**4. Potential applications**

**Figure 4.** Using the rapid uniform heating approach with heating rates in the order of 106 K/s, the undercooled liquid is accessible at any temperature above the glass transition, through the melting point and beyond, where the liquid enters

the lifetime of the mold. Furthermore, the processing is accomplished in the laminar flow regime; therefore, higher quality and reliable parts could be obtained by comparison with the current mold-casting technique [8, 42]. However, the viscosity of the supercooled liquid MGs is much higher than that of the plastics melt, which poses a challenge for mass

In order to improve the thermoplastic formability of supercooled liquid MGs, micro-backextrusion was proposed by Wu et al. [14], and a three-dimensional cup-shaped object with wall thickness of 0.05 mm was successfully fabricated. To reduce the contact area between MGs and mold materials, rolling was developed by Schroers et al. [43] who not only hot-rolled high-quality MG sheets but also replicated micro-patterns with featured size of 300 nm. The micro-replication of MGs through hot-rolling is actually similar to hotembossing process, wherein the high viscosity and interfacial effect are main reasons limit the processability. Subsequently, Schroers et al. [44, 45] developed blow molding (see **Figure 3**), which allows blowing hollow products by using gas pressure to inflate the thermoplastic MGs enclosed in the mold. The low-forming pressure and high-dimensional accuracy indicates that this net-shaping technology could bring economic and environ-

Recently, an ultra-fast MGs' hot-processing technique was probed by Johnson et al. [47], as illustrated in **Figure 4**. When rapidly and uniformly heating a metallic glass at rates of 106

to temperatures spanning the undercooled liquid region, rapid thermoplastic forming of the undercooled liquid into complex net shapes is implemented under rheological conditions typically used in molding of plastics. Owing to the millisecond time window, this method is able to "beat" the intervening crystallization and successfully process even marginal glassforming alloys with very limited stability against crystallization that are not processable by

K/s

the equilibrium state.

production.

14 Metallic Glasses - Properties and Processing

mental benefits.

The above thermoplastic forming techniques endow MGs with superiority in net-shaping precise and versatile structures comprising of macro-/micro-/nano-sized features. Through nanoimprinting, Schroers et al. [5, 8, 9] fabricated metallic glass nanowires with very high aspect ratios (>200); these nanorods not only exhibit enhanced thermal stability [4] but also display superb durability combined with high electrocatalytic activity toward methanol, ethanol oxidation and CO, exhibiting great potential in energy conversion/storage and sensors fields [52]. The superb durability and high-surface area of these MG nano-structures motivate the generation of first functional proton exchange membrane micro fuel cells (MFCs). Such novel MFCs have been identified as a promising alternative power sources for portable electronics [53].

In addition to the potential applications in energy sector, the micro-/nano-gratings hotimprinted on MGs surfaces also exhibit excellent spectroscopic performance [54, 55]. For example, Chu et al. [54] fabricated nano-scale gratings, and Ma et al. [55] hot-embossed micro-scale gratings with fine periodicity on Pd-based MGs surfaces, both surface exhibit beautiful optical properties such as rainbow-like spectrum when shone by fluorescent lamp light, as shown in **Figure 6**. Inoue et al. [56] pointed out that these nano-imprinted MG surfaces exhibit potential applications as anti-reflection materials, electrode materials, hologram technology, next generation ultra-high density of information data storage material and cell culture medium for bio-chips.

By integrating macro-, micro- and nano-scale features in a sequential order, Kumar et al. [13] hot-embossed hierarchical structures and displayed potential applications in optical devices, electrochemical activity and cellular response. Through micro-imprinting, some micro-lens arrays [57], micro-channel geometries [58] have been fabricated, showing potential applications in aspheric lens and fuel cell interconnect plates, respectively. Furthermore, the thermoplastic formed MG components have been used as a master mold (see **Figure 7**) to imprint polymers (such as PMMA) [10, 24, 59, 60], and an integrated PMMA micro-channel part was fabricated, implying that MG is a robust, attractive and viable mold material for thermoplastic imprinting of polymer devices [10]. Bardt et al. [23] thermoplastic formed some complex 3-D micro-topologies and envisaged potential application as high-Q micro-resonators, microwave waveguides, microsurgical tools and devices, connectors for higher frequency operations, micro-scale motors and transmission components, microfluidic arrays, and free-form reflective micro-optics.

The hot-embossed surface micro-components can be used in MEMS, biochips, such as microspring, micro-gear, micro-motor, micro-fan, micro-honeycomb structure, micro-gyroscope and micro-accelerometer structure and micro-turbines; some beautiful surface features such as micro-bats and micro-poetry of Tang Dynasty "Yellow Crane Tower" have also been fabricated by Li et al. [6] through thermoplastic forming. Similar to micro-/nano-scale hot-imprinting, the TPF-based blow molding has also been used to fabricate ultra-smooth and symmetric 3-D metallic glass resonators, which demonstrates precision over 5 orders of magnitude without the use of cleanroom facilities or traditional microfabrication techniques, displaying potential applications in future MEMS vibratory devices, such as accelerometers and gyroscopes, with

**Figure 7.** SEM micrographs of the silicon master (a) with single (c) and continuous bends structure (e); the corresponding

Thermoplastic Forming of Metallic Glasses http://dx.doi.org/10.5772/intechopen.78016 17

The thermoplastic micro-forming technique also exhibits great potential in fabrication superhydrophobic surfaces with long lifespan in service, as demonstrated by Li et al. [62, 63]. Who found that without any modification or post-treatment, superhydrophobic surfaces with good stability could be fabricated by hot-embossing honeycomb patterns on Pd40Cu30Ni10P20 MG [62]. By constructing micro-/nano-hierarchical structures on Zr35Ti30Be26.75Cu8.25 MG surface, Li et al. [63] not only fabricated superhydrophobic MG surface with water contact angle over 150°, but also found that these surfaces exhibit strong adhesion with water droplets. The combined properties of both superhydrophobicity and strong adhesion toward liquid exhibit promising applications as dry adhesives and transport of liquid micro-droplets, as well as desirable mechanical and corrosion resistance showing potential applications in modern industries [64]. Furthermore, Li et al. revealed that MGs surfaces with hot-embossed textures exhibit low friction coefficient especially under dry contact (see **Figure 8**), which indicates that

reduced energy dissipation mechanisms, increased performance and low costs [61].

hot-embossed metallic glass micro-channel structure (b, d, and f) [10].

the lifetime of the textured surfaces could be optimized by minimizing friction [65].

