Microbial Growth in Shrimp Ponds as Influenced by Monosilicic and Polysilicic Acids

doi:10.21203/rs.3.rs-908767/v1

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Microbial Growth in Shrimp Ponds as Influenced by Monosilicic and Polysilicic Acids

https://doi.org/10.21203/rs.3.rs-908767/v1

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published 18 Jan, 2022

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In order to increase shrimp production and minimize detrimental environmental impacts of aquaculture, the maintenance and regulation of the growth and composition of phytoplankton communities and nutritional balance are critical. Silicon (Si) is an essential nutrient for diatoms and other types of microorganisms, but the information about the Si impact on their growth is extremely scarce. Monosilicic and polysilicic acids were tested in several shrimp cultivation systems in Jiangsu Province, China. In pond waters, the concentrations of monosilicic and polysilicic acids sharply reduced by 36–95% and 35–75%, accordingly, as compared with those in supply water sources. The microbial cell abundance was strongly dependent on monosilicic acid, while the correlation with polysilicic acid was absent. In laboratory experiments, monosilicic acid added to pond water or probiotic solution at 1 and 2 mM Si had a significant positive effect on cell abundance. Over three days, the concentrations of monosilicic acid decreased by 81 to 91% in pond water and by 11 to 24% in probiotic solution. In probiotic solutions, the degree of polymerization of silicic acid was more intensive than that in shrimp pond waters. The data obtained demonstrates the importance of systematic studies related to the functions of Si in shrimp aquaculture.

Electronic Materials and Devices

Optical Materials and Devices

bacteria

microbial ecology

monosilicic acid

polysilicic acids

silicon

Shrimp farming production is one of the fastest-growing segments of the world agriculture with a forecast of the compound annual growth rate in the period 2017–2021 being 4.8 to 5.4% (Anderson et al. 2016; 2019). Undoubtedly, shrimp farming management should be based on a systems approach including the application of different materials to provide high productivity and quality and minimize negative impact on the environment (Ray and Lotz, 2017; Sarkar et al. 2019). To develop an efficient management strategy, understanding of the factors that maintain and control shrimp production is required. In the aquaculture system, the microbial community plays a significant role in providing additional feed, enhancing nutrient utilization efficiency, reducing anoxic conditions, and minimizing environmental impacts (Denev et al. 2009).

Silicon (Si) is the second abundant element after oxygen on our planet. The role and functions of Si in plant physiology, veterinary, and medicine have been investigated for more than 200 years (Chepeleva et al. 2018; Szacawa et al. 2019). Much Si-related research was conducted for plants and soil-plant system (Snyder et al. 2016). Over the last decade, Si has been recognized as an essential trace element in the metabolism of higher plants and animals (Michalak & Chojnacka 2018, Peris-Felipo et al. 2020). In plant biology and agriculture, Si has been declared a beneficial element being particularly important for the immune system induction in response to abiotic and biotic stresses (Guerriero et al. 2016). Improved Si nutrition has a multi-effect on plant growth and cell functioning (Ma et al. 2001). Silicon-mediated impacts like sugar formation, DNA stability, transport regulation (activated Fe, B, K, Ca transport and reduced that of Cd, Hg, As, Na) have been reported as a result of Si addition (Bocharnikova et al. 2014; Hernandez-Apaolaza 2014, Adrees et al. 2015).

Several forms of Si are commonly presented in natural water: monosilicic acid, polysilicic acid (oligomers and polymers with large number of Si atoms), and organo-silicon compounds (Iler 1979; Dietzel 2002; Bocharnikova and Matichenkov 2012). However, plants uptake Si only in the form of monosilicic acid (Ma et al. 2001). Although polysilicic acid is soluble, it is not absorbed by plants (Bocharnikova and Matichenkov 2012).

In the aquatic system, Si is recognized as a key nutrient for diatoms and some sponges. It is also consumed by radiolarians, silicoflagellates, several species of choanoflagellates, and some picocyanobacteria (Tréguer and Rocha 2013). Diatoms have silicified frustules and require a high level of Si in the form of monosilicic acid (Leynaert et al. 2009). Diatom populations are responsible for ~ 50% of marine net primary production and provide the base of marine food chains (Nelson 1995). The predominance of diatoms in shrimp ponds is highly desired because of nutritional properties. In the presence of diatoms, biochemical composition of shrimp is characterized by higher proteins, lipids, essential amino acids and unsaturated fatty acids (Brito et al. 2016; de Abreu et al. 2019). Silicon fertilization is a common approach to encourage diatom growth (Martins et al. 2016; Llario et al. 2019).

