, IL-8, and MMPs in MH7A cells (Shibata et al., 2005, Han et al., 2006) in a manner similar to that seen in the parental FLSs, although the immortalized cells grow more rapidly than the parental cells. These results clearly indicate the usefulness of MH7A cells for investigating the regulation of rheumatoid FLSs and the IL-1 signal transduction pathway to develop a future RA therapy.

In what is becoming known as the "post-genomic era", many new technologies and methodologies are being developed to take advantage of the recent progress in genomic research. Among these, gene expression profiling has become an invaluable tool in functional genomics. DNA microarrays, cDNA subtraction, and the serial analysis of gene expression have all emerged as leading transcript profiling technologies for the global analysis of biological systems. One of the high throughput technologies, high-density oligonucleotide genechip microarrays, makes it possible to simultaneously measure the relative abundance of numerous mRNAs within a cell. To better understand the direct IL-1-induced changes in gene expression that might promote inflammatory responses in MH7A cells, in the present study we used an Affymetrix HG-8500 Focus array GeneChip to analyze the patterns of gene expression, and then used the Ingenuity Pathway Analysis (IPA) software program to investigate the signaling pathways leading to the IL-1-induced gene expression. In addition, we used a biotin label-based antibody array to measure the levels of 507 human target proteins, including cytokines, chemokines, growth factors, angiogenic factors, proteases, and soluble receptors, among others, in the cell culture supernatants. We analyzed the expression profiles of both the genes and proteins to identify the key molecules involved in articular rheumatism, which could be related to the induction of inflammation and pannus formation in the synovial tissues of RA patients. In addition, we propose the possibility of a new therapy using linear polarized infrared light, that induces the suppression of inflammation and pain for RA patients with no apparent sideeffects.

#### **2. Microarray analysis**

MH7A human RA-FLSs (Riken Bioresource Center, Ibaraki) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin at 37°C under a 5% CO2 atmosphere. Total RNA from the MH7A cells was treated according to the manufacturer's protocol, and then hybridized to a Human Genome Focus Array HG-8500 GeneChip (Affymetrix Inc.).

Firestein, 2010). However, the original roles of single RA-FLS, and their relationship with macrophage-like type B cells in the rheumatoid synovium are poorly understood, although many case reports of RA patients, and many experimental reports using the synovium tisuues/cells from RA patients have been published. The utilization of cells such as the MH7A human RA-FLSs (Cell Bank, Riken Bioresource Center, Ibaraki, Japan), which are established cells isolated from the knee joint of an RA patient that retain the morphological and functional characteristics of primary synovial cells should provide guidance to clarify the mechanisms of onset of RA and to the development of useful treatments for RA. The MH7A cells are an immortalized RA-FLS line that stains positively for IL-1R, intercellular adhesion molecule-1 (ICAM-1), CD16, CD40, CD80, and CD95 and has been used extensively to investigate the molecular mechanisms underlying RA (Miyazawa et al., 1998). It has been shown, for example, that IL-1 enhances the production of IL-6, IL-8, and MMPs in MH7A cells (Shibata et al., 2005, Han et al., 2006) in a manner similar to that seen in the parental FLSs, although the immortalized cells grow more rapidly than the parental cells. These results clearly indicate the usefulness of MH7A cells for investigating the regulation of rheumatoid FLSs and the IL-1 signal transduction pathway to develop a future RA

In what is becoming known as the "post-genomic era", many new technologies and methodologies are being developed to take advantage of the recent progress in genomic research. Among these, gene expression profiling has become an invaluable tool in functional genomics. DNA microarrays, cDNA subtraction, and the serial analysis of gene expression have all emerged as leading transcript profiling technologies for the global analysis of biological systems. One of the high throughput technologies, high-density oligonucleotide genechip microarrays, makes it possible to simultaneously measure the relative abundance of numerous mRNAs within a cell. To better understand the direct IL-1-induced changes in gene expression that might promote inflammatory responses in MH7A cells, in the present study we used an Affymetrix HG-8500 Focus array GeneChip to analyze the patterns of gene expression, and then used the Ingenuity Pathway Analysis (IPA) software program to investigate the signaling pathways leading to the IL-1-induced gene expression. In addition, we used a biotin label-based antibody array to measure the levels of 507 human target proteins, including cytokines, chemokines, growth factors, angiogenic factors, proteases, and soluble receptors, among others, in the cell culture supernatants. We analyzed the expression profiles of both the genes and proteins to identify the key molecules involved in articular rheumatism, which could be related to the induction of inflammation and pannus formation in the synovial tissues of RA patients. In addition, we propose the possibility of a new therapy using linear polarized infrared light, that induces the suppression of inflammation and pain for RA patients with no apparent side-

MH7A human RA-FLSs (Riken Bioresource Center, Ibaraki) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin at 37°C under a 5% CO2 atmosphere. Total RNA from the MH7A cells was treated according to the manufacturer's protocol, and then hybridized to a Human Genome Focus Array HG-8500

therapy.

effects.

**2. Microarray analysis**

GeneChip (Affymetrix Inc.).

Our aims were to identify potential mediators of RA inflammation and joint destruction induced by the inflammatory cytokine IL-1 and to investigate the mechanisms responsible for their stimulation. Of the 8,746 genes on the Focus array HG-8500 GeneChip, 4,909 from untreated cells and 5,073 from IL-1-treated cells were classified as marginally expressed or present. After the cut-offs for induction (≥2.0-fold) and suppression (≤0.5-fold) by IL-1 were applied, 120 genes (74 upregulated and 46 downregulated genes) were identified as being affected by IL-1. When we then classified the IL-1-inducible gene expression in MH7A cells with respect to six functionalities based on BiologicalProcess words, we found that IL-1 conspicuously increased the expression of inflammation and apoptosis/proliferation-related genes, although the largest group of genes affected by IL-1 was signaling-related (Fig. 1).

The genes that were upregulated (n=74) or down-regulated (n=46) by IL-1 were classified into 6 groups based on the GO-BiologicalProcess terms listed in the GeneSpring 6.2 software program.

Fig. 1. Classification of genes whose expression in MH7A cells was affected by IL-1.

We used IPA to gain a better understanding of how differentially expressed genes were integrated into specific regulatory and signaling networks. A data set containing gene identifiers adhering to the 2-fold (120 genes) change used in microarray experiments was uploaded into the IPA software application. Each gene identifier was mapped to its corresponding gene object and then overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. The networks of these genes were then algorithmically generated based on their connectivity, after which a functional analysis of the networks identified the biological functions that were the most significant.

#### **2.1 IL-1-induced inflammatory signaling pathways**

We attempted to visualize the IL-1-induced genes within the canonical IPA pathway, which involves IL-1, IL-6, and TNF- signaling (Fig. 2A, canonical pathway name: IL-6 signaling). Four IL-1-inducible genes (>2.0-fold), IL-1, IL-6, IL-8, and IB, were situated within this atlas. As shown in Fig. 2A, activation of NF-B signaling is also important for the secretion of IL-8, IL-1 and IL-6. We previously used an ELISA to show that TNF- was not expressed in MH7A cells, regardless of whether they were exposed to IL-1 (0.1 unit/ml) (Shibata et al., 2005). Likewise, the results we obtained using the GeneChip microarray indicated no expression of TNF- in MH7A cells under our experimental conditions. IL-1 did induce its own expression, which may be important for its accumulation during the inflammation in RA. Furthermore, high levels of NF-IL6 (CEBPB) expression were consistently observed.

Profiling Inflammatory Genes

modified from (Shibata et al., 2009a)]

Fig. 3. The effect of Bay11-7085 on the production of IL-8

**2.2 Arithmetic IL-8 production through the PGE2 receptor synthesis**

cells.

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 157

induced IL-8 expression (Matsusaka et al., 1993). We would therefore anticipate that these transcription factors would play an important role in the increased expression of IL-8 mRNA elicited by IL-1, but no increases in the expression for NFKB1 (NF-B), RELA (RelA), or CEBPB (NF-IL-6) were seen in the microarray experiment. On the other hand, in resting cells, NF-B is trapped in the cytoplasm through its interaction with its inhibitor, IkB, and a crucial step in the activation of NF-B is the stimulus-induced phosphorylation of IB by IB kinases (IKKs). To study the extent to which IKKs modulate IL-1 induced IL-8 production, MH7A cells were pretreated with Bay11-7085, an inhibitor of IB phosphorylation, for 1 hr before stimulation with IL-1(Shibata et al., 2009a). As shown in Fig. 3, Bay11-7085 completely blocked the release of IL-8 from IL-1-stimulated MH7A

**A.** The IL-1-induced signal transduction pathways leading to the production of IL-8. Gray symbols indicate genes that were up-regulated by IL-1. White symbols indicate genes whose expression was increased less than 2.0-fold by IL-1. **B.** The effects of Bay11-7085 on the production of IL-8. MH7A cells were first incubated with the IKK/ inhibitor Bay11-7085 for 1 hr, and then with IL-1 for 2 hr. The levels of IL-8 in medium conditioned by the cells were measured using an ELISA. [This figure is

PGE2, a product of the cyclooxygenation of arachidonic acid, is a potent mediator of the immune response and inflammation, and contributes to the pathogenesis of RA (Robinson et al., 1975, Trebino et al., 2003). PGE2 also displays a complex regulatory function affecting IL-8 gene expression, which is dependent on the concentration of PGE2 and on the specific cell type involved. At physiological and pathological concentrations of up to 100 μM, PGE2 is capable of upregulating endogenous IL-8 expression in human colonic epithelial cells (Yu & Chadee, 1998). However, there have been many reports that PGE2 has no effect on neutrophil-derived IL-8 induced by LPS (Wertheim et al., 1993); that it down-regulates IL-8 in response to LPS in human alveolar macrophages and blood monocytes (Standiford et al., 1992); and suppresses the production of chemokines, including IL-8, in human macrophages

**A.** The IL-1 and IL-6 signaling pathways as determined by IPA analysis. Grayed symbols indicate genes that were upregulated by IL-1; white symbols indicate genes whose expression was not increased by IL-1. **B.** A scatter plot of 8,793 genes. Genes whose expression ratio was changed by >2 fold by IL-1 are positioned outside the lines. **C.** Dose-dependent effects of IL-1 on the gene expression. MH7A cells were examined after treatment with fresh medium containing the indicated concentration of IL-1, and the cells were incubated for an additional 2 hr.

