Analysis

Moisture content and porosity determination

The initial moisture content determination of the substrates was achieved by gravimetry. A mass of 20 g of SS and PS previously weighed using an OHAUS brand analytical balance (navigator model) were placed into porcelain capsules of known mass. Subsequently, the capsules were placed in an oven at a temperature of 105 °C. Dehydration was performed until the weight of the samples remained stable. The moisture content (Hu) percentage was determined by using equation 1.

(1)

where mi depicts the SS or PS initial mass (g) and mf the SS or PS final mass (g). The substrates initial moisture content was determined by duplicate.

The porosity for each substrate was calculated by eq. 2 using the bulk density and particle density (Sánchez-Vera et al., 2003).

(2)

where the porosity is expressed in %, bulk density, and particle density in g∙cm-3. The bulk density was determined using equation 3, whereas the particle density was calculated using equation 4. Both values were determined by triplicate.

(3)

where bulk density is expressed in g∙cm-3, substrate dry mass in g, and substrate volume in cm3.

(4)

where particle density is expressed in g∙cm-3, substrate mass in g, and substrate volume in cm3.

Mombaza grass germination time, height, growth rate, and yield calculation

The germination time was determined by measuring from the sowing of the MG seed until the appearance of the first germination shoots in each substrate. On the other hand, the HMG was measured weekly using a scaler. From this data, it was possible to determine the GrMG, using equation 5.

(5)

where GrMG depicts the MG growth rate (cm∙day-1), HMG the MG height (cm), and t the time to reach the corresponding height (days). On the other hand, the YMG was obtained from the monthly harvested mass of MG for each substrate per unit area (equation 6).

(6)

where YMG represents the MG yield per month (MG g∙m-2), mMG the MG dry mass (g) harvested every 30 days, and A the sowing area (m2).

Prior to statistical analysis, normality and homogeneity of variances were evaluated for HMG, GrMG, and YMG using a Shapiro-Wilk test (p > 0.05). After this, a paired t-test was used to compare the means for the variables before mentioned among PS and SS during the experiment. All the differences were statistically significant when p ≤ 0.05.

 

Organic matter and removal quantification

Organic matter (OM) analysis was performed in duplicate for SS and PS. A mass (W) of ~5 g was brought to 105 °C for 2 hours to remove moisture and obtain weight 1 (W1). Once W1 was determined, the samples were heated at 450 °C for 2 hours in the muffle and the weight 2 (W2) was recorded (Handbook, 1992). The OM content was determined by equation 7.

(7)

where OM represents the organic matter content (%), W is the initial mass of the sample, W1 the dehydrated mass (g) of the sample, and W2 the calcined final mass (g).

From the OM content, the organic matter removal (ηOM) was estimated at the end of each cultivation period using equation 8.

(8)

where ηOM represents the organic matter removal (%), OM1 the initial organic matter content (g), and OM2 the final organic matter content (g).

Chemical composition analysis of mombaza grass, sewage sludge, and pattern soil

From previously dehydrated samples, a mass of ~5 g of MG, SS, and PS was pulverized using a mortar with a pestle until obtaining a thin and homogeneous powder, <0.063 mm. Afterwards, the MG, PS, and SS chemical composition determination was carried out. The procedure was performed using a mass of ~10 mg of each sample. It was placed in an aluminum sample holder with double-sided graphite tape. The sample was covered with graphite using the thermal evaporation of carbon. The device utilized for this purpose was a Denton Vaccum brand coater, Desk Carbon Accessory model. The analyzes were carried out using a SEM JEOL IT300-LV equipment, with an EDS Bruker, Quantax: Xflash 6/30.

The SEM-EDS working conditions were escape angle at 35°, 20 keV, and WD = 10 ± 0.5 mm. The chemical mapping was carried out for 60 min in an surface area of ~3.5 mm2.

The SEM-EDS analysis were carried out at Laboratory of Scanning Electron Microscopy and Microanalysis of the Autonomous University of Guerrero.

 

RESULTS AND DISCUSSION

Moisture content in pattern soil and sewage sludge

Table 1 shows the initial average moisture present in the substrates used to germinate MG. It is observed that the PS presented a higher moisture content, ca. 15 % more than SS.

In economic terms, the moisture content in the SS is not a problem. The SS constitutes a waste generated by the WWTPs, therefore, its acquisition has not cost. In this sense, the production costs of the MG would not increase with the use of this nutritional source. However, in the case of using PS or another nutritional additive, it is necessary to consider its monetary value in the total production costs of the MG. Additionally, the variation of the moisture content in the PS affects the purchase price. In this work, 1 kg of PS was purchased at $6.00 MXN.