Among serious problems faced by shrimp farming are infectious diseases and environmental deterioration. Probiotics are successfully used to overcome these challenges (Lakshmi et al. 2013; van Hai & Fotedar 2010). Probiotics improve growth performance, stimulate immune responses, enhance disease resistance of shrimp, inhibit growth of pathogens as well as improve water quality parameters (Hoseinifar et al. 2018). Probiotics usually include different bacteria, bacteriophages, microalgae, and yeast species (Llewellyn et al. 2014; Wang et al. 2019) There are monostrain, multistrain and multispecies probiotics (Das et al. 2017; Van Doan et al. 2017). Information about the effect of Si on the metabolism of microorganisms other than diatoms and its impact on their growth is extremely scarce (Wainwright et al. 1997; Hurst et al. 2007).

Knowledge about the relationship between Si application rates and mono- and polysilicic acid concentrations in pond water, and how fast Si disappears from water as well as the relationship between mono- and polysilicic acid concentrations and microbial growth is critical to manage the microbial community. The aim of this study was to evaluate the concentrations of monosilicic and polysilicic acids in shrimp ponds and to determine the effect of monosilicic acid on microbial growth in pond water and in a probiotic solution.

Field tests

Water samples were collected on 3 shrimp farms located in Jiangsu Province, China, in April. The first farm (near Huan Gangerzu Town; 32^o 30’26” N; 121^o10’11” E) used greenhouse-enclosed raceways for intensive shrimp production. Fresh groundwater was directly pumped into separated ponds under a greenhouse, each individual pond was 6x50 m in size. The shrimp age was 2 months. Water was sampled from well, 6 ponds, and a small creek filled with effluents from ponds.

The second shrimp farm (near Changsha City; 32^o23’31” N; 121^o18’09” E) had a system of open ponds (100 x 50 m each), filled with unsalted water from a local canal. Thee shrimps were 2 months old. Water was sampled from the canal and 6 ponds.

The third shrimp farm (near Yangkou Town; 32^o35’44” N, 120^o59’09” E) took water directly from the Yellow Sea. Open ponds were 250 x 60 m each. Water was collected from 6 ponds, and a canal supplied water to the ponds. The shrimps were 1 month old.

Samples were collected in triplicate in 100 mL plastic bottles early in the morning and were immediately transported to a laboratory to determine monosilicic acid, polysilicic acid, pH and microbial cell abundance.

The pH was analyzed on an Ion Meter PHS-3e (MRC, China). The concentration of monosilicic acid was determined using the modified molybdenum blue method (Mullin and Riley, 1955) with a spectrophotometer V5800 XZBELEC (China). This method tests Si only in the form of monosilicic acid, without interference from phosphorus.

To analyze polysilicic acid, 2 g of NaOH was added to 20 mL of centrifuged water and kept in a refrigerator at + 4^oC for 2 weeks (Matichenkov et al. 1997). During this time all polymers of silicic acid are transformed into monomers. After that monosilicic acid was determined as described above. The concentration of polysilicic acid was calculated by the following formula:

Si_poly = Si_total – Si_mono,

where Si_total is the concentration of monosilicic acid determined after depolymerization;

Si_mono is the concentration of monosilicic acid determined before depolymerization.

Microbial abundance (cells mL^− 1) was estimated on the day of sampling by binocular biological microscope (ML10, Guangzhou Mingmei, China) according to the method proposed by Newell and Newell (2006). The same method was used to calculate the cell density in the laboratory experiment.

Laboratory experiment

A laboratory experiment was conducted with collected farm water summarized samples and 3 commercial probiotics used in shrimp farming: dry probiotics “Ecopro” and “Ecopro Cold” (Ecomicrobial Co, USA) and liquid probiotic “HeJunMei” (Jiangsu Aijiafuru Soil Remediation Co, China). In dry and liquid probiotics, the amounts of bacteria and yeast spores were not less than 1x10¹² and 1x10¹⁰cells kg^− 1, respectively.

To activate dry probiotics, 1 g of probiotic was mixed with 1 L of sterilized distilled water (DW) and kept at + 24^oC for 24 h. The liquid probiotic was diluted 1:10.