**Figure 6.** (a) Photographs of polished BMG plate (left) and BMG grating (right) when fluorescent lamp light shines upon them (b) photographs of Si die (left) and BMG grating (right) under the shine of fluorescent lamp light [55].

#### Thermoplastic Forming of Metallic Glasses http://dx.doi.org/10.5772/intechopen.78016 17

CO, exhibiting great potential in energy conversion/storage and sensors fields [52]. The superb durability and high-surface area of these MG nano-structures motivate the generation of first functional proton exchange membrane micro fuel cells (MFCs). Such novel MFCs have been

In addition to the potential applications in energy sector, the micro-/nano-gratings hotimprinted on MGs surfaces also exhibit excellent spectroscopic performance [54, 55]. For example, Chu et al. [54] fabricated nano-scale gratings, and Ma et al. [55] hot-embossed micro-scale gratings with fine periodicity on Pd-based MGs surfaces, both surface exhibit beautiful optical properties such as rainbow-like spectrum when shone by fluorescent lamp light, as shown in **Figure 6**. Inoue et al. [56] pointed out that these nano-imprinted MG surfaces exhibit potential applications as anti-reflection materials, electrode materials, hologram technology, next generation ultra-high density of information data storage material and cell

By integrating macro-, micro- and nano-scale features in a sequential order, Kumar et al. [13] hot-embossed hierarchical structures and displayed potential applications in optical devices, electrochemical activity and cellular response. Through micro-imprinting, some micro-lens arrays [57], micro-channel geometries [58] have been fabricated, showing potential applications in aspheric lens and fuel cell interconnect plates, respectively. Furthermore, the thermoplastic formed MG components have been used as a master mold (see **Figure 7**) to imprint polymers (such as PMMA) [10, 24, 59, 60], and an integrated PMMA micro-channel part was fabricated, implying that MG is a robust, attractive and viable mold material for thermoplastic imprinting of polymer devices [10]. Bardt et al. [23] thermoplastic formed some complex 3-D micro-topologies and envisaged potential application as high-Q micro-resonators, microwave waveguides, microsurgical tools and devices, connectors for higher frequency operations, micro-scale motors and transmission components, microfluidic arrays, and free-form reflec-

The hot-embossed surface micro-components can be used in MEMS, biochips, such as microspring, micro-gear, micro-motor, micro-fan, micro-honeycomb structure, micro-gyroscope and micro-accelerometer structure and micro-turbines; some beautiful surface features such as micro-bats and micro-poetry of Tang Dynasty "Yellow Crane Tower" have also been fabricated

**Figure 6.** (a) Photographs of polished BMG plate (left) and BMG grating (right) when fluorescent lamp light shines upon

them (b) photographs of Si die (left) and BMG grating (right) under the shine of fluorescent lamp light [55].

identified as a promising alternative power sources for portable electronics [53].

culture medium for bio-chips.

16 Metallic Glasses - Properties and Processing

tive micro-optics.

**Figure 7.** SEM micrographs of the silicon master (a) with single (c) and continuous bends structure (e); the corresponding hot-embossed metallic glass micro-channel structure (b, d, and f) [10].

by Li et al. [6] through thermoplastic forming. Similar to micro-/nano-scale hot-imprinting, the TPF-based blow molding has also been used to fabricate ultra-smooth and symmetric 3-D metallic glass resonators, which demonstrates precision over 5 orders of magnitude without the use of cleanroom facilities or traditional microfabrication techniques, displaying potential applications in future MEMS vibratory devices, such as accelerometers and gyroscopes, with reduced energy dissipation mechanisms, increased performance and low costs [61].

The thermoplastic micro-forming technique also exhibits great potential in fabrication superhydrophobic surfaces with long lifespan in service, as demonstrated by Li et al. [62, 63]. Who found that without any modification or post-treatment, superhydrophobic surfaces with good stability could be fabricated by hot-embossing honeycomb patterns on Pd40Cu30Ni10P20 MG [62]. By constructing micro-/nano-hierarchical structures on Zr35Ti30Be26.75Cu8.25 MG surface, Li et al. [63] not only fabricated superhydrophobic MG surface with water contact angle over 150°, but also found that these surfaces exhibit strong adhesion with water droplets. The combined properties of both superhydrophobicity and strong adhesion toward liquid exhibit promising applications as dry adhesives and transport of liquid micro-droplets, as well as desirable mechanical and corrosion resistance showing potential applications in modern industries [64]. Furthermore, Li et al. revealed that MGs surfaces with hot-embossed textures exhibit low friction coefficient especially under dry contact (see **Figure 8**), which indicates that the lifetime of the textured surfaces could be optimized by minimizing friction [65].

**Conflict of interest**

**Author details**

Wuhan, PR China

\* and Jiang Ma2

10.1016/j.msea.2017.03.062

DOI: 10.1016/j.jallcom.2009.12.174

DOI: 10.1002/adma.200902776

Ning Li1

PR China

**References**

nmat2622

ncomms9157

The authors declare that they have no competing financial interests.

1 State Key Laboratory for Materials Processing and Die and Mould Technology, School of Materials Science and Engineering, Huazhong University of Science and Technology,

Thermoplastic Forming of Metallic Glasses http://dx.doi.org/10.5772/intechopen.78016 19

2 Guangdong Provincial Key Laboratory of Micro/Nano Optomechatronics Engineering, College of Mechatronics and Control Engineering, Shenzhen University, Shenzhen,

[1] Li N, Liu Z, Wang XY, Zhang M. Vibration-accelerated activation of flow units in a Pd-based bulk metallic glass. Materials Science and Engineering A. 2017;**692**:62-66. DOI:

[2] Guo H, Yan PF, Wang YB, Tan J, Zhang ZF, Sui ML, Ma E. Tensile ductility and necking

[3] Jang D, Greer JR. Transition from a strong-yet-brittle to a stronger-and-ductile state by size reduction of metallic glasses. Nature Materials. 2010;**9**:215-219. DOI: 10.1038/

[4] Sohn S, Jung Y, Xie Y, Osuji C, Schroers J, Cha JJ. Nanoscale size effects in crystallization of metallic glass nanorods. Nature Communications. 2015;**6**:8167. DOI: 10.1038/

[5] Kumar G, Desai A, Schroers J. Bulk metallic glass: The smaller the better. Advanced

[6] Li N, Chen W, Liu L. Thermoplastic micro-forming of bulk metallic glasses: Areview.