Mombaza grass germination time, height, and growth rate

The MG germination time in the two substrates used was notably dissimilar. The first germination shoots were observed in the seedbeds using PS as substrate. That happened five days after the MG seeds had sown. On the other hand, using SS as a substrate, the first germination shoots were observed until day 7. These values indicate that the germination of MG using PS requires less time, i.e., the difference among MG germination in the substrates was 2 days. On the other hand, Figure 3 shows the height reached by the MG during the first month of growth. During the first two weeks, it was observed that the HMG grown in PS was slightly higher than the height reached by the MG grown in SS. However, from the third and fourth weeks, the HMG showed a favorable change for the SS. The HMG grown in SS exceeded considerably the height reached in the PS. At the end of the first month, the maximum MG heights reached were 20 and 7 cm in SS and PS, respectively, i.e., the maximum HMG reached in SS was 2.85 times higher than the HMG reached in PS. These differences among both treatments were significant only for weeks 3 and 4 according to the paired t-test (third week: t = -6.348, p = 0.002; fourth week: t = -3.243, p = 0.017).

Unlike the first month (Figure 3), in the second and third months (Figure 4), the initial HMG reached during the first week of every month by the MG grown in the SS as substrate was higher than the HMG reached in PS. This behavior is attributed to the fact that the germination factor was eliminated in these months. That is, the monitoring of the height in these months was from previously germinated MG crops.

In the second month, a linear tendency to increase the height was observed over time in the case of MG grown on SS (Figure 4a). The equation that describes this behavior is y = 6.3x + 20.25 (R² = 0.9657).

However, in the case of MG grown in PS, the tendency to grow was lower, i.e., the slope of the growth curve was lower. This can be observed from the equation that describes this behavior y = 2.16x + 8.55 with R² = 0.8599.

This behavior is attributed to the fact that the MG cultivated on PS began gleaning from the third week of culture, affecting its growth (Figure 5). The HMG of the four weeks belonging to second month was significantly higher in SS compared to PS (paired t-test: p ≤ 0.05).

Soil quality is an important variable in the constitution of plant growth. However, climatic conditions (photoperiod, temperature, and humidity), annual seasons, and even management practices are also responsible for altering plants' flowering and fruiting periods (Velasco et al., 2018). One work carried out to evaluate grass production, dependent on defoliation frequency and height, showed that they also alter flowering (Febles et al., 2009; Márquez, 2014; Velasco et al., 2018). Once the grass is cut or harvested, a metabolic readjustment is generated that promotes a new leaf area formation. The purpose is to reestablish the plant's photosynthetic capacity; this, in turn, could promote flowering if the shoots are large enough or old (Febles et al., 2009). In this work, both crops were carried out under the same climatic and management conditions, therefore, the quality of the PS might be the main factor that caused the growth of MG to stagnate, reaching a plateau at a maximum height of 17 cm. On the other hand, in this second period, the maximum HMG reached in SS was 45 cm (Figure 4a). The HMG cultivated in SS was 2.64 times higher than the recorded in PS.

Finally, in the third month of cultivation, the HMG showed a similar behavior to that of the second month (Figure 4b). The growth curves were y = 8.8x + 7.5 with R² = 0.9954 and y = 2.5x + 6 with R² = 0.8993 for MG grown on SS and PS, respectively. In the third month, the maximum height observed was 42 and 15 cm for MG grown in SS and PS, respectively, i.e., the HMG grown in SS was 2.80 times higher than the height recorded in PS. Additionally, the monthly HMG reached in each substrate is shown in Figure 6. The MG heights among PS and SS were significantly different, both in the second (paired t-test: t = -8.216, p = 0.003) and third month (t = -4.238, p = 0.024).

The GrMG was another important factor that was analyzed. The Figure 7 shows the GrMG determined weekly during the MG cultivation. In the first week of the first month, GrMG was higher in PS. This was because of the MG in PS presented a shorter germination time compared to SS. However, from the second week on, the GrMG observed in SS began to grow linearly with time. In the fourth week, the first harvest or cut of the MG grown in both substrates was carried out. During the first week of the second month, the MG reached its maximum GrMG in each substrate. In case of the MG grown on SS, the maximum GrMG reached was 3.64 cm∙day-1 whereas in PS it was 1.40 cm∙day-1. In the following three weeks, GrMG showed a tendency to decrease in both crops. It was observed that as the HMG increased, the GrMG decreased (Figure 4 and 7). Finally, in the third month of cultivation, the GrMG showed a similar behavior to that of the second month. The Figure 8 shows the monthly GrMG. A paired t-test showed that the monthly GrMG among PS and SS were statistical different in the second (t = -3.287, p = 0.044) and third month (t = -13.98, p = 0.000).