One liter of nutrient solution was prepared with K₂HPO₄ 3.125 g; KH₂PO₄ 3.125 g; (NH₄)₂HPO₄ 3.125 g; MgSO₄.7H₂O 0.25 g; FeSO₄.7H₂O 0.0125 g; MnSO₄.7H₂O 0.00875 g and sucrose 12.5 g (Vitullo et al. 2012). Eighty (80) mL of this nutrient solution was added to each flask. Ten (10) mL of pond water collected on the day of sampling, probiotic solution or DW was added to the flasks. Considering that Farm 3 used sea water, NaCl (35 g L^− 1) was added to flasks with pond water from Farm 3. Then 10 mL of DW or monosilicic acid solution at 10 and 20 mM Si was added to reach the Si concentrations of 0, 1 and 2 mM. Monosilicic acid solutions were prepared from concentrated monosilicic acid (Fisher Scientific, CAS-No 7699-41-4). The pH in each flask was adjusted to 7 by adding 0.1 M HCl or 0.1 M NaCl.

Flasks were kept in a climatic chamber at 24 ± 1^oC. The light/night regime was 12/12 hours with light intensity 600 µmol photons м^-2 s^-1. Flasks were aerated twice a day for 1 h (morning and evening). After 3 days, the concentration of monosilicic acid and the density of microorganisms were determined using the method described above.

Each treatment and each analysis were conducted in triplicate. All data obtained was subjected to a statistical analysis based on comparative methods using Duncan’s multiple range tests for mean separation at the 5% level of significance (Duncan 1957).

The pH, concentration of mono- and poly-silicic acids, and density of microorganisms in tested solutions are presented in Table 1. Monosilicic acid in water supplied to shrimp ponds differed greatly among farms, from 49.3 to 517.0 µM Si, with a higher value in fresh underground water (Farm 1) and minimum value in coastal sea water (Farm 3). Although the maximum polysilicic acid also was in the fresh underground water, its proportion increased: Farm 1 < Farm 2 < Farm 3 and accounted for 3.1; 10.0; and 16.6 %, respectively.

Water was pumped to shrimp pond daily on all farms. In ponds, the concentrations of monosilicic acid were remarkably lower as compared with incoming water: by 26.3 times (517.0 vs 19.6 µM Si), 6.4 times (100.0 vs 15.6 µM Si), and 2.8 times (49.3 vs 17.3 µM Si), respectively on Farm 1, Farm 2, and Farm 3. The concentrations of polysilicic acid decreased as well, but not as significantly.

The cell abundance in ponds of Farm 1 was higher than in others, probably due to more intensive farming system. However, the microbial densities differed, sometimes significantly, between ponds of each farm. For example, on Farm 1, the cell numbers ranged between 2.4 ± 0.1 and 3.9 ± 0.2 x10⁵ mL^− 1, while on Farm 2 and Farm 3 the cell numbers ranged from 1.1 ± 0.1 to 1.6 ± 0.2 x10⁵ mL^− 1 and from 1.4 ± 0.2 to 2.0 ± 0.2 x10⁵ mL^− 1, respectively.

The numbers of microbial cells and soluble forms of Si in the laboratory test are presented in Table 2. Supplementation of monosilicic acid significantly increased the microbial density, up to 60 and 33% in pond water and probiotic solution, respectively.

Over 3 days, the concentration of monosilicic acid decreased in all microorganism-containing solutions in comparison with the corresponding sterile solutions. Remarkable reductions in monosilicic acid were detected in all pond water samples, whereas probiotic solutions demonstrated much smaller changes.

Initial silicic acid contained NaOH to prevent polymerization; therefore, were no polymers. Since in the experiment the pH was 7, the polysilicic acid formation proceeded in both sterile and nonsterile solutions. The process of polymerization was more intense in pond water and especially in probiotic solutions. The polysilicic acid concentration reached up to 230 ± 21 mg L^− 1 Si in liquid probiotic as compared to 10.5 ± 0.3 mg L^− 1Si in the corresponding sterile solution.

In the experiment, the concentrations of dissolved Si (DSi) (monosilicic acid + polysilicic acid) in supply water sources were 533.1 µmol (groundwater, Farm 1), 110.0 µmol (fresh water canal, Farm 2) and 57.53 µmol (sea water, Farm 3). This data is consistent with reported DSi concentrations in groundwater (Khan et al. 2015), surface terrestrial waters (Zhang et al. 2015), and ocean water (Zhao et al. 2000; Wei et al. 2015).