[7] Li N, Chen Q, Liu L. Size dependent plasticity of a Zr-based bulk metallic glass during room temperature compression. Journal of Alloys and Compounds. 2010;**493**:142-147.

[8] Schroers J. Processing of bulk metallic glass. Advanced Materials. 2010;**22**:1566-1597.

Materials. 2011;**23**:461-476. DOI: 10.1002/adma.201002148

Journal of Metals. 2016;**68**:1246-1261. DOI: 10.1007/s11837-016-1844-y

of metallic glass. Nature Materials. 2007;**6**:735-739. DOI: 10.1038/nmat1984

\*Address all correspondence to<EMAIL_ADDRESS>
**Figure 8.** The experimental and theoretically calculated coefficients of friction with the contact area fraction [65].


**2. Natural history**

For appropriate management of RCTs, it is imperative to appreciate the stages of progression of PTTs to FTTs and the contributing factors that lead to symptoms. A thorough patient history includes age, occupation, activities, hand dominance, history of trauma, time since onset of symptoms, history of smoking, diabetes mellitus and patient's expectations. A clinical examination is then supplemented with imaging studies, in particular ultrasound and magnetic resonance imaging (MRI), to further identify the location, size, thickness, retraction of RCTs and any other shoulder pathology such as long head of biceps tendonitis, labral tears, glenohumeral chondrosis, and muscle atrophy.

It is thought that older patients, patients with diabetes mellitus and osteoporosis and smokers will have a less successful repair of a complete tear [3]; however, there is no strong evidence for this. Chronic tears may have poor healing due to associated surrounding fatty infiltration and muscle atrophy [4]. An elderly patient with limited activities may be able to manage activities of daily living with a full-thickness RCT. In contrast, an active young patient may require surgery, even with a small complete RCT.

#### **2.1. Traumatic vs. atraumatic tears**

From the patient history, we can establish whether the RCT is a traumatic or degenerative one. Studies by Hantes et al. [5] and Petersen et al. [6] advocate early surgical treatment of complete traumatic RCTs, regardless of size and patient age, to avoid further RCT progression, with subsequent degenerative changes, fatty infiltration of surrounding muscle and cuff

retraction. Early surgery should be performed to obtain the best functional results. Atraumatic (degenerative) tears usually occur in elderly patients with larger tears, retracted rotator cuff tendons, poor surrounding tissue and fatty infiltration. In addition, they are likely to have fewer demands from their shoulder. Therefore, surgery may provide a less favourable outcome, and treatment may be best managed conservatively.

## **2.2. Partial to full-thickness tears**

tears (RCTs), there is still limited information concerning the natural history and treatment approaches for the disorder. An RCT may initially present as a partial-thickness tear (PTT) that progresses to a full-thickness tear (FTT) in the seventh decade of life [1]. Currently, there are no comprehensive British National Institute of Clinical Excellence (NICE) guidelines and European guidelines on the management of RCTs in general, and conclusions made by the American Academy of Orthopaedic Surgeons (AAOS) show weak evidence. Through understanding the natural history of RCTs, the progression from PTT to FTTs, and the different factors that influence progression such as age and comorbidities that influence progression, we can better advise our patients regarding optimum therapy. Such therapies include rehabilitation, physiotherapy, systemic medications and progression to surgical intervention. Although studies regarding physical therapy and surgical interventions show success in the recovery process, it has become increasingly clear that some biologics may augment the healing of tendon to bone when used as a primary treatment or as an adjunct to surgical procedures [2]. However, there are risks of conservative management, and it is important to identify the indications for transition from conservative to surgical management and appreciate patient satisfaction indices. To do so, the authors performed a critical review of the most recent evidence, providing an overview of the best evidence-based management for

For appropriate management of RCTs, it is imperative to appreciate the stages of progression of PTTs to FTTs and the contributing factors that lead to symptoms. A thorough patient history includes age, occupation, activities, hand dominance, history of trauma, time since onset of symptoms, history of smoking, diabetes mellitus and patient's expectations. A clinical examination is then supplemented with imaging studies, in particular ultrasound and magnetic resonance imaging (MRI), to further identify the location, size, thickness, retraction of RCTs and any other shoulder pathology such as long head of biceps tendonitis, labral tears,

It is thought that older patients, patients with diabetes mellitus and osteoporosis and smokers will have a less successful repair of a complete tear [3]; however, there is no strong evidence for this. Chronic tears may have poor healing due to associated surrounding fatty infiltration and muscle atrophy [4]. An elderly patient with limited activities may be able to manage activities of daily living with a full-thickness RCT. In contrast, an active young patient may

From the patient history, we can establish whether the RCT is a traumatic or degenerative one. Studies by Hantes et al. [5] and Petersen et al. [6] advocate early surgical treatment of complete traumatic RCTs, regardless of size and patient age, to avoid further RCT progression, with subsequent degenerative changes, fatty infiltration of surrounding muscle and cuff

complete RCTs.

16 Advances in Shoulder Surgery

**2.  Traumatic vs. atraumatic tears**

PTTs can be bursal-sided or articular-sided tears. Over the course of time, PTTs enlarge and propagate into FTTs, developing distinct chronic pathological changes due to muscle retraction, fatty infiltration and muscle atrophy. These changes lead to a reduction in tendon elasticity and viability. Although PTT to FTTs are described as a continuum in the literature, these tears can occur without following this natural history path. In its end-state, the glenohumeral joint experiences a series of degenerative alterations known as cuff tear arthropathy.

Maman et al. [7] reported that, based on MRI imaging over an 18-month period for 59 patients, 52% of FTTs will increase in size and were substantially less stable than PTTs. Each shoulder underwent a baseline MRI, and a repeat imaging performed at a minimum interval of 6 months. Progression of tear size was found in 48% of the tears that were followed for at least 18 months compared with just 19% of those followed for less than 18 months. This contrasts with a study by Fucentese et al. [8], who reported seemingly contradictory findings in their report of 24 patients refusing operative treatment for full-thickness supraspinatus tears. They used magnetic resonance arthrography (MRA) as their initial imaging modality and MR without arthrography for their follow-up imaging and reported no increase in the mean size of the RCTs 3.5 years after the initial MRA.

#### **2.3. Small vs. large full-thickness tears**

The risk of tear enlargement is greater for shoulders with more advanced tears and is associated with a greater risk of cuff muscle degenerative changes. This group reported that tear enlargement is also associated with greater risk of pain development across all tear types (50% for FTTs) [9].