Fig. 2. The IL-1 and IL-6 signaling pathways and the results of the RT-PCR analysis

Based on the structural analysis, the chemokine superfamily can be divided into two groups: the C-X-C and the C-C subfamilies (Baggiolini et al., 1994). IL-8 belongs to the first group and acts mainly on neutrophils (Hebert & Baker, 1993). Our microarray experiments showed that IL-8 transcription was the most dramatically upregulated (20.8 fold) in MH7A cells exposed to IL-1. It is also noteworthy that the expression of other chemokines, including CXCL-1 (4.1-fold), -2 (4.0-fold) and -3 (2.7-fold), three potent stimulators of neutrophil activation and tissue infiltration was also enhanced. Leukocyte recruitment from the microvasculature to sites of inflammation is a sequential process that includes rolling, activation, firm adhesion, and finally, transmigration through the vessel wall (King & Hammer, 2001). Although macrophages, neutrophils, and endothelial cells are all thought to be primary sources of IL-8, which is known to be elevated at inflammatory sites such as the synovial fluid of RA patients, fibroblast-like and macrophage-like cells of the synovial membrane, together with the infiltrating leucocytes, are the major source of these mediators in the rheumatoid synovial pannus (Rathanaswami et al., 1993). In the GeneChip microarray experiment, an IL-1 concentration close to the levels seen clinically was used to stimulate the MH7A cells. The gene expression of IL-8, Gro- (CXCL1), Gro- (CXCL2), IL-1, and IL-6 in scatter plots is shown in Fig. 2B. According to a RT-PCR analysis, the levels of IL-8, CXCL1, CXCL2, IL-1, and IL-6 mRNA in MH7A cells were all found to be dosedependently increased by IL-1 (Fig. 2C). Therefore, IL-8 appears to play a major role in the initiation and maintenance of inflammatory responses.

Within the 5'-flanking region of the human IL-8 promoter are a number of motifs with the potential to bind various transcription factors in a cell and stimulus-dependent manner. In particular, the transcription factors NF-B and NF-IL-6 are both involved in stimulus-

**A.** The IL-1 and IL-6 signaling pathways as determined by IPA analysis. Grayed symbols indicate genes that were upregulated by IL-1; white symbols indicate genes whose expression was not increased by IL-1. **B.** A scatter plot of 8,793 genes. Genes whose expression ratio was changed by >2 fold by IL-1 are positioned outside the lines. **C.** Dose-dependent effects of IL-1 on the gene expression. MH7A cells were examined after treatment with fresh medium containing the indicated

Fig. 2. The IL-1 and IL-6 signaling pathways and the results of the RT-PCR analysis

Based on the structural analysis, the chemokine superfamily can be divided into two groups: the C-X-C and the C-C subfamilies (Baggiolini et al., 1994). IL-8 belongs to the first group and acts mainly on neutrophils (Hebert & Baker, 1993). Our microarray experiments showed that IL-8 transcription was the most dramatically upregulated (20.8 fold) in MH7A cells exposed to IL-1. It is also noteworthy that the expression of other chemokines, including CXCL-1 (4.1-fold), -2 (4.0-fold) and -3 (2.7-fold), three potent stimulators of neutrophil activation and tissue infiltration was also enhanced. Leukocyte recruitment from the microvasculature to sites of inflammation is a sequential process that includes rolling, activation, firm adhesion, and finally, transmigration through the vessel wall (King & Hammer, 2001). Although macrophages, neutrophils, and endothelial cells are all thought to be primary sources of IL-8, which is known to be elevated at inflammatory sites such as the synovial fluid of RA patients, fibroblast-like and macrophage-like cells of the synovial membrane, together with the infiltrating leucocytes, are the major source of these mediators in the rheumatoid synovial pannus (Rathanaswami et al., 1993). In the GeneChip microarray experiment, an IL-1 concentration close to the levels seen clinically was used to stimulate the MH7A cells. The gene expression of IL-8, Gro- (CXCL1), Gro- (CXCL2), IL-1, and IL-6 in scatter plots is shown in Fig. 2B. According to a RT-PCR analysis, the levels of IL-8, CXCL1, CXCL2, IL-1, and IL-6 mRNA in MH7A cells were all found to be dosedependently increased by IL-1 (Fig. 2C). Therefore, IL-8 appears to play a major role in the

Within the 5'-flanking region of the human IL-8 promoter are a number of motifs with the potential to bind various transcription factors in a cell and stimulus-dependent manner. In particular, the transcription factors NF-B and NF-IL-6 are both involved in stimulus-

concentration of IL-1, and the cells were incubated for an additional 2 hr.

initiation and maintenance of inflammatory responses.

induced IL-8 expression (Matsusaka et al., 1993). We would therefore anticipate that these transcription factors would play an important role in the increased expression of IL-8 mRNA elicited by IL-1, but no increases in the expression for NFKB1 (NF-B), RELA (RelA), or CEBPB (NF-IL-6) were seen in the microarray experiment. On the other hand, in resting cells, NF-B is trapped in the cytoplasm through its interaction with its inhibitor, IkB, and a crucial step in the activation of NF-B is the stimulus-induced phosphorylation of IB by IB kinases (IKKs). To study the extent to which IKKs modulate IL-1 induced IL-8 production, MH7A cells were pretreated with Bay11-7085, an inhibitor of IB phosphorylation, for 1 hr before stimulation with IL-1(Shibata et al., 2009a). As shown in Fig. 3, Bay11-7085 completely blocked the release of IL-8 from IL-1-stimulated MH7A cells.

**A.** The IL-1-induced signal transduction pathways leading to the production of IL-8. Gray symbols indicate genes that were up-regulated by IL-1. White symbols indicate genes whose expression was increased less than 2.0-fold by IL-1. **B.** The effects of Bay11-7085 on the production of IL-8. MH7A cells were first incubated with the IKK/ inhibitor Bay11-7085 for 1 hr, and then with IL-1 for 2 hr. The levels of IL-8 in medium conditioned by the cells were measured using an ELISA. [This figure is modified from (Shibata et al., 2009a)]

Fig. 3. The effect of Bay11-7085 on the production of IL-8

### **2.2 Arithmetic IL-8 production through the PGE2 receptor synthesis**

PGE2, a product of the cyclooxygenation of arachidonic acid, is a potent mediator of the immune response and inflammation, and contributes to the pathogenesis of RA (Robinson et al., 1975, Trebino et al., 2003). PGE2 also displays a complex regulatory function affecting IL-8 gene expression, which is dependent on the concentration of PGE2 and on the specific cell type involved. At physiological and pathological concentrations of up to 100 μM, PGE2 is capable of upregulating endogenous IL-8 expression in human colonic epithelial cells (Yu & Chadee, 1998). However, there have been many reports that PGE2 has no effect on neutrophil-derived IL-8 induced by LPS (Wertheim et al., 1993); that it down-regulates IL-8 in response to LPS in human alveolar macrophages and blood monocytes (Standiford et al., 1992); and suppresses the production of chemokines, including IL-8, in human macrophages

Profiling Inflammatory Genes

and Signaling Pathways in Rheumatoid Synoviocytes for RA Light Therapy 159

**A.** The gray symbols indicate the IL-1-inducible genes (>2.0-fold) in the apoptosis signaling pathway. **B.** The time-dependent effects of IL-1 on the gene expression of cIAP (BIRC3), which negatively

It has been suggested that RA is driven by a network of closely connected interdependent pathogenic mechanisms involving innate and adaptive immunity that ultimately lead to synovial inflammation and aggressive synovial overproliferation during the terminal destructive phase (Firestein, 2003). Furthermore, in a chronically inflamed microenvironment, these destructive changes are driven by invasive synovial hyperplasia and neovascularization (pannus formation) at the interface between the synovium and intraarticular space. During the process of synovial pannus formation, FLSs actively contribute to the inflamed and destructive local microenvironment by secreting a variety of inflammatory mediators, which lead to tissue degradation. In response to IL-1 and TNF-, FLSs also secrete angiogenic growth factors, such as vascular endothelial growth factor (VEGF) (Nagashima et al., 1999) and basic fibroblast growth factor (bFGF), which are essential for the pathophysiological neovascularization in RA joints. In addition, we have identified that BIRC3 is upregulated, making it a candidate mediator of FLS overproliferation in RA. BIRC3 encodes cIAP, which is a key negative regulator of apoptotic signaling in FLSs. The resulting inhibition of FLS apoptosis in the inflamed area would be expected to contribute to the

A RayBio biotin label-based human antibody array I (RayBiotech, Inc.) was used to measure the expression levels of 507 proteins in culture medium conditioned by MH7A cells. Table 1 presents the results of the biotin label-based antibody array analysis of secreted proteins that categorized interleukins, and molecules involved in ECM metabolism. IL-12 p70 and IL-8 were

regulates apoptotic signaling, were examined using MH7A cells.