In tested shrimp ponds, DSi ranged from 14.0 to 60.7 µM Si on Farm 1, from 17.8 to 23.5 µM Si on Farm 2 and from 19.7 to 26.4 µM Si on Farm 3 which corresponds to the available data. For example, DSi in low salinity shrimp ponds in Alabama ranges between 3.4 and 196 µM Si with an average of 37.0 µM (Prapaiwong and Boyd 2014). It is important to note that we observed very fast reductions in pond water Si, in spite of daily water exchange. The monosilicic acid concentration decreased more than the polysilicic acid (Table 3). It is well known that higher plants take up Si only in the form of monosilicic acid. Perhaps algae, being phototrophic organisms like higher plants, have the same mode of Si uptake. With decreasing monosilicic acid, equilibrium between soluble forms of Si shifts, resulting in acceleration of depolymerization, which is typical for the systems with low concentrations of monosilicic acid, in turn leading to decreasing polysilicic acid (Iler 1979).

The correlation coefficients between soluble forms of Si and cell abundance evidence that the number of microorganisms in ponds correlates positively with monosilicic acid (R = 0.80–0.84) (Table 4). There was no correlation between cell abundance and polysilicic acid. Therefore, unlike polysilicic acid, monosilicic acid is an essential factor in the regulation of microbial growth in the shrimp pond.

The laboratory experiment has shown that monosilicic acid affected beneficially microbial population in pond water and probiotics solution (Table 2). In pond water, Si may be consumed mainly by different algae species, including diatoms. The tested probiotics contained only bacteria having less need for Si, though additional Si benefitted their growth as well. The increase in polysilicic acid with the addition of monosilicic acid could be the result of polymerization (Table 2). Monosilicic acid can be physically adsorbed on the cell wall leading to initiation of the polymerization process (Matichenkov et al. 2001). The formation of polymers was higher in probiotic solutions. Although a significance of this process in shrimp cultivation is unknown, Si polymers generally possess high adsorption properties to organic and inorganic molecules (Parida et al. 2006; Xia et al. 2008). Thus, new formed silica-gel (polymerized silicic acid) may adsorb organic compounds and nutrients promoting attraction of microorganisms and floc formation. This hypothesis requires further confirmation.

As is well known, diatoms require high level of DSi for growth (Sun et al. 2013; Tantanasarit et al. 2013; Matsumoto et al. 2017). Minimum DSi required for diatom growth was shown to be 0.1 mg L^− 1 (3.57 µM Si) (Reynolds 2006). Early research reported that the monosilicic acid concentrations of 8.1–81.0 µM Si benefit the cultivation of diatoms (Mast and Pace 1937). Soluble Si at 714 µM stimulated respiration of diatoms (Lewin 1955). As recently shown, suitable Si concentration in a growth medium for diatoms amounts to 470 µM as Na₂SiO₃*5H₂O (Ali et al. 2017). Optimal Si concentrations for diatoms depend on the species, as well as the content of other elements. For example, Adams and Bugbee (2014) found that the maximum dry mass density of diatoms (Chaetoceros gracilis) was observed at the Si concentrations of 200–400 µM and 600–800 µM, respectively in highly salted water (400 mM Na) and low salted water (50 mM Na).

With decreasing the Si concentration, other phytoplanktonic algae that do not need so much Si can replace diatoms (Boyd 2014). Among undesirable algae species, blue-green algae are of particular concern. Blooms of blue-green algae cause a lack of dissolved oxygen, off-flavors problems, and toxin formation, thus deteriorating water quality and declining shrimp productivity (Jescovitch et al. 2018). Evidently, the abundance of silicic acid is an essential requirement to achieve desirable diatom domination in algal communities. However, no systematic studies have been conducted showing the Si limitation and influence of its addition on shrimp production.

The obtained data has demonstrated that all tested shrimp ponds were characterized by extremely low concentrations of monosilicic acid, while the supply waters originally were high in DSi. Monosilicic acid applied to shrimp pond water or probiotic solution significantly increased the microbial cell abundance. It is important to distinguish monomeric and polymeric forms of DSi, because these substances affect microbial population in aquaculture in different ways.