The same study by Fucentese et al. [8] concludes that small isolated FTTs of the supraspinatus in patients under the age of 65 do not necessarily progress over time. Yamaguchi et al. [10] reported no increase in tear size over 5 years in 23 patients evaluated by ultrasound. This contrasts with a larger case series of 51 patients by Safran et al. [11] which reports that FTTs tend to increase in size in approximately half of patients aged 60 years or younger.

## **2.4. Demographics**

#### *2.4.1. Age*

Advancing age has been considered the most important prognostic factor for surgical outcome. Gumina et al. [12] reported that patients older than 60 years of age were twice as likely to develop a tear that was likely to progress to full-thickness and larger tears (54% of tears in patients older than 60 years showed such progression compared to only 17% of tears in those younger than 60 years). A cohort of patients younger than 60 years who were treated non-operatively for FTTs was found to have a higher rate of tear progression than older patients. Of the 61 tears, 49% increased in size according to the findings of ultrasound imaging [11].

*2.4.7. Family history*

*2.4.8. Posture*

impingement.

*2.4.9. Mental health*

*2.4.10. Symptoms and pain*

opment of symptomatic RCTs [23].

health in patients with complete RCTs [28].

There is limited evidence regarding the genetic predisposition and hereditary component for RCTs; however, a study examining the genealogical database in Utah, USA by Tashjian et al. [22] a population-based controlled study of 3091 patients, with a subgroup analysis of 652 patients diagnosed before 40 years of age, showed a significant association between individuals with rotator cuff disease in close and distant relations (reportedly up to third cousin relations). This study was included in the systematic review conducted by Dabija et al. [23], which includes the study by Harvie et al. [24], concluding that siblings of patients diagnosed with RCTs were twice as likely to develop complete RCTs. In addition, they identified single-nucleotide polymorphisms (SNPs) associated with RCTs, indicating the future risk for development of RCTs to enable prophylactic rehabilitation techniques and to avoid the devel-

Complete Rotator Cuff Tear: An Evidence-Based Conservative Management Approach

http://dx.doi.org/10.5772/intechopen.70270

19

The relationship between posture and shoulder pathology is still under investigation. For example, Gumina et al. [25] found a reduced subacromial space in 47 patients over the age of 60 years with hyperkyphosis, as compared to a control group. Yamamoto et al. [26] observed RCTs in 65.8% of patients with kyphotic-lordotic postures, 54.3% of patients with flat-back postures, and 48.9% with sway-back postures, whereas only 2.9% of patients with ideal alignment had symptomatic or asymptomatic RCTs. It is hypothesised that the reduced subacromial space is due to less posterior tilting and dyskinesis of the scapula, resulting in extrinsic

Cho et al. [27] demonstrated that as many as 82% of patients with chronic shoulder pain had sleep disturbance and that rates of depression were significantly increased in patients with more than 3 months of shoulder pain. Educational level, employment status, pain levels and patient perception of percentage of shoulder normalcy were most predictive of emotional

There is disagreement in the literature regarding the correlation between tear size and pain. These studies tend to be cross-sectional as opposed to prospective observational studies [11]. For example, in a prospective study of 50 patients, Moosmayer et al. [29] reported that 40% of asymptomatic RCTs became symptomatic and anatomically deteriorated, and that an increase in tear size and a decrease in muscle quality correlated with the development of symptoms. Mall et al. [30] who compared asymptomatic and symptomatic RCTs, determined that many with asymptomatic FTTs will develop symptoms with time and that pain development is associated with an increase in tear size and deterioration of shoulder function and active range of motion (ROM). In the study, this was primarily seen with larger tears and required significant time for progression to occur and for glenohumeral and scapular mechanical dysfunction to become apparent. There


**1. Introduction**

118 Lipid Metabolism

Zhao, Y., Kuge, Y., Zhao, S., Strauss, W.H., Blankenberg, F.G., & Tamaki, N. (2008). Prolonged high-fat feeding enhances aortic 18F-FDG and 99mTc-annexin A5 uptake in apolipoprotein E-deficient and wild type C57BL/6J mice. *J Nucl Med.* 49, 1707-1714.

> Bacteria of the Corynebacterineae, a suborder of the Actinobacteria, comprise *Mycobacterium*, *Corynebacterium*, *Nocardia*, *Rhodococcus* and other genera. This suborder of high GC gram-positive bacteria includes a number of important human pathogens, such as *Mycobacterium tuberculosis*, *Mycobacterium leprae* and *Corynebacterium diphtheriae,* the causative agents of tuberculosis, leprosy and diphtheria, respectively. *M. tuberculosis* is the most medically significant species, a devastating human pathogen infecting around onethird of the entire human population and responsible for more than 1 million deaths annually. The Corynebacterineae also includes non-pathogenic species such as *Mycobacterium smegmatis*, a saprophytic species, and *Corynebacterium glutamicum*, an industrial workhorse for the production of amino acids and other useful compounds. These relatively fast-growing species serve as useful models to study metabolic processes essential to the growth and survival of the slow-growing pathogens.

> All these bacteria share a common feature, a distinctive multilaminate cell wall composed of peptidoglycan, complex polysaccharides, and both covalently linked lipids and free lipids/lipoglycans (Fig. 1). Among them, mycolic acids are the hallmark of these species. These long chain α-branched, β-hydroxylated fatty acids are covalently linked to the arabinogalactan polysaccharide layer. This mycolic acid layer is complemented by a glycolipid layer to form an outer "mycomembrane" analogous to the outer membrane of Gram-negative bacteria. [1, 2]. The outer leaflet of the mycomembrane is composed of a variety of lipids including trehalose dimycolates (TDMs), glycopeptidolipids (GPLs), phthiocerol dimycocerosates (PDIMs), sulfolipids, phenolic glycolipids (PGLs), and lipooligosaccharides. Some of these lipids are widely distributed while others are restricted to particular species. For example, TDMs and their structural equivalents are found in both mycobacteria and corynebacteria, while PDIMs and PGLs are restricted to a subset of

© 2013 Crellin et al., licensee InTech. This is an open access chapter 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. © 2013 The Author(s). Licensee InTech. 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.

mycobacteria. The structure and hydrophobic properties of the cell wall make it a potent permeability barrier that is responsible for intrinsic resistance of mycobacteria to an array of host microbiocidal processes, many antibiotics and sterilization conditions [3, 4]. Many of the cell wall components of pathogenic mycobacterial species are essential for pathogenesis and *in vitro* growth, hampering efforts to characterize the function of individual proteins in their assembly. In contrast, some non-pathogenic species such as *C. glutamicum* can tolerate the loss of major cell wall components, making them useful model systems for delineating processes involved in the assembly of core cell wall structures.