Fig. 4. An atlas of an apoptosis signaling pathway

formation of the pannus in the RA joint.

**3. Protein chip analysis**

**3.1 ECM degradation**

(Takayama et al., 2002). Furthermore, PGE2 alone had little detectable effect on IL-8, although a small enhancement of the mRNA and protein levels of IL-6 has been observed in human synovial fibroblasts (Agro et al., 1996). In RA research, while PGE2 alone has limited effects on synovial cell production of IL-8, its effects are significant in the context of IL-1 stimulation; endogenous PGE2 may alter the cytokines secreted by mesenchymally-derived cells.

A variety of transcription factors, including NF-κB, NF-IL6, activator protein-1 and octamer-1, have been shown to regulate IL-8 gene transcription (Matsusaka et al., 1993). Caristi *et al*  (Caristi et al., 2005) have shown that, in human T cells, PGE2 induces IL-8 synthesis through an NF-B-independent pathway via its EP1- and EP4-type receptors (PGE2EP1 and PGE2EP4, respectively). We have demonstrated that PGE2 at physiological, as well as pathological, concentrations induced IL-8 production thorough the increased expression of PGE2EP4 receptors in synovial fibroblasts of RA *in vitro* (Shibata et al., 2009b). The present results were of particular interest, since the effect of PGE2 on IL-8 production was found in the IL-1-pretreated MH7A cells. We demonstrated that the newly expressed-PGE2 receptors are involved in this process.

These findings show the complexity with which PGE2 regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions. Therefore, we proposed that, in IL-1-stimulated synovial fibroblasts in RA patients, PGE2 induces IL-8 mRNA transcription by the activation of different signal transduction pathways from the conventional IL-1-stimulated pathways, including PGE2 receptor EP4-triggered pathways. IL-1 enhanced the gene expression of IL8 and PTGER4 (Shibata et al., 2009b), subsequently IL-8 production was enhanced by IL-1 and PGE2 from environmental neutrophils/macrophages in the synovial tissues. These results may therefore highlight a new important role for PGE2 in regulating IL-8 production by the synovial fibroblasts of RA patients, confirming the pro-inflammatory activity of this prostaglandin.

#### **2.3 Apoptotic signaling**

Inflammation of the synovial membrane results in the development of aggressive granulation tissue, called "pannus". Pannus tissue is composed mainly of inflammatory cells such as macrophages and FLSs. By a microarray analysis, the apoptosis/proliferation-related molecules included those involved in processes associated with negative/positive regulation of cell proliferation, apoptosis/anti-apoptosis, cell death, cell proliferation and regulation of the cell cycle, and a large number of these genes were influenced by IL-1 (Fig. 1). Interestingly, whereas the expression of the gene encoding the apoptosis inhibitor cIAP (BIRC3) was enhanced (4.8-fold), the expression of those encoding the cell death inducing molecule FOSL2 (FOS-like antigen 2, 0.4-fold)) and the cell proliferation-related molecule EDNRA (endothelin receptor type A, 0.2-fold) were both suppressed. We attempted to apply the IL-1-inducible genes to a typical pathway atlas involving apoptotic signaling (Fig. 4A). The expression of BIRC3, which encodes the anti-apoptotic protein cIAP, implies that IL-1 signaling leads to inhibition of apoptotic signaling, as expression of cIAP should exert an inhibitory effect on caspase 3 activity (Rothe et al., 1995, Budihardjo et al., 1999). To confirm the increased cIAP production in MH7A cells, we found that levels of the BIRC3 transcript were substantially increased in the IL-1-induced MH7A cells (Fig. 4B).

(Takayama et al., 2002). Furthermore, PGE2 alone had little detectable effect on IL-8, although a small enhancement of the mRNA and protein levels of IL-6 has been observed in human synovial fibroblasts (Agro et al., 1996). In RA research, while PGE2 alone has limited effects on synovial cell production of IL-8, its effects are significant in the context of IL-1 stimulation; endogenous PGE2 may alter the cytokines secreted by mesenchymally-derived

A variety of transcription factors, including NF-κB, NF-IL6, activator protein-1 and octamer-1, have been shown to regulate IL-8 gene transcription (Matsusaka et al., 1993). Caristi *et al*  (Caristi et al., 2005) have shown that, in human T cells, PGE2 induces IL-8 synthesis through an NF-B-independent pathway via its EP1- and EP4-type receptors (PGE2EP1 and PGE2EP4, respectively). We have demonstrated that PGE2 at physiological, as well as pathological, concentrations induced IL-8 production thorough the increased expression of PGE2EP4 receptors in synovial fibroblasts of RA *in vitro* (Shibata et al., 2009b). The present results were of particular interest, since the effect of PGE2 on IL-8 production was found in the IL-1-pretreated MH7A cells. We demonstrated that the newly expressed-PGE2

These findings show the complexity with which PGE2 regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions. Therefore, we proposed that, in IL-1-stimulated synovial fibroblasts in RA patients, PGE2 induces IL-8 mRNA transcription by the activation of different signal transduction pathways from the conventional IL-1-stimulated pathways, including PGE2 receptor EP4-triggered pathways. IL-1 enhanced the gene expression of IL8 and PTGER4 (Shibata et al., 2009b), subsequently IL-8 production was enhanced by IL-1 and PGE2 from environmental neutrophils/macrophages in the synovial tissues. These results may therefore highlight a new important role for PGE2 in regulating IL-8 production by the synovial fibroblasts of RA patients, confirming the pro-inflammatory activity of this

Inflammation of the synovial membrane results in the development of aggressive granulation tissue, called "pannus". Pannus tissue is composed mainly of inflammatory cells such as macrophages and FLSs. By a microarray analysis, the apoptosis/proliferation-related molecules included those involved in processes associated with negative/positive regulation of cell proliferation, apoptosis/anti-apoptosis, cell death, cell proliferation and regulation of the cell cycle, and a large number of these genes were influenced by IL-1 (Fig. 1). Interestingly, whereas the expression of the gene encoding the apoptosis inhibitor cIAP (BIRC3) was enhanced (4.8-fold), the expression of those encoding the cell death inducing molecule FOSL2 (FOS-like antigen 2, 0.4-fold)) and the cell proliferation-related molecule EDNRA (endothelin receptor type A, 0.2-fold) were both suppressed. We attempted to apply the IL-1-inducible genes to a typical pathway atlas involving apoptotic signaling (Fig. 4A). The expression of BIRC3, which encodes the anti-apoptotic protein cIAP, implies that IL-1 signaling leads to inhibition of apoptotic signaling, as expression of cIAP should exert an inhibitory effect on caspase 3 activity (Rothe et al., 1995, Budihardjo et al., 1999). To confirm the increased cIAP production in MH7A cells, we found that levels of the BIRC3 transcript were substantially increased in

cells.

prostaglandin.

**2.3 Apoptotic signaling**

the IL-1-induced MH7A cells (Fig. 4B).

receptors are involved in this process.

**A.** The gray symbols indicate the IL-1-inducible genes (>2.0-fold) in the apoptosis signaling pathway. **B.** The time-dependent effects of IL-1 on the gene expression of cIAP (BIRC3), which negatively regulates apoptotic signaling, were examined using MH7A cells.

Fig. 4. An atlas of an apoptosis signaling pathway

It has been suggested that RA is driven by a network of closely connected interdependent pathogenic mechanisms involving innate and adaptive immunity that ultimately lead to synovial inflammation and aggressive synovial overproliferation during the terminal destructive phase (Firestein, 2003). Furthermore, in a chronically inflamed microenvironment, these destructive changes are driven by invasive synovial hyperplasia and neovascularization (pannus formation) at the interface between the synovium and intraarticular space. During the process of synovial pannus formation, FLSs actively contribute to the inflamed and destructive local microenvironment by secreting a variety of inflammatory mediators, which lead to tissue degradation. In response to IL-1 and TNF-, FLSs also secrete angiogenic growth factors, such as vascular endothelial growth factor (VEGF) (Nagashima et al., 1999) and basic fibroblast growth factor (bFGF), which are essential for the pathophysiological neovascularization in RA joints. In addition, we have identified that BIRC3 is upregulated, making it a candidate mediator of FLS overproliferation in RA. BIRC3 encodes cIAP, which is a key negative regulator of apoptotic signaling in FLSs. The resulting inhibition of FLS apoptosis in the inflamed area would be expected to contribute to the formation of the pannus in the RA joint.