The environmental temperature and those of the substrates was monitored during MG cultivation (Figure 9). In general, the environmental temperature was higher, in a range of 34 – 42 °C during the twelve weeks of experimentation. This recorded temperature is within the range established for the optimal growth temperature for MG: tropical climate. This climate characterizes by a monthly average temperature of ~24 °C and no frosts. Also, in the arid tropical climate, temperatures of 40 °C can be recorded, similar to those reached in this experiment (Papadakis, 1980).

On the other hand, in the first week of cultivation, it was observed that the temperature in both substrates was higher than that recorded in the environment. This is attributed to the fact that there was not a considerable HMG that prevented the supports from absorbing heat.

The environmental temperature was higher than the temperature measured directly on the supports from the second week on. It can be because each substrate already presented a significant MG growth density. Another aspect to highlight is that, in general, the PS temperature was always higher than the temperature determined in SS. The heat capacity of the substrates, related to the amount of water that each one retains can be the main reason because of this difference in temperatures was observed. The PS has a noteworthy effect absorbing water, whereas in SS the water percolates to the bottom of the seedbed. The PS has a silty structure, and the water retention phenomenon is larger than SS. A 60 % of the particles size of PS were <0.125 mm. On the other hand, the SS has a sandy structure (84 % of particles have a size >1 mm and 14.5 % have a size between 0.5 and 1 mm) and the porosity is bigger than the porosity observed by PS, 60 and 38 %, respectively. Thus, the defined structure present in PS leads to retaining more water in the porous space whereas the SS does not have a defined structure (Figure 2). Also, the porous structure and composition of SS promoted the evapotranspiration process. In PS the evapotranspiration phenomenon was noticeable lower than SS (Figure 2). In other words, the presence of water in the substrates affects their ability to absorb and/or release heat. Unlike other materials, water has a high heat capacity value. The heat capacity of water is 4187 kJ∙m-3 °C-1, and its specific heat 1000 cal (kg °C)-1. Specific heat represents the amount of heat that water requires to raise its temperature by one degree (AQS, 2005).

 

Mombaza grass yield

The YMG was calculated monthly (Figure 10, Table 2). The first month showed a maximum YMG of 30 and 146 MG g∙m-2 in PS and SS, respectively. In the first month, both crops needed about a week to germinate. For this reason, the YMG was lower compared to subsequent months. From the second month on, the MG on SS showed a YMG significantly higher than that observed in PS. It reached an average YMG of 416 MG g∙m-2 whereas PS reached an average YMG 72 MG g∙m-2. In the case of the MG cultivation on PS, in the second month the grass began to glean, this phenomenon affected the HMG and GrMG and consequently the YMG. The YMG values obtained from months 1 and 2 were analyzed using a paired t-test, which showed a statistically significant difference among SS and PS (p < 0.05). The third month was not possible to analyze because of the absence of replicates. However, in the third month, the HMG and GrMG values for MG cultivated in SS showed significant differences with the values obtained for the MG cultivated in PS (Figure 6 and 8). Moreover, the YMG depends on variables such as the HMG and GrMG. Therefore, the YMG value for third month showed by MG cultivated in SS would represent a difference statistically significant.

On the other hand, the YMG accumulated throughout the three months of cultivation was calculated (Table 2). The results showed a cumulative YMG ca. 1.0 MG kg∙m-2 using SS as substrate whereas in PS the YMG was 174 MG g∙m-2.

 

Organic matter removal

Throughout the MG cultivation time the OM content was monitored. It was determined that the initial OM content in the evaluated substrates were 36 and 15 % for SS and PS, respectively. This difference in OM content is one of the factors related to the quality of the substrate that could have affected the growth of MG in the PS. In terms of ηOM determined for PS, it was practically not observed. This value is consistent with the observed YMG (Table 2). On the other hand, a significant ηOM was observed in SS (Table 3). Once again, this value was closely related to the estimated monthly and quarterly YMG (Table 2). In general, the cultivation of MG on SS contributes with the ηOM reached in SS, ca. 12 %. Nevertheless, it is important to consider the OM natural degradation. It is another factor that contributes with the ηOM in soils. The substrates used showed a bulk density of 0.4217±0.0218 and 0.5313±0.0150 g∙cm-3 for PS and SS, respectively. The SS depicts a high and major porosity than PS, 60 and 38 %, respectively. The sandy soils such as SS substrate have a better aeration than PS. Hence, the rate of organic matter decomposition influences by chemical and microbial processes in SS is bigger than PS (Clapp et al., 1986). Thus, the major ηOM reached by SS was expected (Terry et al., 1979).