Metabolism of Plasma Membrane Lipids in Mycobacteria and Corynebacteria 121

pivotal site for the metabolism of lipids. At the same time, this membrane must perform many other functions associated with energy production, nutrient uptake, protein export, and various sensing/signaling reactions. Studies on how these metabolic and cellular processes might be organized within bacterial plasma membranes are in their infancy. Understanding the homeostasis of the plasma membrane is particularly important in Corynebacterineae organisms because this structure must support the high biosynthetic demands of sustaining such a lipid-rich cell wall. In this chapter, we focus our discussion on processes of lipid metabolism that are critical for the biogenesis and maintenance of the plasma membrane, and illustrate the recent progress on our understanding of plasma

In this section we will describe the functions of plasma membrane lipids. First, we will describe the functions of major structural phospholipids. We will then describe quantitatively minor lipids, which have important metabolic/physiological functions. Lastly, we will discuss the functions of neutral lipids because their biosynthesis is closely linked to phospholipid metabolism and neutral lipid storage is a critical part of plasma membrane homeostasis.

Major structural components of the mycobacterial plasma membrane are phospholipids such as cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and glycosylated PIs (*i.e.* phosphatidylinositol mannosides (PIM), lipomannans (LM) and lipoarabinomannans (LAM), see below). The ratio of these phospholipids may vary depending on the species and growth conditions [6-8]. For example, one study indicated that CL, PE, and PI/PIMs represent about 37, 32, and 28%, respectively, of the total phospholipids in the plasma membrane in *M. smegmatis* [9], while another reported the ratio in *Mycobacterium phlei* to be about 50, 10, and 40% [10]. Phosphatidylglycerol (PG), which is abundant in other bacteria, is a relatively minor species in mycobacteria. Deletion of the PI biosynthetic gene has been shown to be lethal in *M. smegmatis* [9], indicating that PI or glycosylated PIs are essential for mycobacterial viability. In *M. tuberculosis*, putative PI synthetase (*Rv2612c*) and PGP synthetase (*Rv2746c*, involved in CL synthesis) genes are predicted to be essential [11], while the PS synthetase gene (*Rv0436c*, involved in PE synthesis) is not [12]. In corynebacteria, major species of phospholipids are PI, PG, CL, and

CL is widely found in both prokaryotes and eukaryotes. It forms aggregates within the membrane bilayer. Nonyl acridine orange (NAO) is a fluorescent dye which is proposed to bind the hydrophobic surface created by the CL cluster [14], allowing microscopic visualization of CL domains. Indeed, using NAO, CLs were found to be enriched in septa and poles of actively dividing *M. tuberculosis* and *M. smegmatis* cells [15, 16]. CL has a non-

membrane biogenesis in mycobacteria and corynebacteria.

**corynebacteria**

**2.1. Structural lipids**

**2. Functions of plasma membrane lipids in mycobacteria and**

acylphosphatidylglycerol (APG) [13], and PE appears to be absent.

**Figure 1. Mycobacterial plasma membrane and cell wall with flow of key metabolic pathways**. Some of the metabolites are exported to the mycomembrane. SLD, small lipid droplet; LD, lipid droplet; FA-CoA, fatty acyl-CoA. See text for other abbreviations used in the figure.

Studies on mycobacteria and corynebacteria provide a unique opportunity to illustrate the complexity and diversity of lipid metabolic pathways in bacteria. They have a significantly higher lipid content than other bacteria with cell wall lipids comprising ~40% of the dry cell mass. *M. tuberculosis* produces a diversity of lipids unparalleled in bacteria, from simple fatty acids to highly complex long chain structures such as mycolic acids. It has devoted a significant proportion of its coding capacity to lipid metabolism and produces about 250 enzymes dedicated to fatty acid metabolism, which is around five times the number produced by *Escherichia coli* [5]. Lipid biosynthesis places a significant metabolic burden on the organism but is ultimately advantageous, allowing *M. tuberculosis* to survive and replicate in the inhospitable environment of host macrophages. While capable of *de novo* synthesis, these bacteria also scavenge and degrade host cell membrane lipids to acetyl-CoA, via broad families of β-oxidation and other catabolic enzymes, for incorporation into their own metabolic pathways and to fuel cellular processes.

The plasma membrane provides the platform for lipid metabolism. While some lipid metabolic reactions take place in the cytoplasm or cell wall, the plasma membrane is the pivotal site for the metabolism of lipids. At the same time, this membrane must perform many other functions associated with energy production, nutrient uptake, protein export, and various sensing/signaling reactions. Studies on how these metabolic and cellular processes might be organized within bacterial plasma membranes are in their infancy. Understanding the homeostasis of the plasma membrane is particularly important in Corynebacterineae organisms because this structure must support the high biosynthetic demands of sustaining such a lipid-rich cell wall. In this chapter, we focus our discussion on processes of lipid metabolism that are critical for the biogenesis and maintenance of the plasma membrane, and illustrate the recent progress on our understanding of plasma membrane biogenesis in mycobacteria and corynebacteria.


Assuming a constant (24/365) median well flow rate of 2.8 L/s.

**Figure 7.** Spatial distribution of the percentage of paddy field area (**A**) and area with crop rotation (**B**) at hamlet level.

Crop rotation is a vital paddy farming practice to reduce the intensity of pests and diseases, and to improve soil fertility. Different crops rotated with paddy included corn, beans, taro, chilli, and other crops. Villages in Gondang Wetan Sub-district tended toward crop rotating more often compared to those in Winongan Sub-district. The area of crop rotation reached 30.1 ha, and 26.6 ha of it (88.5%) was found in Gondang Wetan Subdistrict. Brambang Village in Gondang Wetan Sub-district was the village with the largest area of crop rotation with 10.9 ha, while Penataan Village in Winongan Sub-district had only 0.34 ha, the smallest area compared to other villages (Table 5). At the hamlet level, Brambang Barat Hamlet in the village of Brambang had the largest area of crop rotation, reaching 34.6% of the total paddy field area (Figure 7B). Hamlets in Gondang Wetan Subdistrict had a percentage of area with crop rotation between 4.3–11%. Other hamlets were classified as having a low rotation area, which was less than or the same as 4.2% and it was relatively common in Winongan Sub-district.