*Communication* **Toward the Transition of Agricultural Anaerobic Digesters into Multiproduct Biorefineries**

**David Bolzonella \* , Davide Bertasini, Riccardo Lo Coco, Miriam Menini, Fabio Rizzioli, Anna Zuliani, Federico Battista, Nicola Frison, Aleksandra Jelic and Giovanna Pesante**

> Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy **\*** Correspondence<EMAIL_ADDRESS>Tel.:+ 39-045-8027965

**Abstract:** Anaerobic digestion allows for the proper management of agro-waste, including manure. Currently, more than 18,000 anaerobic digestion plants are under operation in EU, 80% of which are employed in the rural context. Tariff schemes for power generation from biogas produced during anaerobic digestion of agricultural feedstocks in Germany, Italy and Austria are coming to an end and new approaches are needed to exploit the existing infrastructures. Digesters in the rural context can be implemented and modified to be transformed into sustainable multi-feedstock and multi-purpose biorefineries for the production of energy, nutrients, proteins, bio-chemicals such as carboxylic acids, polyesters and proteins. This paper describes how the transition of agricultural anaerobic digesters into multi-products biorefineries can be achieved and what are the potential benefits originating from the application of a pilot scale platform able to treat cow manure and other crop residues while producing volatile fatty acids, polyhydroxyalkanoates, microbial protein material, hydrogen, methane and a concentrated liquid stream rich in nitrogen, potassium and phosphorus.

**Keywords:** agricultural waste; anaerobic digestion; biobased products; biogas; nutrients; polyhydroxyalkanoates; proteins; volatile fatty acids

### **1. Introduction**

The European Union is an international leader in the bioeconomy sector: Germany, France and Italy are the greatest global producers of crops, meat, fish and processed food, which determine the production of large amounts of residual organic streams, mainly livestock manure. The total European production of livestock effluents accounts for 1.4 billion tonnes per year, with France, Germany and Italy producing 276, 197 and 103 million tonnes per year, respectively [1]. The production of agricultural residues derived from crop cultivation accounted for 21 million tonnes in 2020 in the European Union [2]. These streams, collectively named agro-waste, are a rich untapped source of carbon and nutrients and should not be disposed of massively but rather properly valorized within the circular economy concept with the potential to support human well-being and saving resources [3].

Anaerobic Digestion (AD) is a robust technology for the management and valorization of agro-waste in the rural context: in fact, AD enables the bioconversion of the organic matter present in manure, residual crops, and other residual organic streams of the food processing chain while recovering biogas for power or biomethane production [4,5] and a digestate containing considerable quantities of valuable nutrients [6].

Because of its benefits and the incentives schemes applied in different Countries, AD is largely diffused in Europe [3]: more than 18,000 plants with an installed capacity of 8000 MWel are globally under operation in Europe according to the European Biogas Association [7]. In 2021, the EU produced 3 billion m3 of biomethane [8]. However, the EU production should increase to 35 billion m3 by 2030, to be able to comply to the new EU communication COM/2022/108, implementing the "RE. PowerEU" plan.

**Citation:** Bolzonella, D.; Bertasini, D.; Lo Coco, R.; Menini, M.; Rizzioli, F.; Zuliani, A.; Battista, F.; Frison, N.; Jelic, A.; Pesante, G. Toward the Transition of Agricultural Anaerobic Digesters into Multiproduct Biorefineries. *Processes* **2023**, *11*, 415. https://doi.org/10.3390/pr11020415

Academic Editors: Sonia Heaven, Sigrid Kusch-Brandt and Charles Banks

Received: 21 December 2022 Revised: 24 January 2023 Accepted: 25 January 2023 Published: 30 January 2023

**Copyright:** © 2023 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/).

As reported above, besides biogas, AD plants generate also digestate, a sort of renewable fertilizer [3,6]. The use of digestate provides macro (nitrogen, phosphorus, potassium, etc.) and micro (cobalt, selenium, etc.) nutrients to the soil. Moreover, it determines the supplementation of stable carbon to the fields, thus increasing the carbon sink capability of soils [9].

In the last 20 years, the number of AD plants has increased considerably in Western and Central Europe because of favorable incentive schemes dedicated to the supporting of power and biomethane generation [10]. However, the incentive schemes for power generation in Germany, Italy and Austria are rapidly coming to an end, therefore the use and exploitation of rural anaerobic digesters should be reconsidered.

In perspective, an obvious upgrade of this technology is the transformation of anaerobic digesters into a multi-feedstock, multipurpose and multiproduct biorefinery able to valorize agro-waste into high added value biobased products such as nutrients and chemicals, thus creating social and economic benefits at local level [11–13].

In recent years, several studies showed the possibility to generate biofuels such as hydrogen and methane, biobased chemicals such as polyhydroxyalkanoates, produce microalgae, bacteria or yeast, convert CO2 and recover nutrients from organic wastes.

It is therefore possible to imagine anaerobic digestion at the center of a future biorefinery approach where agro-wastes and food processing waste are converted into high added value biobased products other than biofuels. This new bioeconomy approach is crucial for the rural renaissance of Europe.

In this framework, the Horizon Europe project AgriLoop will study the upcycling of agro- and food-processing waste and byproducts to high value biobased products. A pilot scale platform biorefinery will be operated where cow manure, crops silage and other agrowaste will be transformed into biofuels, carboxylic acids, and polyesters, while nutrients will be recovered in concentrated forms.

In this paper, we will provide evidence of this global approach, here implemented in a pilot scale biorefinery able to convert agro-waste into valuable products working in a real environment. For the purpose of this study, agricultural wastes, by-products and coproducts are defined as plant or animal residues that are not (or not further processed into) food or feed. This large amount of undervalued biomass is further called agro-waste as it is intended to be fully integrated as resource in optimised cascading biorefinery and therefore revoked as waste, to become multiple levers of the transition away from a fossil-based carbon-intensive economy.

#### **2. Materials and Methods**

The general approach proposed in this study envisages that livestock effluents, crop silage and other agro-waste are transformed into biofuels and biobased products such as volatile fatty acids (VFAs) and polyhydroxyalkanoates (PHAs), while nutrients and microbial proteins are recovered. All these activities will be carried out treating real substrates and in a real environment: the pilot scale platform is placed in "La Torre" farm, Isola della Scala, Verona, in the northeast of Italy.

This biorefinery platform will allow for the treatment of different types of feedstocks and their bioconversion into biobased products.

#### *2.1. Pilot Plant Description*

The biorefinery plant is an upgrade and modification of the one presented by Righetti and colleagues [13], first developed in the framework of the project NoAW, No Agro-Waste-Innovative approaches to turn agricultural waste into ecological and economic assets (http://noaw2020.eu/), accessed on 20 December 2022, and it can be divided into the following main areas:

*Processes* **2023**, *11*, x FOR PEER REVIEW 3 of 12

agro-waste pre-treatment and preparation;

the following main areas:

duced;

assets (http:// noaw2020.eu/), accessed on 20 December 2022, and it can be divided into

acidogenic fermentation where volatile fatty acid production and hydrogen are pro-

**Figure 1.** Scheme of the unit operations involved in the studied pilot scale process. **Figure 1.** Scheme of the unit operations involved in the studied pilot scale process.

The 2 m3 storage tank for feedstock mixing and preparation is equipped with a weighing system for the control of the amount of material fed into the system. The tank has a mixer to homogenize the substrates. Mixed material is then ground by means of a special centrifuge pump model TM451 (Vidotto Dissipatori, Eraclea, Italy, Italy) and pumped to the anaerobic acidogenic reactor. The fermentation unit is a 5 m3 unit with an adjustable volume. The fermenter is temperature controlled with warm water and can operate both in mesophilic and thermophilic conditions. The 2 m3 storage tank for feedstock mixing and preparation is equipped with a weighing system for the control of the amount of material fed into the system. The tank has a mixer to homogenize the substrates. Mixed material is then ground by means of a special centrifuge pump model TM451 (Vidotto Dissipatori, Eraclea, Italy, Italy) and pumped to the anaerobic acidogenic reactor. The fermentation unit is a 5 m3 unit with an adjustable volume. The fermenter is temperature controlled with warm water and can operate both in mesophilic and thermophilic conditions.

The operational conditions applied to the reactor are shown in Table 1. The operational conditions applied to the reactor are shown in Table 1.

**Table 1.** Operational conditions of the anaerobic acidogenic fermenter and anaerobic digester. **Table 1.** Operational conditions of the anaerobic acidogenic fermenter and anaerobic digester.

The fermentation process operates in mesophilic conditions with an average organic loading rate in the range of 15–25 kgCOD/m3 d, a desired OLR of approximately 18 kgCOD/m3 d and a hydraulic retention time of 3 to 5 days to improve the conversion of particulate COD into short chain VFAs to be fed to the PHA bioreactor. The operational pH of the anaerobic fermenter is determined by the characteristics of fed substrates and it The fermentation process operates in mesophilic conditions with an average organic loading rate in the range of 15–25 kgCOD/m3 d, a desired OLR of approximately 18 kgCOD/m3 d and a hydraulic retention time of 3 to 5 days to improve the conversion of particulate COD into short chain VFAs to be fed to the PHA bioreactor. The operational pH of the anaerobic fermenter is determined by the characteristics of fed substrates and it is typically around 5.5. Operational conditions such as OLR can be set in order to tune the abundance of different short chain VFAs in the liquid phase.