Funding

This study was supported by the Ministry of Science and Higher Education of RF, theme #АААА-А17-117030110137-5 and #AAAA-A17-117030110139-9. This research also has been funded by Jiangsu Province's "provincial entrepreneurship and innovation talent plan" and Nantong's "Jianghai talent plan".

Conflict of interest

Dr. Ruiping Zhang declares that she has no conflict of interest.
Dr. Elena Bocharnikova declares that she has no conflict of interest.

Prof. Vladimir Matichenkov declares that he has no conflict of interest.

Availability of data and materials

All data availability statement is present within the text of the manuscript

Code availability

Not available

Authors’ contributions

Dr. Ruiping Zhang participate in sample collection, analysis and manuscript writing, Dr. Elena Bocharnikova is participated in the analysis, laboratory test and manuscript writing, Prof. Vladimir Matichenkov is participated in the sample collection, laboratory experiments and manuscript preparation.

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors

Consent to participate

All authors are agreed to be listed as authors in current version of manuscript.

Consent for publication

All authors are agreed for publication of tis manuscript in the Applied Microbiology and Biotechnology.

Conflict of Interest

There is no conflict of interest

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Table 1

Mono- and poly-silicic acids, pH and abundance of microbial cells in waters sampled on shrimp farms.
Sample, pond #	pH	Monosilicic acid	Polysilicic acid	x10⁵ cells mL^− 1
Sample, pond #	pH	--------------Si, µM ------------		x10⁵ cells mL^− 1
Farm 1
Supply water	7.27 ± 0.11	517.0 ± 25.4	16.1 ± 0.3	n/d
1	7.36 ± 0.13	8.6 ± 0.4	5.4 ± 0.2	2.5 ± 0.2
2	7.50 ± 0.12	52.1 ± 2.4	8.6 ± 0.2	3.9 ± 0.2
3	8.44 ± 0.13	13.2 ± 1.3	4.3 ± 0.1	2.4 ± 0.1
4	7.36 ± 0.13	17.5 ± 1.5	3.9 ± 0.1	3.2 ± 0.2
5	7.58 ± 0.11	13.5 ± 1.4	8.6 ± 0.2	3.4 ± 0.3
6	8.25 ± 0.11	12.5 ± 0.5	9.3 ± 0.3	2.6 ± 0.1
Average for ponds	7.74	19.6 ± 2.1	6.7 ± 0.2	3.1
Farm 2
Supply water	8.43 ± 0.12	100.0 ± 15.4	10.0 ± 0.3	0.2 ± 0.1
1	8.65 ± 0.11	20.0 ± 1.1	3.5 ± 0.2	1.6 ± 0.2
2	8.58 ± 0.10	15.4 ± 1.2	4.3 ± 0.2	1.4 ± 0.2
3	8.55 ± 0.10	12.8 ± 1.0	5.0 ± 0.2	1.1 ± 0.3
4	8.65 ± 0.12	19.3 ± 1.3	4.3 ± 0.1	1.3 ± 0.2
5	8.53 ± 0.12	15.0 ± 1.2	5.4 ± 0.2	1.2 ± 0.2
6	8.43 ± 0.11	11.4 ± 1.1	6.4 ± 0.2	1.1 ± 0.1
Average for ponds	8.56	15.6 ± 1.4	4.8 ± 0.1	1.25
Farm 3
Supply water	8.32 ± 0.11	49.3 ± 3.5	8.2 ± 0.3	0.1 ± 0.1
1	8.46 ± 0.12	16.1 ± 1.3	3.6 ± 0.2	1.5 ± 0.2
2	8.34 ± 0.13	15.4 ± 1.3	3.6 ± 0.2	1.7 ± 0.2
3	8.38 ± 0.11	21.8 ± 1.4	4.6 ± 0.3	2.0 ± 0.2
4	8.42 ± 0.10	18.2 ± 1.2	5.3 ± 0.3	1.8 ± 0.3
5	8.43 ± 0.11	15.4 ± 1.1	4.3 ± 0.2	1.4 ± 0.2
6	8.37 ± 0.11	17.1 ± 1,3	3.9 ± 0.2	1.5 ± 0.2
Average for ponds	8.40	17.3 ± 1.2	4.2 ± 0.1	1.60