**Table 5.** Distribution of rice yield (ton/ha), fertiliser application (kg/ha), number of type of pesticides applied, water sources (% respondent), and intensity of rodents (% respondent) in each village in two sub-districts.

#### *3.3. Water Sources and Irrigation Systems*

Rivers and artesian wells were two primary water sources to irrigate paddy fields in Gondang Wetan and Winongan Sub-districts. Water from those two sources flowed into the irrigation channel, but in some cases, the artesian wells were located inside the paddy fields itself. The total number of artesian wells reached 318 points spread across 11 villages, and 63% of it was found in Gondang Wetan Sub-district (Table 4). In line with this distribution, 89% of respondents in Winongan Sub-district mentioned that the river was more dominant than artesian wells as a water source to irrigate their paddy fields, while in Gondang Wetan Sub-district, they were only 61% of respondents (Table 4). Brambang Village was the village with the lowest percentage of respondents that mentioned river water as the primary source. Therefore, one paddy field could use river water and more than two artesian wells (shared with other farmers) (Figure 8D). At the hamlet level, most artesian wells were found in Podokaton Hamlet of Bayeman Village of Gondang Wetan Sub-district. Some hamlets were identified to have artesian wells between 8 to 17 wells. Other hamlets had less than eight wells. Nuso Hamlet at Wonosari Village was the only hamlet without artesian wells for irrigation, relying almost 100% on river water to irrigate paddy fields (Figure 8A,C).

**Figure 8.** *Cont.*

**Figure 8.** Spatial distribution of the number of artesian wells (**A**), drainage density (**B**), the hamlets that use the river as the main water resource (**C**), and the number of water resources (**D**) at hamlet level.

Winongan Sub-district had more extended irrigation channels compared to Gondang Wetan Sub-district, reaching 76.6 km in length (Table 4). Mendalan Village in Winongan Sub-district was the area with the most elongate irrigation channels (20.3 km). Kebon Candi Village in Gondang Wetan Sub-district had the shortest channels compared to other villages (7.27 km). At hamlet level, hamlets in Winongan Sub-districts such as Mendalan, Sukun, Kurban, Gading, Kalongan, Kletek Lor, Kletek Kidul, Putat, Krajan, Gondang, and two hamlets in Gondang Wetan Sub-district such as Karangasam and Ngemplak were classified as the highest drainage density, i.e., 8.5–11.3 km/km2 (Figure 8B).

#### *3.4. Rice Yield and Fertiliser Application*

Farmers in Gondang Wetan Sub-district tended to apply a higher dose of fertiliser compared to farmers in the Winongan Sub-district (Table 5). The average dose of N fertiliser (Urea) applied by farmers in Gondang Wetan was 436 kg/ha/season and 322 kg/ha/season for compound fertiliser (NPK), while farmers in Winongan Sub-district applied N fertiliser about 343 kg/ha/season and compound fertiliser about 266 kg/ha/season. However, Gondang Wetan and Winongan Sub-districts had the same average of rice yield of 4.9 tons/ha/season, with distribution between 4.1-6.7 tons/ha/season (Table 5). Bayeman in Gondang Wetan and Gading in Winongan were villages that had the highest average of yield. Tenggilis Rejo in Gondang Wetan Sub-district was the village with the highest use of fertiliser, which was almost 600 kg/ha/season for N fertiliser and 370 kg/ha/seasons for compound fertiliser. It was indicated that high yield did not necessarily depend on high fertiliser rates (Table 5).

At the hamlet level, Krajan in Bayeman Village and Kurban in Gading Village were hamlets with the highest rice yield (almost 8 tons/ha/season) (Figure 9A). Karang Asem and Krajan in Tenggilis Rejo Village, and Kebon Sawo in Kebon Candi Village were some hamlets with the highest use of urea (above 540 kg/ha/season) and compound fertiliser (more than 348 kg/ha/season) (Figure 9B,C). Hamlets with low yield were Masangan, Karang Asem, Wulu, Bicaan, and Kebon Candi (Figure 9A).

**Figure 9.** Spatial distribution of rice yield (**A**), urea application (**B**), and compound fertiliser (**C**) at hamlet level.

### *3.5. Intensity of Pest/Rodents and Number of Type of Pesticides Applied*

Regarding the main problem of paddy cultivation, farmers in both Gondang Wetan and Winongan Sub-districts agreed that the high intensity of rodent attacks was the major problem in need of immediate attention. However, the respondents in Winongan Subdistrict mentioned that rodents were the main problem, and their condition was worse than in Gondang Wetan (Table 5). Figure 10A shows that most paddy fields in Winongan Subdistrict suffered from rodents. In Gondang Wetan Sub-district, only paddy fields in hamlets or villages next to Winongan Sub-district had a high intensity of rodent attacks, especially during rainy season. Until this study was undertaken, farmers were still struggling with how to deal with rodents. In terms of pests and disease control, there were more than 100 brands of pesticides used by farmers. Farmers in Gondang Wetan and Winongan Subdistricts used at least three brands, but some applied more than seven brands of pesticides in one season. In general, farmers in Gondang Wetan Sub-district applied more brands of pesticides compared to farmers in Winongan Sub-district (Figure 10B).

**Figure 10.** Spatial distribution of the intensity of pest/rodents (**A**) and number of types of pesticide applied (**B**) at hamlet level.

#### *3.6. Characteristic of Paddy Farming*

The twelve parameters (paddy field area, drainage density, number of artesian wells, the dose of N fertiliser, the dose of compound fertiliser, number of pesticide types, yield, the area with crop rotation, the intensity of rodents attack, number of water sources, river as the primary water source and presence of water regulatory officer) are independent to each other. The cluster analysis of the twelve parameters and the elbow method (Figure 11) allows us to have five clusters of paddy fields in eleven villages of Gondang Wetan and Winongan Sub-districts. Figure 12 presents the map of the resulting characteristic of paddy farms in eleven villages, and Tables 6 and 7 describe the characteristics of each cluster. Cluster 1 was in Gondang Wetan Sub-district, cluster 2 was mostly in Winongan Sub-district, and cluster 5 was spread evenly in both sub-districts (Figure 12). Meanwhile, clusters 3 and 4 consisted of only 1 hamlet, which was located in Gondang Wetan Sub-district.