The fermenter effluent is treated in a horizontal screw press unit (Sepcom, WAM Group, Modena, Italy) for the solid/liquid separation. The screw press treatment capacity is 5 m3/h.

The liquid fraction obtained through solid/liquid separation is then pre-filtered at 500 micron and then micro-filtered in a rotating ceramic unit. The permeate rich in short chain VFAs is then sent to the mixed microbial biomass selection unit.

The first bioreactor is a 2 m3 volume system for the selection of PHA-accumulating microorganism, while the second reactor, 1 m3 in volume, is dedicated to the accumulation of PHAs.

Both bioreactors can operate in batch or continuous mode.

The sludge rich in PHAs produced in the second bioreactor, after settling and concentration, is quenched with sulfuric acid and sent to a chemical extraction unit.

The separated solid fraction obtained from the screw press is sent to a mesophilic anaerobic digester designed to operate at an optimal organic loading rate of 4 kgVS/m3 d and with a hydraulic retention time of approximately 30 days. The anaerobic digester has a working volume of 1 m3 . The operational pH of the anaerobic digester is typically self-buffered around 8. Table 1 reports the expected ranges of operation for both OLR and HRT.

The anaerobic digestion effluent undergoes to solid/liquid separation and the liquid stream is sent to a screening and filtration unit with mesh of 250 µm and then to a double stage filtration unit where ultra-filtration is followed by reverse osmosis for nutrient recovery and concentration. The permeate (clean water) is used to dilute the treated feedstock in the initial storage tank. The ultra-filtration unit has a ceramic disc filter with a porosity of 0.22 µm, with a maximum flow of 10 L/h. The reverse osmosis unit has a membrane porosity of less than 1 nm, with a maximum flow of 200 L/h.

#### *2.2. Analysis*

In order to carry out the mass balances for the system and for each operation unit, the feedstocks and the effluents of the reactors were monitored once or more times per week through the analysis of total and volatile solids (TS and TVS), chemical oxygen demand (COD), Total Kjeldahl Nitrogen (TKN) and total phosphorus (TP) concentrations. The process stability parameters for the anaerobic fermenter and digester, namely pH, VFAs content and speciation, total and partial alkalinity and ammonia (NH3-N) were regularly checked daily. All the analyses, except for the VFAs, were carried out in accordance with the standard methods for water and wastewater analysis [14].

The biogas flowrate was measured by a volumetric counter (Ritter Gmbh, Schwabmünchen, Germany), while biogas composition in terms of CH4 and CO2 concentrations was measured using a portable gas monitor (GAS Counter BIOGAS 5000), equipped with a hydraulic guard to remove H2S. Hydrogen content was determined by a gas chromatograph (GC Agilent Technology 6890 N) equipped with the column HP-PLOT MOLESIEVE, 30 × 0.53 mm ID × 25 µm film, using a thermal conductivity detector and an argon as gas carrier.

#### *2.3. Mass Balance Equation and Yields of the Pilot Plant*

The mass balance of the pilot scale plant is based on the chemical characteristics of the influent and effluent streams and the relative flowrates. The general equation for mass balances for a given chemical compound considers influent and effluent mass, generated and consumed mass and a term for accumulation according to the general formula:

$$\text{M influent} - \text{M effluent} + \text{M generated} - \text{M consumed} = \text{M accumulated in the system.} \tag{1}$$

The general equation can be then applied considering flowrate and concentration for any chemical compound according to the following equation:

$$\mathbf{V} \cdot \frac{d\mathbf{C}}{dt} = \mathbf{Q}\_{\text{in}} \cdot \mathbf{C}\_{\text{in}} - \mathbf{Q}\_{\text{out}} \cdot \mathbf{C}\_{\text{out}} - \mathbf{V} \cdot \mathbf{R}\_{\text{c}} + \mathbf{V} \cdot \mathbf{R}\_{p\text{s}} \tag{2}$$

where

*Qin* is influent flowrate, m3 per day;

*Cin* is influent concentration of a given compound, kg per m3 ;

*Cout* is effluent concentration of a given compound, kg per m3 ;

*Qout* is effluent flowrate, m3 per day;

*C* is compound concentration in the bioreactor, kg per m3 ;

*V* is volume of the bioreactor, m3 ;

*Rp* is specific production rate in the system, kg per m3 per day;

*Rc* is specific consumption rate in the system, kg per m3 per day.

In steady state conditions, the concentration of a given component, *C*, is constant and Equation (2) equals zero.

Yields for a given product are determined considering the quantity of product per time unit, typically per day, over the quantity of substrate entering the system at the same time.

The mass balance and yields of the system discussed below are determined according to the description reported here.

#### **3. Expected Results and Discussion**

As described above, existing AD plants can be converted into multipurpose biorefineries able to produce a portfolio of different biobased products.

In this study, we will consider the conversion of a mix of cow manure, straw and residual crops into different bioproducts:

The scheme of the general approach is shown in Figure 1.

Specific details for the different biobased products are illustrated and discussed below.

#### *3.1. Biofuels: Hydrogen and Methane Production*

Hydrogen and methane have gained particular interest in the scientific community in recent years due to the need of decarbonisation to fight climate change and to the increasing price of natural gas. This has led to the further development and implementation of the twostage anaerobic digestion technology, a biological process which allows for the simultaneous production of hydrogen and methane from organic residues. The two anaerobic reactors treating agro-waste are dedicated to dark fermentation and methanogenesis, respectively. The first step consists in hydrogen and carbon dioxide production by the acidogenic microorganisms, through the hydrolysis of complex organic compounds into soluble macro-molecules and then into short chain VFAs. During the methanogenic step, in the second reactor, VFAs are degraded to carbon dioxide and methane, while part of the carbon dioxide is bio-converted to methane through its reaction with hydrogen [15]. The resulting gaseous blend can be upgraded to eliminate undesired compounds such as hydrogen sulphide, ammonia and carbon dioxide [16] in order to obtain the hydrogen–methane blend bio-hythane.

In the AgriLoop project, we will operate the two reactors as described in Table 1 and we expect yields of approximately 20 L H2 and 180 L CH4 per kgVS treated. Besides the gaseous effluents, the process produces digestate, a mix of solid and liquid parts.

#### *3.2. Volatile Fatty Acids Production*

VFAs are short chain carboxylic acids from acetate (C1) to caproate (C6), usually derived from fossil sources. The biological production of VFAs is receiving great attention to favour the transition from a linear to a circular economy model. Through anaerobic fermentation, agro-waste can be converted into valuable VFAs with high economic value, ranging from 800 to 2500 €/tonne [17]. In our project, agro-waste will be converted into VFAs for the synthesis of PHAs and single cell proteins (SCP, see Section 3.5).

The VFAs yields depend on the chemical and physical nature of the organic substrates: in this case, the mixed material will be composed of both hard-to-degrade organic material (straw) and easily biodegradable cellulose and hemi-cellulose, therefore expected yields are in the range of 0.2–0.3 gVFA/gVS. These values are in line with literature data: agro-food wastes rich in simplest compounds, for example, winery waste such as lees, having high content of glucose, fructose and ethanol, have typical VFAs yields of 0.5–0.7 gVFA/gCOD; olive pomace, rich in lipids and recalcitrant lignocellulosic compounds, have lower yields [18]. In order to increase the process yield, lignocellulosic materials represented by rice, corn, wheat straws and by-products from energy crops are usually pre-treated under steam explosion to favour the degradation of the long and complex carbon chains into smaller organic fractions, which can be easily converted by the mixed microbial culture [19], increasing the VFAs yield to 0.45–0.55 gVFA/gCOD [20].

In this specific study, chemical pre-treatments to increase bioconversion of recalcitrant organic matter will be applied; in particular, the use of soda will be tested.

In particular, crop residues and manure with straw will be added with soda at 36% to match up a final concentration in the pre-treatment vessel of 1% and digested overnight at environment temperature.

In terms of concentration, VFAs in the fermentation broth are usually in the range of 15–40 g/L [21] depending on both bioconversion capability and dilution. To be exploitable for industrial purpose, VFAs need to be separated from the other organic compounds and concentrated in order to have a minimal concentration level of 100 g/L [22].

Several techniques such as adsorption on solid matrix [23], distillation and evaporation [22], extraction by solvent [24], electrodialysis [25] and pressure-driven membrane processes [26] have been implemented for VFAs recovery. Traditional recovery techniques by solvents and distillation are known to be high in cost and energy, but are still largely applied in order to separate VFA from fermentation medium. It was demonstrated that the maximum VFA recovery by this technique falls in the range of 61–98% [27]. Filtration techniques, such as nanofiltration and reverse osmosis, are becoming attractive alternatives to traditional recovery techniques, being capable of recovering over 90% of the VFAs content from the fermentation medium [28].