Table 2

Abundance of microbial cells and silicic acid concentration after 3-day incubation, laboratory experiment.
	x10⁵ cells mL^− 1	Cell increase, %	Monosilicic acid	Polysilicic acid
	x10⁵ cells mL^− 1	Cell increase, %	--------------- Si, µM ---------------
Sterile solution
Control	n/d	-	1.5 ± 0.3	0.1 ± 0.1
Si, 1 mM	n/d	-	994.5 ± 0.7	0.3 ± 0.1
Si, 2 mM	n/d	-	1983.5 ± 1.5	10.5 ± 0.3
Farm 1
Control	2.1 ± 0.1	-	1.4 ± 0.2	0.2 ± 0.1
Si, 1 mM	2.5 ± 0.1	19.0	151.4 ± 13.1	15.3 ± 0.7
Si, 2 mM	2.9 ± 0.2	38.1	202.3 ± 15.3	47.5 ± 0.9
Farm 2
Control	2.0 ± 0.1	-	1.2 ± 0.2	0.3 ± 0.1
Si, 1 mM	2.4 ± 0.1	20.0	142.3 ± 10.1	27.4 ± 0.9
Si, 2 mM	2.7 ± 0.1	35.0	183.3 ± 10.3	48.5 ± 1.4
Farm 3
Control	1.5 ± 0.1	-	1.1 ± 0.1	0.2 ± 0.1
Si,1mM	1.9 ± 0.2	26.7	186.4 ± 11.2	18.4 ± 0.5
Si, 2 mM	2.4 ± 0.2	60.0	254.2 ± 16.8	57.5 ± 0.8
Ecopro
Control	1.3 ± 0.1	-	0.3 ± 0.1	0.1 ± 0.1
Si,1 mM	1.4 ± 0.1	7.7	755 ± 45	92 ± 12
Si, 2 mM	1.6 ± 0.2	23.1	1618 ± 120	133 ± 21
Ecopro Cold
Control	1.2 ± 0.1	-	0.2 ± 0.1	0.1 ± 0.1
Si, 1 mM	1.4 ± 0.1	16.6	816 ± 27	25 ± 1.5
Si, 2 mM	1.6 ± 0.1	33.3	1633 ± 127	150 ± 15
HeJunMei
Control	1.4 ± 0.1	-	0.4 ± 0.1	0.1 ± 0.1
Si, 1 mM	1.6 ± 0.2	14.3	833 ± 32	167 ± 14
Si, 2 mM	1.8 ± 0.2	28.6	1770 ± 125	230 ± 21

n/d – not detected

Table 3

Reductions of monosilicic and polysilicic acids in pond water in comparison with those in the corresponding supply water, %.
Pond, #	Reduction, %
	Monosilicic acid	Polysilicic acid	Monosilicic acid	Polysilicic acid	Monosilicic acid	Polysilicic acid
	Farm 1		Farm 2		Farm 3
1	98.3	66.4	80.0	65.0	67.3	63.4
2	89.9	46.6	84.6	60.0	68.8	56.1
3	97.4	73.3	87.2	50.0	55.8	43.9
4	96.6	75.8	80.7	57.0	63.1	35.4
5	97.5	46.6	85.0	46.0	68.8	47.6
6	97.6	42.2	88.6	36.0	65.3	52.4
Average	96.2	58.4	84.4	52.0	64.9	48.8

Table 4

Correlation coefficients (R) between cell abundance and soluble forms of Si in pond water.
Farm #	Monosilicic acid	Polysilicic acid
1	0.80	0.41
2	0.83	-0.87
3	0.84	0.52

Table 5

Changes in monosilicic and polysilicic acids in pond waters and probiotic solutions in comparison with the corresponding sterile solutions, %.
Solution	Si, 1 mM		Si, 2 mM
	Monosilicic acid	Polysilicic acid	Monosilicic acid	Polysilicic acid
Farm 1	-84.8	+ 5000	-89.8	+ 352
Farm 2	-85.7	+ 9033	-90.8	+ 362
Farm 3	-81.2	+ 6033	-87.2	+ 447
Ecopro	-24.1	+ 30567	-18.4	+ 2090
Ecopro Cold	-17.9	+ 8233	-17.7	+ 1328
HeJunMei	-16.2	+ 55566	-10.7	+ 2090

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published 18 Jan, 2022

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Microbial Growth in Shrimp Ponds as Influenced by Monosilicic and Polysilicic Acids

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