**Figure 12.** Characteristic of paddy farming in Gondang Wetan and Winongan Sub-districts.

**Table 6.** The result of cluster analysis and characteristic results for the 12 parameters.

**Table 7.** Description of each paddy field type.

#### **4. Discussion**

#### *4.1. Relevance of Reducing Groundwater Use in Lowland Zone*

Our first question was seeking quantitative evidence that the lowland practices are co-responsible for the decrease of the Umbulan spring's discharge. The estimated total outflow of the artesian wells (over 2400 L/s in 450 measured wells; currently there may be 600 wells) does not fully account for the observed decline in the Umbulan spring flow record (from 5000 to 3500 L/s with a declining trend). The water balance results of Table 3 suggest that the lowland paddy production through its reliance on unconstrained artesian wells has been the major contributor to the observed decline of the Umbulan spring, but changes in the upper and middle zone also contribute. The water balance model includes some interactions between surface and groundwater flows, but models at a higher temporal resolution that include seasonal patterns of rainfall could refine the results in future. The attribution of effects as 55% lowlands, 49% upland and 4% interaction is, at the current level of detail in the analysis, only indicative; however, it is consistent with process understanding. Models that operate on a daily time step and at higher spatial resolution will be needed [31,32], but tend to require parametrization efforts that challenge current data availability.

#### *4.2. Groundwater-Wasting Irrigation Methods: Understanding Farmer Decisions*

The second question sought relevant geographic variation between villages and hamlets in the farmer's practices in managing land and water in cultivating paddy. The shift from rainfed sugarcane to irrigation-based paddies in Pasuruan district got a boost when relatively cheap groundwater drilling with bore holes of 10–100 m (or more) provided additional water, year-round. Without any control valve to manage the well's flow, excess groundwater was channelled back to the river and then lost to the sea. Agronomically, the water supply is considerably in excess of crop demand, with the average 10 mm/day of well supply per unit paddy area, two to three times the potential evapotranspiration rate, even without accounting for rainfall and river-based irrigation water. The high crop frequency, approaching three crops per year and currently reaching five crops per two years leaves short time for a break between crops. As the landscape as a whole is permanently saturated, the rice crop does not ripen off well and the harvested product is only of medium quality. Crop rotations with other crops are applied to only a small part of the area. Despite abundant water availability and intensive fertilization, however, rice yields of around 5 ton per ha in the survey were below the 5.8 ton per ha reported for the district [19] and representing a nearly 50% yield gap relative to the potential yield for irrigated rice in Indonesia of around 9.5 ton per ha [33]. Within our data, there was no indication that variation in yields were related to variation in fertilizer level (either Urea or compound fertilizer, the two were strongly correlated). Rodents were widely seen by farmers as the main yield-reducing factor, and despite ample use of pesticides, could not be controlled at farmer level. It appears that the current intensification pathway is reaching a dead end, where they produce large volumes of a medium quality product at considerable environmental costs and, even if these externalities are ignored, modest farmgate profitability.

Investment in closing the existing wells and replacing them by wells with improved design that can be turned off when not needed appears to be a cost-effective way for external stakeholders to recover the flow at the Umbulan spring (and the millions of households that can thus be supported). In addition, the irrigation from artesian wells only during the night can avoid water wasting from evaporation process during the day. A substantial reduction of groundwater wasting seems to be feasible without risking water shortages in critical periods for the crop. However, for a free flowing well the only decision is in its construction, whereas a controlled well requires agreements among farmers about when it will be opened.

#### *4.3. Collective Action Aspects of Solutions*

The third question was whether a participatory survey of paddy cultivation and spatial data analysis for the development of characteristic can identify options by context for upscaling sustainable paddy cultivation. Sustainable agriculture within a sustainable landscape context is beyond food production, it safeguards the increasing capacity of rural people to be self-reliant and resilient when facing changes and shocks and building strong rural institutions, including landscape governance, and their economies [34]. A wellknown analysis of the Bali water temples in Indonesia, or '*subak*' in the local language, highlights the importance of local institutions that secured synchronous rice planting [35]. Synchronous rice planting ensured landscape-wide breaks between cropping seasons in the traditional system, that effectively controlled rodents. When the water temples were abandoned and technical irrigation allowed for an increased cropping frequency, rodent problems came to the fore in Bali. It appears that with the unconstrained artesian wells in Rejoso, an even easier year-round availability of habitat and food supports rat populations beyond control.

Following this line of interpretation, we suggest that in the Rejoso watershed context, the lack of strong farmer institutions imply an inability to synchronise planting calendars and that this has become one of the principal causes of aggravating rodent pest attack. When farmers can collectively dry their paddy field, this cycle of fallow can reduce the rodent pressures. Thus, the management of rodent pest problems (which directly link to yield and income) can be considered as a collective driver to strengthen local institutions, which at the end leads to better water management and may allow win–win solutions of water-saving and yield increase to be feasible.

#### *4.4. Sustainable Paddy Cultivation and Its Relevance to Global Agenda and Practices*

Our efforts to understand existing constraints to a sustainable production landscape in the Rejoso watershed showed that among the eleven villages in the Gondang Wetan and Winongan Sub-districts, a substantial variation in farmer's practices in managing the land could already be found. Given this variation, a one-size-fits-all solution is not likely to work. Our current understanding of such variations of the paddy field enables the landscape managers and decision-makers to identify the potential area for upscaling specific solutions and to ensure that the adoptions of interventions run smoothly because the process has embraced potential constraints and solutions according to the perspectives of smallholders and local communities.

Our approach aligns with a global agenda. The innovations of sustainable production landscape management consider the dual goals of reducing environmental impacts while increasing productivity. Discussion of the interconnected dimensions of sustainable production landscape is not new, while understandings and solutions towards actions to transform the environmentally sustainable food production systems are still unresolved [36].

The research by Pretty et al. [37] on the adoption of practices and technologies for environmentally sustainable with substantial benefits for the rural poor hold promising advances. The 208 projects were derived from 52 countries of the South resulted in approximately 8.98 million household farming 28.92 million ha representing 3.0% of the 960 million ha of arable and permanent crops in Africa, Asia and Latin America, adopted and practised sustainable agriculture. Knowledge from the literature confirms that increasing paddy productivity while reducing environmental impacts is doable. Likewise, it is attainable that farmers practice more efficient water use [38] and emit less CH4 and N2O [39,40].