VFA recovery has other advantages if applied directly on the liquid phase of the fermenter: it can enhance anaerobic fermentation because (i) it shifts the balance toward VFAs biosynthesis, (ii) it alleviates product-induced inhibition, and (iii) it maintains stable alkalinity levels, allowing further waste degradation [29].

In order to obtain a functional cascade of integrated membrane separation processes that allow the recovery, purification and optimal concentration of VFAs from waste-derived AD effluents, it is essential to have pre-treatment processes able to deeply remove suspended solids and colloids. To ensure the long-term application of a membrane filtration system, it is necessary to take into account the factors that influence filtration operations, such as pH and solids content (TS and TVS) [28]. The solids content is the limiting factor for the application of specific membranes, since it can cause damage to the membrane itself. Consequently, pre-treatments such as solid/liquid separations with a centrifuge or vibrating screen must be applied [17].

In the AgriLoop project, because of the bio-recalcitrant nature of treated agro-waste, mainly due to straw, expected yields are low, in the range of 0.1–0.2 kgVFA per kg of VS fed to the anaerobic fermenter, while the fermentation broth will show VFA concentrations in the range of 10–15 g/L. Several recovery techniques will be tested at lab scale with a preference for membrane contactors.

#### *3.3. Polyhydroxyalkanoates Production*

Polyhydroxyalkanoates are a family of thermoplastic polyesters of hydroxyacid (HA) monomers connected by an ester bond [18]. They can be produced from different renewable sources by bacterial fermentation, as a form of intracellular carbon and energy storage. The process of PHA production by mixed microbial culture applied in this biorefinery platform

is conducted in three independent stages [30]. In the first step, the complex organic substrate is fermented to obtain a stream rich in VFAs; the second step is based on the natural principles of selection and competition of PHA-accumulating microorganisms against microorganisms that are unable to accumulate PHAs, by applying transient conditions in sequential batch reactors (SBR) [30]. It has been shown that conditions of external substrate excess (feast) and limitation (famine) select microbial populations with an increased capacity to store PHAs [31]. Finally, in the third phase, when the cells reach maximum PHAs content, they are harvested and sent to downstream extraction processes [18]. The downstream processes for extracting the polymer from the cells are among the most important factors affecting the overall cost of PHAs production, and they represent the bottleneck of the whole process. So far, the most studied methods for the recovery of PHAs can be grouped into two categories: solvent extraction and digestion of the non-polymeric material. Solvent extraction is the predominant method for PHAs extraction, used when high polymer purity is desired [32]. It typically involves soaking the PHA-containing biomass in an appropriate solvent or solvent mixture to dissolve the granules, followed by the addition of a precipitating agent to recover the polymers in crystalline form [33]. In the dissolution step, chlorinated solvents are typically used, e.g., chloroform and dichloromethane. For the precipitation step, methanol and ethanol are used [33]. Solvent extraction in large-scale applications is generally not an environmentally friendly method due to the harmful characteristics of the most used solvents, especially chlorinated solvents such as chloroform, and the massive amounts of antisolvent used (approximately 10 volumes per volume of PHAs solution) in order to precipitate the polymer. However, PHA-producing microorganisms can accumulate these polymers in quantities of up to 70% g PHA/gVSS [18], therefore it is possible to remove the small fraction of non-PHA cellular mass (NPCM) surrounding the PHA rather than extracting the PHA by solubilizing it in a suitable solvent. For this reason, several methods have been developed to release the PHAs granules by solubilizing the surrounding NPCM. Various chemical compounds can be used: sodium hypochlorite, surfactants, acid and alkaline compounds. All these chemicals can be used in the aqueous phase, thus avoiding the energy consumption required to dry the biomass when solvent extraction is applied [33].

Although the mass balance indicates higher productions, the expected final PHA productions for this pilot scale biorefinery process can reach a maximum of 2 kg PHA per day with a yield estimated in 0.01 kgPHA per kgVS in the feedstock because of the complexity of the biomass treated in this specific context, mainly due to straw, and because of the set-up of the extraction and purification phase.

#### *3.4. Nutrient Recovery*

The main by-product of the AD process is represented by a solid-liquid mixture, the digestate, which contains the stabilized organic matter not converted into biogas and considerable amounts of macro- and micro-nutrients. Digestate is now receiving great attention from the agricultural community because it is rich in nitrogen and phosphorous, and because of the high costs of fossil fertilizers. For example, when livestock effluents are treated, TKN concentration can reach values in the range of 2.5–9 g per kg of fresh matter with an ammonium content exceeding 50–60% *w*/*w* [17]. Agricultural digestate have also a good content of phosphorous, typically in the range of 0.5–1.5 g per kg fresh matter, a small part of which is in the soluble form.

There are currently several technological options for digestate treatment and nutrients recovery available on the market. All the options require a common step of solid/liquid separation of the agricultural digestate. These two phases, solid and liquid, can be then further processed to obtain concentrated nutrient streams minimizing the transport costs [16,34].

The most developed technologies at industrial scales for nitrogen and phosphorus recovery are ammonia stripping and struvite precipitation, respectively, which allow both an average removal and recovery yields of 90–95%. However, they have very high environmental and energetical costs [17]. For this reason, new technologies are emerging to

reduce the use of chemicals, such as pressure-driven membranes, ultrafiltration and reverse osmosis (RO).

Membrane processes are physical-based technologies, where the agricultural digestate is treated under sequential solid/liquid separations. The solid fraction from each stage is called concentrate or retentate, while the liquid phase is named permeate. Usually, the first separation stage consists in a preliminary removal of the coarse solids by means of a centrifuge or a vibrating screen. Then, the liquid is treated in several steps: (i) microfiltration (MF), having membrane with pore size >0.1 µm under a trans-membrane pressure of 0.1–3 bar, (ii) ultrafiltration (UF, pore size >0.001 µm, pressure 2–10 bar) and a (iii) RO (pore size <1 nm, pressure 10–100 bar), to obtain a nutrient-rich retentate and water as permeate. In this way, it is possible to obtain a fertilizer rich in N and P (8.2–12.0 kg/tonne TN; 5.6–10.4 kg/tonne P2O5) [19,20].

In this study, we will treat the liquid fraction of digestate obtained after solid/liquid separation via a screw press in a train of operation consisting of filtering/sieving, microfiltration and reverse osmosis to obtain a retentate characterized by relatively high concentrations of nitrogen, potassium and phosphorus, as well as other important minerals such as iron, magnesium and sulfur, with the characteristics of distillation vinasse which can be used as soil amendment. Expected typical concentrations for nitrogen are in the range of 7–10 gN/L [35] while phosphorus is mainly recovered in the form of fine microfiltered sludge before the RO unit.

#### *3.5. Proteins Production*

Due to the growing human population, expected to reach 10 billion by 2050 [36], proteins are a sought-after resource not only as a source of food or animal feed, but also as the starting material for other high-value products such as protein hydrolysates, biostimulants for agriculture, wood adhesives, flocculants, surfactants and protein-based plastics [37]. Animal- and vegetal-based proteins are at present mainly produced through low-efficiency processes with a high environmental impact in terms of greenhouse gas emissions, land and water use, and soil and air pollution [38,39]. However, AD plants are an underexploited source of proteins, which could be used in place of traditional protein sources, with a lower ecological footprint. AD plants, which at present are either run at a cost or in need of subsidies, could be turned into profitable or at least neutral-cost establishments, once equipped for the upcycling of proteins.

Proteins can be recovered from AD plants through different routes as described in Figure 2:

2. CH4, CO2 and H2 produced by methanogenesis are generally turned into thermal or electrical energy. However, these gases can be upcycled into microbial protein production by methanotrophic and hydrogenotrophic microbial strains, while the digestate can also be utilised as a source of nitrogen in the form of ammonia [42,43]. In the AD context, the CO2 used as feedstock for bacterial strains can be generated from the upgrading of biogas into biomethane, or from the CO2 emitted as a bioproduct of the

3. A more straightforward route for the recycling of proteins in AD plants is the utilisation of the microbial biomass produced in the digesters directly as MP source. This biomass containing mixed microbial cultures can be richer in protein than pure cul-

In this study, route 1 is followed: specifically, VFA generated in the acidogenic anaerobic fermenter is used to produce PHA-accumulating microbial biomass which can be employed for feeding purposes, especially in the aquaculture sector. Expected yields are high and interesting with microbial biomass production in the range of 0.3–0.5 kg VSS per

tures, and has many applications, mainly in the animal feed sector [44].

combustion of biogas.

kg COD consumed.

**Figure 2.** Schematic representation of the routes through which proteins can be recovered from AD plants. **Figure 2.** Schematic representation of the routes through which proteins can be recovered from AD plants.

*3.6. Expected Mass Balance for the Overall Biorefinery Process* The overall biorefinery process allows for the recovery of biofuels (hydrogen and methane, VFA, PHA and nutrients). PHA-rich microbial mass can be used as protein microbial material instead of as a source of polyesters. Considering the feeding of 1 m3 per day at 20% dry matter, the expected productions In this study, route 1 is followed: specifically, VFA generated in the acidogenic anaerobic fermenter is used to produce PHA-accumulating microbial biomass which can be employed for feeding purposes, especially in the aquaculture sector. Expected yields are high and interesting with microbial biomass production in the range of 0.3–0.5 kg VSS per kg COD consumed.