The removal value shown by SS (ca. 12 %) indicates that there is still enough OM that can be used to cultivate MG and that the crop can continue to be growing and obtain similar YMG to the values obtained if the physical space for the root growth was not limited.

It has been reported, the OM from SS exert significant influence on the physical, chemical, and biological properties of soils. The OM improves the soil's productive capacity, and its structure in terms of porosity and bulk density. Also, the water, air, and heat transmission are impacted. Finally, the soil strength is modified and translated into desirable physical conditions (Clapp et al., 1986). Nevertheless, a detailed chemical analysis must be carried out to know the SS composition prior to agricultural soils application. This type of analysis will prevent the dispersion and accumulation of potentially hazardous compounds such as trace heavy metals and toxic organic compounds (Clapp et al., 1986; González-Flores et al., 2011).

In addition to the previously described important aspects, another difference was observed in the MG cultures: the coloration grass. The MG grown on PS showed a solid green color. In contrast, the MG grown on SS showed a yellow coloration. The yellow color emergence in the leaves of MG is known as chlorosis (Durán Quiroz et al., 1998). This phenomenon originates in the leaf tissue due to the lack of chlorophyll. There are several causes associated with this color deficiency. Some of them are insufficient drainage, damaged roots, compacted roots, high alkalinity, and nutritional deficiencies of the plant. Nutritional deficiencies can occur because the soil is not rich in nutrients or nutrients are not available. Nutrients such as Ca, Mg, Co, Cu, Fe, Mn, Mo, and Zn can be present, although associated to five fractions according to a classification established by Tessier et al. (1979) for particulate trace metals. The particulate trace metals associated with fraction 1 (exchangeable) can be released by a simple change in water ionic composition, whereas the fraction 2 (bound to carbonates) is a pH function. A slightly acid rain phenomenon can lead the trace metals releasing (Krauskopf and Bird, 2003). The particulate trace metals associated with fraction 2 can be released when the environmental conditions reach a pH value between 6.5–7.5. In fraction 3, the particulate trace metals are bound to iron and manganese oxides and under anoxic conditions they are thermodynamically unstable (low Eh value). On the other hand, the particulate trace metals bounded with organic matter (faction 4) requires oxidizing conditions to release soluble nutrients through the organic matter degradation. The SS usually can contain large quantities of trace metals associated to organic matter and the organic matter oxidation control their release (Clapp et al., 1986). Finally, the particulate trace metals associated with fraction 5 (residual) are not expected to be released in solution. The releasing of particulate trace metals from fraction 5, needs conditions normally not encountered in a natural environment. In other words, the nutrients solubility and geodisponibility such as particulate trace metals are in function of environmental conditions (Tessier et al., 1979; González-Flores et al., 2011). Fe, Zn, or Mn inadequacy can cause the chlorosis phenomenon (Oliveira Prendes et al., 2006). Alvira Serrano (2019) carried out a chemical-mineralogical characterization of SS of the Taxco de Alarcón WWTP. In his research results, he reported a pH of 7.1 for the SS and total concentrations of 19.38, 3.13, and 1.83 g of Fe, Zn, and Mn per kg of SS, respectively. However, in fraction 4, concentrations of 6.25, 0.70, and 0.20 g per kg of SS were found for Fe, Zn, and Mn, respectively. Also, in fraction 3, important concentrations were reported: 0.13, 0.70, and 0.50 g per kg of SS for Fe, Zn, and Mn, respectively. Finally, in fractions with major geodisponibility (fractions 1 and 2), the sum reached the following values: 4.33, 1.72, and 1.14 g per kg of SS for Fe, Zn, and Mn, respectively. Hence, pH and availability of nutrients could not be part of the reasons that cause chlorosis in the MG grown in SS.

In the case of PS, the growth of MG kept a relationship with the quantity and size of the roots, i.e., the growth of the roots was abundant, fibrous, or fasciculate and they developed near the surface (Figure 11a). In the case of MG grown in SS, its roots were thicker and longer. Root mass per unit area was lower in SS. The roots dispersed in all directions within the SS. The length of these roots extended to the seedbeds depth where a drainage system was lacking (Figure 11b). In this area, due to the texture of the SS (sandy structure) and porosity (60 %) when the water irrigation was carried out, the water infiltrated and accumulated mainly at the bottom. From this, the MG cultivated in SS developed roots longer than in PS. Thus, the main cause of chlorosis observed in MG grown in SS can be related to the drainage lack in seedbeds.