#### *4.5. Potential of Development of Paddy Farming Characteristic for Intervention Scenario and Upscaling*

The five clusters identified here represent variation of farmer's practices in managing the land and cultivating paddy. Each cluster provides unique information with different degrees of the constituent parameters, yet still presenting the whole targeted landscape. Intervention scenarios might include incentives for sustainable cultivation, such as insurance of stable agricultural inputs, microcredit, agricultural insurance, market transparency, and capacity strengthening in farmer group management. Table 8 presents the analysis and the risks for intervention and upscaling in each paddy field cluster. Clusters with high risk, when targeted for conducting innovative interventions, will provide 'gold standards' of success, compared to the ones with low risks.

The proposed characteristic of paddy farming that considers the variation of farmer's practices in managing the land and cultivating paddy corresponds to the requirement to implement an 'options by context' approach [8]. Rather than selections based on proximity to the central power, or 'low-hanging fruit' ones, we expect that through the 'options by context', a more robust selection of locations and intervention scenarios allow for higher adoption rates with expected results and calculated risks of the innovations at each cluster. However, ongoing implementation efforts will have to provide the test of effectiveness. We identified some limitations in the methods applied.

**Table 8.** Analysis and risk for intervention and upscaling of each paddy field cluster, based on feedback in local focus group discussions.

#### *4.6. Implications for Methodology*

From the survey of paddy cultivation, identification of respondents by combining available information of farmer group and its member and a snowball technique is an ideal approach. However, the unavailability of up-to-date farmer group data was a challenge, and the impacts were on the length of time in finding a respondent and level of respondent representation in each hamlet. During the data analysis, the domicile of the farmer and the location of the paddy field can be unmatched. The domicile of some respondents can be outside of the eleven villages. Considering that the interventions would be delineated according to the sub-district jurisdictional boundary, the data of seven percent of the respondents who stayed outside the eleven villages were eliminated. Hence, we suggested that the filtering process in selecting respondents should be better from the beginning, and the number of respondents in each hamlet should be adequately represented if resources allow. Apart from the above limitation, we ensured that information gained from the farmer representatives were well obtained, and a structured questionnaire complemented the interviews.

Analysis of spatial data based on aerial photograph/drones produced a high-resolution image. However, the direct georeferencing method that we applied referred that orthorectification was processed without ground control point (GCP) and independent checkpoints (ICP), resulting in a mild shift of location and take effect of Root Mean Square Error (RMSE) value. Although the shifting is about 2–2.5 m compared to orthorectification using GCP and ICP [41], in the future, we suggested applying GCP and ICP for similar analysis to increase geometric accuracy when resources allow. Other analyses to increase the degree of spatial data accuracy focused on verification of the area of paddy fields with annual crop rotation. Multi-temporal images should be used, or a detailed survey of farmers should be conducted to obtain more accurate area estimates of paddy fields with annual crop rotation.

#### **5. Conclusions**

The production landscape of the Rejoso watershed has problems of unsustainable agricultural development, particularly on its lowland part, where paddy is the primary land use. The unrestricted use of artesian wells to irrigate rice paddies is reducing the pressure on and water yield of artesian wells for urban water users, while the actual rice yields achieved are below potentials achieved elsewhere. The introduction of water-saving technology, with modification of conventional paddy cultivation, and better design and management of artesian wells more control over the wells, can target yield improvement as well as positive environmental impacts. Considerable variation was found to exist within this paddy-dominant production landscape. Our analysis of the variation of farmer's practices in managing the land and cultivating paddy was based on a survey of paddy cultivation and spatial data analysis complemented and verified by the participatory approach indicating the application of landscape-approach in its development. The characteristics of paddy farming as the results reflect the requirement to implement an 'option by context' approach within a landscape for targeting effective, yet efficient, interventions and upscaling technological improvement. The characteristic of paddy farming encompassed five clusters of paddy farms. Clusters were characterised based on the relative area of paddy fields, the density of irrigation networks, area with crop rotation, rice yield, the dose of fertiliser, number of pesticide types, water sources, and the intensity of pests/rodents. Hamlets within a cluster are similar in characteristics of farmer's practices and have unique, contextual conditions. We discussed the potentials and risks of such characteristics for further implementation and upscaling. Clusters with high risk, when targeted for conducting innovative interventions, will provide 'gold standards' of success, compared to the ones with low risks. The information is expected to be useful for the landscape managers and decision-makers in targeting, considering, and budgeting the interventions that are relevant for sustainable landscape management. Future applications of a spatially differentiated intervention approach to innovate in the direction of sustainable paddy cultivation based on water saving is expected to reduce environmental impacts while increasing productivity. On-the-ground empirical action research activities engaging research organisations, private sectors and financing institutions are ongoing.

**Author Contributions:** N.K., L.T. and B.L. designed the study. L.D.Y.P. collected and analysed spatial data and was supervised by A.H. and M.T.Z. F.M. and E.P. collected paddy cultivation data and were supervised by L.T. L.T. analysed paddy cultivation data. N.K., L.T., L.D.Y.P., A.H., M.T.Z. writing the original draft. N.K., B.L., L.T. and M.v.N. shaped the original draft into the manuscript, which was approved by all co-authors, A.T. provided the ecohydrological backgrounds of the Rejoso watershed to improve the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by the Danone Ecosystem Fund through "Sustainable, Low Carbon Emissions and Water-Efficient Agriculture of Rejoso Watershed" project.

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

#### **Appendix A. Calibrating the Annual Water Balance Model**

Data for mean of annual precipitation (P) were obtained from 13 rainfall stations in lower zone (7 stations), middle zone (5 stations) and upper zone (1 station) (Table A1). Precipitation for each zone (lower zone < 100 m a.s.l., middle zone 100–1000 m a.s.l. and upper zone m a.s.l.) was then generated based on correlation of elevation and annual mean of rainfall (Figure A1). Potential evapotranspiration Epot were generated using Thornthwaite equation using temperature data of Accu weather (Table A2).

\*

) 1990–2015.

**Table A1.** Mean of annual rainfall for 13 rainfall stations.

Parameter estimates for actual/potential evapotranspiration (ε) and runoff coefficient (ρ) were derived as area-weighted average from estimate for the specific land use types in the GenRiver model, calibrated for a number of Indonesian watersheds [29].

#### **References**

*Article*


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Lufuno Makhado\*, Angelina Maphula, Thendo Gertie Makhado and Ofhani Prudance Musekwa University of Venda, Thohoyandou, South Africa

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