#### of hydrogen, methane, VFAs, PHAs, nitrogen and phosphorus are those reported in Table 2. *3.6. Expected Mass Balance for the Overall Biorefinery Process*

The overall feedstock is anaerobically fermented and can produce some 40 kg of short chain fatty acids, where relative concentrations of acetic, propionic, butyric and valeric acids can be tuned using different operational conditions in terms of HRT and OLR. Dur-The overall biorefinery process allows for the recovery of biofuels (hydrogen and methane, VFA, PHA and nutrients). PHA-rich microbial mass can be used as protein microbial material instead of as a source of polyesters.

ing the anaerobic process, up to 2 m3 of hydrogen is produced. Produced VFAs can be considered a product itself, or, after solid/liquid separation Considering the feeding of 1 m3 per day at 20% dry matter, the expected productions of hydrogen, methane, VFAs, PHAs, nitrogen and phosphorus are those reported in Table 2.

and further purification, can be fed to the PHA-accumulating bioreactors. If transferred to this section, VFAs can produce up to 20 kg of microbial biomass containing up to 8 kg of **Table 2.** Expected quantitative products obtained from the overall AgriLoop process.

The overall feedstock is anaerobically fermented and can produce some 40 kg of short chain fatty acids, where relative concentrations of acetic, propionic, butyric and valeric acids can be tuned using different operational conditions in terms of HRT and OLR. During the anaerobic process, up to 2 m3 of hydrogen is produced.

Produced VFAs can be considered a product itself, or, after solid/liquid separation and further purification, can be fed to the PHA-accumulating bioreactors. If transferred to this section, VFAs can produce up to 20 kg of microbial biomass containing up to 8 kg of PHAs. Because of the recovery procedure, the PHAs effectively recovered will be clearly lower than the calculated mass.

The solid fraction recovered after the solid/liquid separation step, equivalent to 60% of the total solids in the feedstock, is sent to the anaerobic digestion unit where up to 18 m3 methane can be produced. Anaerobic digestate will be then treated to separate solids and recover nutrients from the liquid phase via screening, microfiltration and ultrafiltration: 0.6 kg and 0.05 kg of nitrogen and phosphorus, respectively, can be recovered.

Table 2 summarizes the expected quantities of products.

#### **4. Conclusions**

Currently, more than 18,000 anaerobic digestion units are under operation in EU, 80% of which are employed in the agricultural sector. Because of the need for the sustainable production of biobased products from agro-waste and since the tariff schemes are rapidly approaching their end in several countries, there is the urgent need for a reconfiguration of these plants. In particular, these can be transformed into biorefineries able to transform agro-waste into valuable bio-based products.

In this study, a complete biorefinery platform for the treatment of agro-waste (especially cow manure) was designed and implemented at pilot scale in a real farm environment: biofuels (methane and hydrogen), VFAs, PHAs or microbial mass proteins were the main products. The high flexibility of the process allows for the different production of biofuels or biochemicals depending on the operator choices.

Considering the treatment of 1 m3 of slurry at 20% dry matter every day, for biofuels, up to 2 and 18 m3 of hydrogen and methane, respectively, can be produced. Then, 40 kg of VFA can be produced and up to 8 kg PHA after extraction and purification. Moreover, 0.6 kg and 0.05 kg of N and P, respectively, can be recovered in a concentrated form, easy to transported.

The practical set up of this platform will be further studied and its applicability will be analyzed especially through the environmental, social and economic benefits and sustainability performance that will be quantitatively assessed during the AgriLoop project implementation.

**Author Contributions:** Conceptualization, D.B. (David Bolzonella); data curation; writing—original draft preparation, writing—review and editing, D.B. (David Bolzonella), D.B. (Davide Bertasini), R.L.C., M.M., F.R., A.Z., F.B., N.F., A.J. and G.P.; funding acquisition, D.B. (David Bolzonella). All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors acknowledge financial support from the Project AgriLoop. This project has received funding from the European Union's Horizon Europe research and innovation programme under grant agreement No. 101081776. Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.

**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**

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obtained before the start of the study. The questionnaire survey enabled the identification of the main socio-demographic characteristics. In this study, thermal stress was evaluated by the PHS ISO 7933 index "Analytical determination and interpretation of heat stress using calculation of the predicted heat strain" [10]. The physical parameters used to calculate this index come from measurements of the environment as well as from the estimation of the equivalent metabolism in accordance with the ISO 8996 "Ergonomics of the thermal environment - Determination of

*Ergonomic Evaluation of Thermal Stress in a Tunisian Steel Industry*

*DOI: http://dx.doi.org/10.5772/intechopen.98697*

The ISO 7933 standard provides a main advantage of the PHS model related to the reorganization of work, by interposing of short periods of rest during work and the distribution of drinks so as to compensate the water losses taking into account the interindividual differences in physiological responses to heat [1]. This model has been validated using data from 672 laboratory experiments and 237 field experi-

Thermal comfort is defined as a state of satisfaction against a person's thermal environment evaluated by six primary parameters providing this comfort [1]. This comfort was estimated by the PMV and PPD indices described by the ISO 7730 standard [8]. The results of this study were convincing, more than a half (65

It should be noted that the thermal stress is also estimated using the WBGT index described by the ISO 7243 standard [9]. This index has a fairly limited field of application, in particular while evaluating thermal stress during very short periods or conditions close to the thermal comfort [1]. The PHS model seems to be more discriminating than the WBGT index in defining the severity of thermal stress during work situation and organizing work in order to minimize or eliminate thermal stress. Metabolism value estimated for the calculation of this index comes from the results of the recording of the heart rate which gives it an accuracy of the order of 10% and then an accurate estimation [14]. Therefore, only results of the

The climatic conditions category was 3 in 68.18% of subjects (Long-term constraint: discomfort and risk of dehydration after several hours of exposure) and 4 (Short-term constraint: health risk after 30 to 120 minutes of exposure) in 30.3% of workers. The use of this index enabled the assessment of the thermal stress and the management of work in heat [15]. Thus, workers were advised to drink water at 10° C after an average working time of 19.4 5.49 min. A possible alternative is to let employees spontaneously choose their space of work or to allow them to stop

In this study population, the core temperature could reach 38°C after an average working time of 131.3 60.89 min. The World Health Organization (WHO) Technical Report No. 412 published in 1969 stated that: "*It is not recommended that the core body temperature exceeds 38°C for prolonged daily exposure to heavy work*..." [4]. It should be emphasized that, at a rectal temperature of 38°C, an employee does not run any health risk. This limit value concerns the "average" subject and is intended to protect employees who, due to a greater intolerance to heat, evolve to

In this study, more than half of workers (58.75%) know nothing about the risks associated with working in the heat, while 10 employees have already had accidents due to heat (cramps, faintness, stroke. heat). Employees should be informed and trained to recognize signs of thermal stress and prevent the occurrence of these

This study has limitations that warrant mentioning. The thermal stress evaluation was made by physical objective measurements that did not take into account operators' differences. Moreover, the investigation was limited to one company located in the central-eastern region of Tunisia. Therefore, findings cannot be

operators) were not in a situation of thermal comfort.

PSH evaluation will be discussed in this work.

working as soon as they feel some symptoms of strain [1, 16].

higher temperatures, under the same conditions [1].

energy metabolism".

ments [1, 11–13].

accidents [1, 17].

**73**

### **Table 2.**

*Physical parameters of thermal stress.*

#### **Table 3.**

*Thermal stress evaluation.*

In addition, the mean sweat flow was 699.4 160.02 g/h and the mean total water loss was estimated to be 6083.7 1737.29 g.

In accordance with the recommendations of ISO 7933 "Analytical determination and interpretation of thermal stress based on the calculation of the foreseeable thermal strain", it would be imperative to offer all workers oral rehydration by water at 10°C after a calculated average working time of 19.4 5.49 min.

According to these estimates, after 8 hours of work, the average core temperature would reach 38.6 1.03°C. Excessive water loss was reported among 80.3% of workers and would be reached after 292.6 74.64 min. The core temperature would be 38°C, based on these evaluations, among 51.5% of workers who received a heart rate recording after 131.3 60.89 min.

In total, on the basis of these results of the estimation of thermal stresses, 68.18% of subjects would be concerned by a category 3 of climatic condition, namely "Long-term stress: discomfort and risk of dehydration after several hours of exposure" and 30. 3% of them by a category 4 of climatic condition equivalent to a "Short-term constraint: risk to health after 30 to 120 minutes of exposure".

#### **4. Discussion**

This study included measurements of the physical parameters of thermal stress followed by an evaluation of thermal stress. Informed consent from workers was

#### *Ergonomic Evaluation of Thermal Stress in a Tunisian Steel Industry DOI: http://dx.doi.org/10.5772/intechopen.98697*

obtained before the start of the study. The questionnaire survey enabled the identification of the main socio-demographic characteristics. In this study, thermal stress was evaluated by the PHS ISO 7933 index "Analytical determination and interpretation of heat stress using calculation of the predicted heat strain" [10]. The physical parameters used to calculate this index come from measurements of the environment as well as from the estimation of the equivalent metabolism in accordance with the ISO 8996 "Ergonomics of the thermal environment - Determination of energy metabolism".

The ISO 7933 standard provides a main advantage of the PHS model related to the reorganization of work, by interposing of short periods of rest during work and the distribution of drinks so as to compensate the water losses taking into account the interindividual differences in physiological responses to heat [1]. This model has been validated using data from 672 laboratory experiments and 237 field experiments [1, 11–13].

Thermal comfort is defined as a state of satisfaction against a person's thermal environment evaluated by six primary parameters providing this comfort [1]. This comfort was estimated by the PMV and PPD indices described by the ISO 7730 standard [8]. The results of this study were convincing, more than a half (65 operators) were not in a situation of thermal comfort.

It should be noted that the thermal stress is also estimated using the WBGT index described by the ISO 7243 standard [9]. This index has a fairly limited field of application, in particular while evaluating thermal stress during very short periods or conditions close to the thermal comfort [1]. The PHS model seems to be more discriminating than the WBGT index in defining the severity of thermal stress during work situation and organizing work in order to minimize or eliminate thermal stress. Metabolism value estimated for the calculation of this index comes from the results of the recording of the heart rate which gives it an accuracy of the order of 10% and then an accurate estimation [14]. Therefore, only results of the PSH evaluation will be discussed in this work.

The climatic conditions category was 3 in 68.18% of subjects (Long-term constraint: discomfort and risk of dehydration after several hours of exposure) and 4 (Short-term constraint: health risk after 30 to 120 minutes of exposure) in 30.3% of workers. The use of this index enabled the assessment of the thermal stress and the management of work in heat [15]. Thus, workers were advised to drink water at 10° C after an average working time of 19.4 5.49 min. A possible alternative is to let employees spontaneously choose their space of work or to allow them to stop working as soon as they feel some symptoms of strain [1, 16].

In this study population, the core temperature could reach 38°C after an average working time of 131.3 60.89 min. The World Health Organization (WHO) Technical Report No. 412 published in 1969 stated that: "*It is not recommended that the core body temperature exceeds 38°C for prolonged daily exposure to heavy work*..." [4]. It should be emphasized that, at a rectal temperature of 38°C, an employee does not run any health risk. This limit value concerns the "average" subject and is intended to protect employees who, due to a greater intolerance to heat, evolve to higher temperatures, under the same conditions [1].

In this study, more than half of workers (58.75%) know nothing about the risks associated with working in the heat, while 10 employees have already had accidents due to heat (cramps, faintness, stroke. heat). Employees should be informed and trained to recognize signs of thermal stress and prevent the occurrence of these accidents [1, 17].

This study has limitations that warrant mentioning. The thermal stress evaluation was made by physical objective measurements that did not take into account operators' differences. Moreover, the investigation was limited to one company located in the central-eastern region of Tunisia. Therefore, findings cannot be

In addition, the mean sweat flow was 699.4 160.02 g/h and the mean total

**Mean air temperature (Ta) (°C)** 29,66 2,57 27,99 1,87 **Mean air speed (Va) (m/s)** 0,58 0,82 0,23 0,26 **Mean relative humidity (RH) (%)** 59,56 10,98 68, 56 7,78 **Mean Globe temperature (Tg) (°C)** 34, 15 2,87 40,21 7,95

**PMV** 1,5 4,7 3,2 0,47 **PPD (%)** 50 100 97,3 3,7 **WBGT (°C)** 20 29,9 27,5 2,62 **Limit WBGT (°C)** 26,5 30,2 28,4 0,81

**Sweat loss (g/h)** 230 1130 699,4 160,03 **Total water loss (g)** 1780 10600 6083,7 1737,3

**Central temperature after 8 hours of work (°C)** 37,3 42 38,6 1,03 **Time to reach excessive water loss (min)** 160 480 292,6 74,64

and interpretation of thermal stress based on the calculation of the foreseeable thermal strain", it would be imperative to offer all workers oral rehydration by water at 10°C after a calculated average working time of 19.4 5.49 min.

In accordance with the recommendations of ISO 7933 "Analytical determination

**In door Out door**

**Minimum Maximum Mean Standard**

3 60 48 13,19

10 55 19,4 5,49

46 385 131,3 60,89

**deviation**

According to these estimates, after 8 hours of work, the average core temperature would reach 38.6 1.03°C. Excessive water loss was reported among 80.3% of workers and would be reached after 292.6 74.64 min. The core temperature would be 38°C, based on these evaluations, among 51.5% of workers who received a

In total, on the basis of these results of the estimation of thermal stresses, 68.18%

This study included measurements of the physical parameters of thermal stress followed by an evaluation of thermal stress. Informed consent from workers was

of subjects would be concerned by a category 3 of climatic condition, namely "Long-term stress: discomfort and risk of dehydration after several hours of exposure" and 30. 3% of them by a category 4 of climatic condition equivalent to a "Short-term constraint: risk to health after 30 to 120 minutes of exposure".

water loss was estimated to be 6083.7 1737.29 g.

**Time interval Indicated to drink 200 cc of water**

**The central temperature reaches 38°C at the end**

heart rate recording after 131.3 60.89 min.

**4. Discussion**

**72**

**Table 2.**

*Physical parameters of thermal stress.*

*Occupational Wellbeing*

**Work limit time/60 min**

**(min)**

**at 10 °C (min)**

**of (min)**

*Thermal stress evaluation.*

**Table 3.**

extended to all of Tunisian companies, especially with the huge differences in weather, in characteristics and resources between sectors of activity, between industrial process and between operators.

**References**

p. 0001-0012.

380-387.

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*DOI: http://dx.doi.org/10.5772/intechopen.98697*

*Ergonomic Evaluation of Thermal Stress in a Tunisian Steel Industry*

[10] International Standard

[11] Malchaire J, Kampmann B, Mehnert P, Gebhardt H, Piette A, Havenith G, et al. Évaluation du risque de contrainte thermique lors du travail en ambiances chaudes. Méd Hyg Trav

Ergon 2001;XXXVIII:101–112.

[13] Malchaire J, Kampmann B, Mehnert P, Gebhardt H, Piette A, Havenith G, et al. Assessment of the risk of heat disorders encountered during work in hot conditions. Int Arch Occup Environ Health 2002;75:153–162.

[14] Jacques MALCHAIRE, Francesca Romana d'AMBROSIO ALFANO and Boris Igor PALELLA. Evaluation of the metabolic rate based on the recording of the heart rate. Ind Health. 2017; 55(3):

[15] Pongjan Yoopat, K. Vanwonterghem and K. Intaranont, An assessment of workload in the Thai steel industry, Applied Ergonomics, 1998: 29 (4) 267-271

[17] Kaija Tuomi, Sinikka Vanhala,Erkki

Organizational practices, work demands and the well-being of employees: a follow-up study in the metal industry and

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retail trade. Occup Med 2004; 54:

115-121.

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[2] C Martinet, JP Meyer. Travail à la chaleur et confort thermique. Note scientifiques et techniques 184 (1999). www.inrs.fr/dms/inrs/Publication/ NOETUDE-3641-01/ns184.pdf consulted 25 february 2017

[3] Directive 89/654/CEE du Conseil, du 30 novembre 1989, concernant les prescriptions minimales de sécurité et de santé pour les lieux de travail (première directive particulière au sens de l'article 16 paragraphe 1 de la directive 89/391/CEE). Journal Officiel n L 393 du 30/12/1989.

[4] Norme ISO 7726. Ergonomie des ambiances thermiques - Appareils de mesure des grandeurs physiques. 1998.

[5] Malchaire J. Occupational Heat Stress Assessment by the Predicted Heat Strain Model. Industrial Health 2006; 44:

[6] International Standard Organization, ISO 8996, Determination of metabolic

Organization 9920. Ergonomics of the thermal environment — Estimation of thermal insulation and water vapour resistance of a clothing ensemble. 2007.

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rate, Genève: ISO; 2004.

[7] International Standard

[8] International Standard

comfort criteria. 2005.

**75**

[9] International Standard


*2.3. First-Order Delta–Sigma (*ΔΣ*) Modulator (*ΔΣ*M) ADC*

The ΔΣM topology emerged to avoid the shortcomings of the ΔM by moving the integrator from the feedback to the forward path. As illustrated in Figure 3a, in which the local quantizer (ADC) can again simply be a single comparator, the integrator operates over the error difference instead of the signal estimation, as in the ΔM case.

**Figure 3.** First-order ΔΣM ADC: (**a**) block diagram and (**b**) illustrative magnitude of STF, NTF and *Smax*.

This architecture relies essentially on an analog filter (integrator), a quantizer, and a DAC [4,11]. Using the linear additive white-noise model for the quantizer, this system can be represented in the Z domain, resulting in the following STF and NTF:

$$STF(z) = \frac{H(z)}{1 + H(z)}\tag{1}$$

$$NTF(z) = \frac{1}{1 + H(z)}\tag{2}$$

where *H*(*z*) is the integrator transfer function. The NS effect is more effective for higher *H*(*z*) filter orders, promoting higher-resolution converters; however, extra complexity is added to the system. These functions are represented in Figure 3b.